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the dots should be next to submerged

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replace with: LOONY's

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the dots should be next to caress, not in a new line

Could we add a refence to my book here, where I write about this issue? Also, if we want to keep our initials for the notes, this would end with: (LS). The reference to my book is: See L. Scuriatti, Mina Loy's Critical Modernism (Gainesville: University of Florida Press, 2019), pp. 113-116.

add space. It should read: museums and

replace "souls to" with: soul's

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replace with: anticipate

replace "for autumn" with: for the autumn

replace with: "Has to have"

replace with: DIANA

replace with: Futurism

Replace with HOUSELESS LOONY

replace with LADY DIANA

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There does not seem to be a line break in Crangle's edition, but it is not clear. Is there a break in The Dial?

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dots should be next to "you", and not in a new line

the three dots should not be on a new line, but next to frost

it should read "into the Arabic" - although it sounds wrong. Either we correct it and add [sic], or insert an excision sign to show that we have intervened onto the text

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Italics. "With" should be "with"

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hyphenate: steam-heating

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Capitalized: People

Differentiator - add container above about why this approach works

layout as columns?

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XD this sounds fun

Such is the risk of limiting beliefs.

"He who looks for external validation is not properly grounded in life." -- Marcus Aurelius (20 June 2024 future edit, this must be Epictetus)

In other words, do not care about what others think... Heed their advice, take it into account, but ultimately you must make the decision yourself.

YAML is not generally considered easily editable by humans

DataCite is not a data repository.

doing something that is different from what people consider to be normal or acceptable

doing something that is different from what people consider to be normal or acceptable

AIM for Climate Investments

PREPARE and VACS: $50 million announced at COP28

Agriculture Innovation Mission AIM for Climate

PREPARE and the Vision for Adapted Crops and Soils multi-donor funding platform

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and frustrated?

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Love this!

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Plus

You said people join for community Highlight main benefit What's the final impact on the biz: growth?

Target customer pain point Desire Then introduce

nn.CrossEntropyLoss是PyTorch中的一个类，它实现了交叉熵损失函数。交叉熵损失函数常用于多分类问题，它可以度量模型的预测概率分布与真实概率分布之间的差异。

reduction='none'是一个参数，它指定了如何对每个样本的损失进行聚合。'none'表示不进行聚合，即返回一个损失值的向量，向量的每个元素对应一个样本的损失。其他可能的值包括'mean'（返回所有样本损失的平均值）和'sum'（返回所有样本损失的总和）。

在 train_ch3 → train_epoch_ch3 中内置优化器是 l.mean().backwar()

在这个例子中，我们选择'none'是因为我们想要在后续的计算中手动处理每个样本的损失，例如，我们可能想要计算每个样本损失的平均值，或者只关注损失最大的几个样本。

Résumé de la vidéo [00:00:05][^1^][1] - [00:22:49][^2^][2] : La vidéo présente une recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique" menée à l'Université Saint-Louis. Elle aborde les défis de l'échec universitaire et les moyens de le surmonter en réévaluant les méthodes pédagogiques et en soutenant les étudiants de manière plus personnalisée.

Points forts : + [00:00:05][^3^][3] Contexte et objectifs de la recherche * Lancement de la recherche dans le cadre de la fusion des universités * Objectif de comprendre et d'adresser l'échec étudiant * Financement par l'université pour une approche collective + [00:08:01][^4^][4] Méthodologie et résultats préliminaires * Suivi d'une cohorte d'étudiants sur trois ans * Identification de profils d'étudiants et de leurs chances de réussite * Importance de l'adaptation des méthodes pédagogiques aux besoins des étudiants + [00:13:00][^5^][5] Profil des étudiants à l'entrée de l'université * Étudiants investis mais inégalement informés et préparés * Confiance en la capacité de réussir malgré des préparations diverses * Six profils d'étudiants identifiés avec des chances de réussite variables + [00:19:00][^6^][6] Expérience des étudiants à l'université * Difficultés d'adaptation à l'autonomie universitaire * Variabilité dans l'appréciation des méthodes d'enseignement * Nécessité d'ajuster les méthodes de travail pour la réussite

Résumé de la vidéo [00:22:51][^1^][1] - [00:44:26][^2^][2] : La vidéo aborde la recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle examine les expériences des étudiants durant le premier quadrimestre à l'université, leurs attentes envers les enseignants et l'institution, et l'impact des profils d'entrée sur la réussite académique.

Points forts : + [00:22:51][^3^][3] Expérience du premier quadrimestre * Transition difficile pour les étudiants * Questions sur les attentes et la manière de répondre à ces attentes * Gestion de l'autonomie et attentes envers les enseignants + [00:23:48][^4^][4] Rôle de l'enseignant * Doit être un expert et proche des étudiants * Importance de l'empathie et de l'accompagnement * Les cours doivent être utiles, structurés et bien soutenus + [00:27:00][^5^][5] Hétérogénéité des expériences étudiantes * Différences dans la façon de vivre l'expérience universitaire * Le profil d'entrée n'influence pas directement l'expérience universitaire * Importance de l'assistance et des ressources institutionnelles + [00:32:01][^6^][6] Facteurs de réussite académique * Objectifs académiques et heures de travail personnel * Impact des profils d'entrée sur la réussite * Nécessité de changer les paramètres de fonctionnement de l'université + [00:37:31][^7^][7] Satisfaction et sens dans l'expérience universitaire * Satisfaction non corrélée à la réussite * Les étudiants les plus satisfaits sont souvent parmi les profils les plus fragiles * Importance de la clarté des attentes et de la communication avec les étudiants + [00:42:07][^8^][8] Recommandations pour l'amélioration * Travailler en amont avec l'enseignement obligatoire * Informer sur l'hétérogénéité des publics * Intégrer des tests réflexifs dans le cursus académique

Résumé de la vidéo [00:44:28][^1^][1] - [01:05:38][^2^][2]:

La vidéo présente une recherche-action sur la lutte contre l'échec scolaire et la redéfinition de la relation pédagogique. Elle aborde les propositions pour améliorer le suivi des étudiants, l'importance de ne pas présumer de leur autonomie, et la nécessité de restructurer le calendrier universitaire.

Points saillants: + [00:44:28][^3^][3] Suivi individualisé des étudiants * Proposition de suivis universels mais personnalisés * Importance d'accompagner les étudiants vers l'autonomie * Nécessité d'évaluer la compréhension des consignes + [00:45:26][^4^][4] Apprentissage du travail personnel * Focalisation sur le travail personnel en dehors des cours * Proposition d'organiser des semaines types pour guider les étudiants * Importance de la quantité et de la qualité du travail personnel + [00:46:24][^5^][5] Réorganisation du cadrimestre * La première session arrive trop tard pour les étudiants * Proposition de découper le premier cadrimestre en deux parties * Importance des retours et feedbacks après les mini-sessions + [00:51:01][^6^][6] Débat politique sur l'éducation * Discussion entre journalistes et politiciens sur les thèmes abordés * Échanges sur l'encadrement des étudiants et l'orientation scolaire * Débat sur l'impact du décret paysage et le calendrier universitaire

Résumé de la vidéo [01:05:39][^1^][1] - [01:23:45][^2^][2]:

La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des stratégies pour combattre les inégalités dans l'éducation, notamment en améliorant l'orientation des étudiants et en gérant mieux la transition entre l'enseignement secondaire et supérieur. Ils soulignent l'importance d'une analyse à long terme pour suivre les progrès des étudiants et abordent le rôle des facteurs socio-économiques dans la réussite éducative.

Points saillants: + [01:05:39][^3^][3] Propositions du PS pour l'éducation * Mettre en place un observatoire de la vie étudiante * Améliorer l'orientation et la transition entre les niveaux d'enseignement * Attaquer les déterminants socio-économiques de l'échec + [01:08:27][^4^][4] Approche du PTB sur la taille des classes * Réduire la taille des classes pour une meilleure attention individuelle * Proposer des classes de 15 à 17 élèves jusqu'à 8 ans et environ 20 élèves après * Lier la taille des classes à la diminution des inégalités et à la réussite + [01:12:01][^5^][5] Coût des études universitaires selon le PTB * Réduire les frais pour atténuer les inégalités * Proposer des repas à 2 € et diminuer le prix des côtes * Tendre vers la gratuité du minerval + [01:14:43][^6^][6] Formation des enseignants et assistants * Améliorer la formation des enseignants pour traiter les lacunes du secondaire * Utiliser les compétences des professeurs de promotion sociale * Assurer un suivi adéquat des étudiants pour leur réussite

Résumé de la vidéo [01:23:46][^1^][1] - [01:44:37][^2^][2] : La vidéo présente un débat politique sur la recherche-action intitulée "Lutter contre l'échec, repenser la relation pédagogique". Les intervenants discutent des moyens d'améliorer l'enseignement supérieur en Belgique, notamment en réformant le financement, en intégrant des tests d'orientation non contraignants et en renforçant l'aide à la réussite dans les cursus universitaires.

Points forts : + [01:23:46][^3^][3] Financement de l'enseignement supérieur * Nécessité de refinancer et d'ouvrir l'enveloppe budgétaire * Financement basé sur le nombre d'étudiants * Importance de la pédagogie dans l'orientation + [01:25:46][^4^][4] Tests d'orientation et aide à la réussite * Tests pour identifier les lacunes des étudiants * Aide à la réussite intégrée au cursus * Premier quadrimestre avec modules pédagogiques transversaux + [01:27:19][^5^][5] Conditions de vie des étudiants * Impact de la précarité étudiante sur la réussite * Propositions pour soutenir les étudiants financièrement * Sortie de l'enveloppe fermée pour réduire la concurrence entre établissements + [01:30:00][^6^][6] Évaluation des acquis de base et contrat d'aide à la réussite * Proposition d'évaluation obligatoire mais non contraignante en juillet * Contrat d'aide à la réussite axé sur la remédiation * Importance de l'orientation et de l'évaluation précoce pour la réussite + [01:34:19][^7^][7] Formation et encadrement pédagogique des enseignants * Nécessité de former les enseignants à la pédagogie * Coordination entre l'enseignement secondaire et supérieur * Inclusion et co-enseignement dans les classes précaires + [01:37:04][^8^][8] Vision des étudiants et utilisation des nouvelles technologies * Importance de la réflexivité et du regard critique des enseignants * Utilisation des outils contemporains comme chat GPT dans l'enseignement * Adaptation de l'enseignement aux enjeux actuels et aux besoins des étudiants

Résumé de la vidéo [01:44:39][^1^][1] - [02:04:25][^2^][2] : La vidéo aborde la recherche-action "Lutter contre l'échec, repenser la relation pédagogique" et discute des défis de l'enseignement supérieur, notamment la nécessité d'adapter les méthodes pédagogiques à l'hétérogénéité des classes et l'importance de la formation des enseignants. Elle souligne également le rôle de l'enseignement supérieur dans la promotion de la justice sociale et l'égalité des chances, ainsi que les implications des examens d'entrée et du financement sur la réussite des étudiants.

Points saillants: + [01:44:39][^3^][3] Défis de l'enseignement supérieur * Adaptation aux grandes classes hétérogènes * Formation continue des enseignants * Impact des examens d'entrée sur la diversité étudiante + [01:47:29][^4^][4] Justice sociale et égalité des chances * Rôle de l'enseignement supérieur dans la réduction des inégalités * Importance du soutien aux étudiants sans antécédents universitaires * Nécessité d'un financement adéquat pour l'enseignement + [01:49:00][^5^][5] Financement et réforme de l'enseignement * Débat sur le refinancement de l'enseignement supérieur * Réforme des rythmes académiques et de la pédagogie * Propositions pour améliorer l'orientation et l'aide à la réussite + [01:59:02][^6^][6] Innovations pédagogiques et accueil des étudiants * Nouvelles approches pour l'accueil et l'intégration des étudiants en première année * Importance de l'auto-réflexion et de la compréhension du sens de l'éducation universitaire * Initiatives pour renforcer l'aide à la réussite et l'engagement étudiant

Résumé de la vidéo [02:04:27][^1^][1] - [02:06:15][^2^][2] :

La vidéo présente une discussion sur la fondation d'une université, soulignant l'importance de la diversité dans son conseil et la nécessité de financements privés pour compléter les fonds publics. Elle met en avant la collaboration avec des entreprises privées pour renforcer la position de l'université dans la région bruxelloise.

Points forts : + [02:04:27][^3^][3] Diversité du conseil * Représentation de la diversité étudiante * Membres variés comme Joseph Chovanek et Akima d'Armouche + [02:04:50][^4^][4] Financements privés et regard extérieur * Apport de fonds complémentaires * Challenge des méthodes et positionnement régional + [02:05:28][^5^][5] Fondation de l'université * Projet aligné sur les objectifs inclusifs * Booster complémentaire au soutien public + [02:05:44][^6^][6] Convergence politique * Accord sur les besoins et envies malgré les différences * Motivation collective pour relever les défis

Résumé de la vidéo [00:00:04][^1^][1] - [00:24:22][^2^][2] : La vidéo présente une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle aborde le cadre du dispositif "La vie, la mort, on en parle", initié au printemps 2021, qui est un portail de ressources sur la mort et le deuil pour les enfants et adolescents. La vidéo met en lumière l'importance de la recherche et de l'éducation sur ces sujets délicats.

Points forts : + [00:00:04][^3^][3] Introduction de la journée d'étude * Remerciements et contexte du dispositif "La vie, la mort, on en parle" * Présentation des objectifs et du programme de la journée + [00:02:01][^4^][4] Développement du dispositif de recherche * Lancement d'un dispositif de recherche sur la confrontation des jeunes à la finitude * Études sur la scolarisation des jeunes en situation palliative et des jeunes orphelins + [00:06:07][^5^][5] Présentation des recherches en cours * Focus sur les jeunes aidants endeuillés et l'impact du deuil sur la scolarité * Projets de recherche futurs et collaborations + [00:14:44][^6^][6] Intervention de partenaires et spécialistes * Contributions de la Fondation SIRP et de l'association Jeune Aident Ensemble * Importance du soutien et de la reconnaissance des jeunes orphelins

Résumé de la vidéo [00:24:24][^1^][1] - [00:47:01][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle explore les défis auxquels sont confrontés les enfants et les parents après la perte d'un proche, notamment en termes de scolarité et de soutien social.

Points forts : + [00:24:24][^3^][3] Impact sur les parents et la scolarité * Les parents partagent leurs stratégies pour aider leurs enfants à l'école * Certains informent l'école, d'autres changent d'école ou de maison * Les enfants peuvent être distraits ou avoir des comportements modifiés + [00:27:30][^4^][4] Perspective des enfants et changements observés * Les enfants expriment le manque, la tristesse et le vide ressenti * La perte affecte leur attention, concentration et comportement à l'école * Certains enfants se renferment ou ont des difficultés à suivre les cours + [00:30:45][^5^][5] Camarades comme ressources * Les amis peuvent devenir un soutien émotionnel important * Les enfants partagent des expériences similaires et renforcent les liens * L'identification et la proximité avec les pairs qui ont vécu des pertes similaires + [00:34:11][^6^][6] Conséquences à long terme et rôle des enseignants * La mort d'un parent a un impact sur la réussite scolaire et professionnelle * Les enseignants peuvent se sentir mal à l'aise et démunis face à la mort * Il est crucial de soutenir l'enfant et de créer une relation de confiance avec le parent

Résumé de la vidéo [00:47:03][^1^][1] - [01:07:56][^2^][2]:

La vidéo aborde les besoins des jeunes aidants et des jeunes endeuillés, en mettant l'accent sur la méconnaissance de ces besoins dans différents contextes sociaux et l'importance de la discussion et de la reconnaissance pour répondre à ces besoins.

Points forts: + [00:47:03][^3^][3] Méconnaissance des besoins * Manque de connaissance des besoins des enfants * Nécessité de discussions entre famille, école et loisirs * Importance de partager et d'exprimer les besoins + [00:52:01][^4^][4] Expérience des enseignants avec les orphelins * Les enseignants décrivent l'expérience comme déstabilisante * Ambivalence émotionnelle et besoin d'ajustement relationnel * Impact marquant sur les enseignants et les élèves + [00:57:01][^5^][5] Étude sur les représentations des enseignants * Exploration des attitudes des enseignants envers les jeunes orphelins * Manque de préparation et de formation pour gérer le deuil * Importance du soutien des collègues et des ressources disponibles

Résumé de la vidéo [01:07:57][^1^][1] - [01:31:51][^2^][2] : La vidéo présente une journée d'étude sur le vécu des jeunes aidants et des jeunes endeuillés. Elle aborde l'importance de travailler avec les familles, en particulier avec le parent restant, pour mieux comprendre et accompagner les enfants endeuillés. Les intervenants discutent de l'impact du deuil sur le développement et l'éducation des enfants, ainsi que de la nécessité d'une approche plus dynamique et intégrée pour soutenir les enfants et les familles touchées par le deuil.

Points saillants: + [01:08:00][^3^][3] Travailler avec les familles * Nécessité de collaboration avec le parent restant * Comprendre les comportements des enfants endeuillés * Prévenir les répercussions, notamment scolaires + [01:10:14][^4^][4] Anticipation de la prise en charge * Importance de l'anticipation dans les soins palliatifs * Impact de la préparation sur le deuil des enfants * Différences selon le type de décès et l'anticipation psychique + [01:20:00][^5^][5] Développement et éducation des enfants * Intégration de la dynamique de développement dans l'approche * Impact du deuil sur la trajectoire éducative des enfants * Nécessité d'opérationnaliser le modèle théorique pour la pratique + [01:26:13][^6^][6] Influence du milieu social sur le deuil * Conséquences socioéconomiques du deuil sur les familles * Variabilité de l'impact du deuil selon le milieu social * Relation entre le deuil et les facteurs sociaux et économiques

Résumé de la vidéo [01:31:53][^1^][1] - [01:55:59][^2^][2]:

Cette partie de la vidéo aborde la gestion du deuil chez les jeunes, en particulier dans le contexte scolaire. Les intervenants discutent des défis rencontrés par les enseignants et les professionnels de la santé pour soutenir les élèves endeuillés, ainsi que de l'importance de la communication et du soutien familial.

Points forts: + [01:32:00][^3^][3] La communication familiale * L'importance de rassembler la famille pour discuter * Les mécanismes de protection peuvent entraver la communication * Les entretiens familiaux permettent d'aborder les non-dits + [01:34:07][^4^][4] L'utilisation de la littérature jeunesse * Peut servir de média pour initier la conversation sur le deuil * Utile pour les échanges entre élèves et enseignants * Ne suffit pas seul, mais aide à structurer la parole + [01:34:39][^5^][5] Le rôle des enseignants * Souvent les premiers à prendre en charge l'orphelinage * La bienveillance des enseignants est cruciale * La temporalité du deuil peut affecter les apprentissages + [01:37:26][^6^][6] Le deuil à l'école * Le deuil fait partie de la vie de l'école * Nécessité de sensibiliser et former le personnel * L'impact du deuil sur les apprentissages est significatif + [01:45:01][^7^][7] La professionnalisation du personnel * Formation sur l'accompagnement du deuil en milieu scolaire * Intégration de la thématique dans l'adaptation à l'emploi * Élargissement de la formation à d'autres membres de la communauté scolaire + [01:50:07][^8^][8] Évaluation des pratiques professionnelles * Évaluation exploratoire sur l'accompagnement des jeunes en deuil * Importance de la formation pour réduire l'appréhension * Proposition de groupes de parole pour les élèves endeuillés

Résumé de la vidéo [01:56:01][^1^][1] - [02:01:05][^2^][2]:

Cette partie de la vidéo aborde l'expérience des jeunes aidants et endeuillés, mettant en lumière l'importance de reconnaître et d'intégrer la mort et le deuil dans le contexte scolaire. L'intervenant souligne la nécessité de former et de sensibiliser les éducateurs pour mieux accompagner les élèves dans leur parcours scolaire et de vie.

Points forts: + [01:56:01][^3^][3] L'importance de la formation * Nécessité d'augmenter les compétences * Importance de la persévérance malgré les défis financiers * Réinscription du programme dans le plan académique + [01:57:02][^4^][4] La mort et le deuil à l'école * Reconnaissance de la mort comme sujet scolaire * Importance de la sensibilisation et de la formation des éducateurs * Intégration de la mort dans le parcours de vie des élèves + [01:58:02][^5^][5] L'égalité des chances et l'inclusion * Inscription du programme dans les orientations ministérielles * Promotion de l'école inclusive pour tous les enfants * Accès à une scolarité diversifiée pour tous + [01:59:01][^6^][6] Sensibilisation des chefs d'établissement * Difficulté à engager les chefs d'établissement * Importance de la politique d'établissement pour la santé * Déploiement potentiel dans d'autres académies + [02:00:04][^7^][7] Accompagnement des élèves endeuillés * Mise en place de moyens supplémentaires pour l'accompagnement * Liaison avec les équipes des lycées pour un suivi continu * Objectif d'un accompagnement pérenne pour les élèves

Résumé de la vidéo [00:00:04][^1^][1] - [00:23:49][^2^][2]:

Cette vidéo présente la deuxième partie d'une journée d'étude sur l'expérience des jeunes aidants et des jeunes endeuillés. Elle se concentre sur les défis et les impacts de ces rôles sur le bien-être et la scolarité des jeunes.

Points forts: + [00:00:04][^3^][3] Définition et rôle des jeunes aidants * Un jeune aidant est un enfant ou adolescent qui aide régulièrement un membre de sa famille souffrant d'une maladie ou d'un handicap. * Ils peuvent effectuer des tâches ménagères, administratives, ou fournir un soutien moral et des soins personnels. * L'aide apportée est évaluée sur un continuum de faible à très importante. + [00:04:37][^4^][4] Évolution de la reconnaissance des jeunes aidants en France * La prise de conscience en France a commencé en 2014, avec des initiatives clés et la création de l'association nationale Jeunes Aidants Ensemble. * En 2019, le gouvernement a identifié les jeunes aidants dans la stratégie "Agir pour les aidants". + [00:07:05][^5^][5] Recherche sur les jeunes aidants * Un programme de recherche vise à identifier les jeunes aidants en France, étudier les facteurs de protection et de vulnérabilité, et développer des interventions pour les aider. * Des études ont été menées pour comprendre les caractéristiques, les besoins et les difficultés des jeunes aidants. + [00:20:26][^6^][6] Conséquences sur la santé et la scolarité * Les jeunes aidants rapportent une moins bonne qualité de vie et ont souvent eux-mêmes des problèmes de santé. * Ils ont tendance à redoubler plus souvent et à choisir des formations à distance pour rester proches de leurs proches aidés.

Résumé de la vidéo [00:21:00][^1^][1] - [01:40:17][^2^][2]:

Cette partie de la vidéo se concentre sur les jeunes aidants en France, leur identification, les défis auxquels ils sont confrontés et les efforts pour développer des interventions de soutien. La présentation souligne l'importance de reconnaître et d'accompagner ces jeunes, qui fournissent souvent des soins à un membre de la famille souffrant de maladie ou de handicap.

Points forts: + [00:21:00][^3^][3] Recherche sur les jeunes aidants * Identification des jeunes aidants en France * Étude des facteurs de protection et de vulnérabilité * Développement d'interventions pour les soutenir + [00:41:00][^4^][4] Limites de la recherche actuelle * Échantillon limité ne permettant pas de généraliser * Manque d'informations sur le contexte scolaire des jeunes + [00:57:00][^5^][5] Expérience dans une école d'ingénieurs * Mise en place d'entretiens pour comprendre les besoins des étudiants endeuillés * Adaptation des aménagements scolaires en fonction des situations individuelles + [01:18:00][^6^][6] Difficultés systémiques dans l'éducation * L'épuisement des jeunes aidants affecte leur capacité à s'engager dans les démarches administratives * Besoin d'un soutien pour naviguer dans les processus institutionnels

Résumé de la vidéo [01:21:00][^1^][1] - [01:23:09][^2^][2]:

Cette partie de la vidéo se concentre sur les résultats d'une étude qualitative exploratoire en psychologie, nommée "jadisp", qui examine la perception des interférences entre le processus de deuil et la scolarité chez les jeunes aidants. L'étude a été soutenue financièrement par la Fondation Osirp et vise à comprendre les conséquences de l'apport des jeunes aidants, en particulier sur leur scolarité.

Points forts: + [01:21:00][^3^][3] Présentation de l'étude jadisp * Recherche qualitative exploratoire en psychologie * Soutien financier de la Fondation Osirp * Focus sur les jeunes aidants et le deuil + [01:22:18][^4^][4] Définition des jeunes aidants * Clarification sur l'apport des jeunes aidants * Conséquences multiples, y compris sur la scolarité * Importance de la prise en compte de ces incidences + [01:22:55][^5^][5] Résultats de l'étude * Interférences entre deuil et scolarité * Perception des jeunes aidants sur ces interférences * Objectif de mieux comprendre et accompagner les jeunes aidants

1 стадия изучения 2 влюбленность подкреплять стабильность. и классное в 1 момент нужно сделать легкую яму 3.начало любви

The lopsided asymmetry here is charring. Imagine putting in time like that, where responses are instantly generated. You'll feel a) that this is worth something because you spent time on this, and we equate such investment in others with depth, but here there's no other b) there will always be a response by generated personas, and you will feel a likely 'social' pressure to respond in kind.

And of course there's lots of money in this sociopathic scheme. likely because the funders come from the same strain.

wholesome? 'could help people connect with others such as AIs' An AI is not an other and there is no connection as it has no concept nor memory of you.

It might well be a narcissist honey pot.

Where on the spectrum is this guy? No they couldn't have been AIs, or you wouldn't have built meaningful connections. Relations aren't just something you experience for you to feel good over, but a thing in itself playing out between two or more people. This sounds sociopathic.

Will humans and AI be distinguishable? _butterfly who's a woodworker' no it isn't. It is generating stuff statistically matching what a woodworker might post, and all it generates is fantasy. There is no woodworker, there is no 'latest creation' of actual wood, just a generated image. Can we please stop this utter crap?

whose creativity? what actual relationships? More seductive to project you mean.

Yet this too is just a generative AI chatbot, with nice pictures.

What? a Butterfly is an interactive persona, and supposedly people want to interact with it? Each one has "backstories, opinions and emotions" no they don't. Each one generates text that people then project upon to perceive a past, opinions and emotions. It even doesn't need much for it, see Eliza.

These descriptions are very uncanny valley. Imagine a community where each AI friend has its own unique digital life, ready to share moments, create memories, post images just like real friends Butterflies is more than just a social network; it’s a fresh approach to connection Imagine a place where every friend understands you perfectly,

eLife assessment

This important study examines the relationship between expiratory airflow and vocal pitch in adult mice during the production of ultrasonic vocalizations and also identifies a molecularly defined population of brainstem neurons that regulates mouse vocal production across development. The evidence supporting the study's conclusions that expiratory airflow shapes vocal pitch and that these brainstem neurons preferentially regulate expiratory airflow is novel and compelling. This work will be of interest to neuroscientists working on mechanisms and brainstem circuits that regulate vocal production and vocal-respiratory coordination.

Reviewer #1 (Public Review):

Summary:

In this important work, the authors propose and test a model for the control of murine ultrasonic vocalizations (USV) in which two independent mechanisms involving changes in laryngeal opening or airflow control vocal tone. They present compelling experimental evidence for this dual control model by demonstrating the ability of freely behaving adult mice to generate vocalizations with various intonations by modulating both the breathing pattern and the laryngeal muscles. They also present novel evidence that these mechanisms are encoded in the brainstem vocalization central neural pattern generator, particularly in the component in the medulla called the intermediate reticular oscillator (iRO). The results presented clearly advance understanding of the developmental nature of the iRO, its ability to intrinsically generate and control many of the dynamic features of USV, including those related to intonation, and its coordination with/control of expiratory airflow patterns. This work will interest neuroscientists investigating the neural generation and control of vocalization, breathing, and more generally, neuromotor control mechanisms.

Strengths:

Important features and novelty of this work include:

(1) The study employs an effective combination of anatomical, molecular, and functional/ behavioral approaches to examine the hypothesis and provide novel data indicating that expiratory airflow variations can change adult murine USV's pitch patterns.

(2) The results significantly extend the authors' previous work that identified the iRO in neonatal mice by now presenting data that functionally demonstrates the existence of the critical Penk+Vglut2+ iRO neurons in adult mice, indicating that the iRO neurons maintain their function in generating vocalization throughout development.

(3) The results convincingly demonstrate that the iRO neurons encode and can generate vocalizations by modulating both breathing and the laryngeal muscles.

(4) The anatomical mapping and tracing results establish an important set of input and output circuit connections to the iRO, including input from the vocalization-promoting subregions of the midbrain periaqueductal gray (PAG), as well as output axonal projections to laryngeal motoneurons, and to the respiratory rhythm generator in the preBötzinger complex.

(5) These studies advance the important concept that the brainstem vocalization pattern generator integrates with the medullary respiratory pattern generator to control expiratory airflow, a key mechanism for producing various USV types characterized by different pitch patterns.

Weaknesses:

A limitation is that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow has not been fully worked out, requiring future studies.

Reviewer #2 (Public Review):

Summary:

Both human and non-human animals modulate the frequency of their vocalizations to communicate important information about context and internal state. While regulation of the size of the laryngeal opening is a well-established mechanism to regulate vocal pitch, the contribution of expiratory airflow to vocal pitch is less clear. To consider this question, this study first characterizes the relationship between the dominant frequency contours of adult mouse ultrasonic vocalizations (USVs) and expiratory airflow using whole-body plethysmography. The authors also include data from a single mouse that combines EMG recordings from the diaphragm and larynx with plethysmography to provide evidence that the respiratory central pattern generator can be re-engaged to drive "mini-breaths" that occur during the expiratory phase of a vocal breath. Next, the authors build off of their previous work characterizing intermediate reticular oscillator (iRO) neurons in mouse pups to establish the existence of a genetically similar population of neurons in adults and show that artificial activation of iRO neurons elicits USV production in adults. Third, the authors examine the acoustic features of USV elicited by optogenetic activation of iRO and find that a majority of natural USV types (as defined by pitch contour) are elicited by iRO activation and that these artificially elicited USVs are more likely than natural USVs to be marked by positive intonation (positive relationship between USV dominant frequency and expiratory airflow).

Strengths:

Strengths of the study include the novel consideration of expiratory airflow as a mechanism to regulate vocal pitch and the use of intersectional methods to identify and activate the iRO in adult mice. The establishment of iRO neurons as a brainstem population that regulates vocal production across development is an important finding.

Weaknesses:

The conclusion that the respiratory CPG is re-engaged during "mini-breaths" throughout a given vocal breath would be strengthened by including analyses from more than one mouse.

Author response:

The following is the authors’ response to the original reviews.

In the revised manuscript we have included an additional study that significantly contributes to the conclusions and models of the original version. Briefly, Figure 3 now describes our characterization of the diaphragm and laryngeal muscle activities (electromyography, EMG) during endogenous vocalizations. These EMGs also serve as representations of the brainstem breathing central pattern generator (CPG) inspiratory and post-inspiratory generating neurons, respectively. In our original submission, we found that many of the vocalizations had changes in pitch that mirrored the change in expiratory airflow (we termed positive intonation), and we proposed that the coordination of breathing muscles (like the inspiratory muscles) and larynx patterned this. This mechanism is akin to our findings for how neonatal cries are rhythmically timed and produced (Wei et al. 2022). The newly presented EMG data re-inforces this idea. We found that for vocalizations with positive intonation, the inspiratory diaphragm muscle has an ectopic burst(s) of activity during the expiration phase which corresponds to a decrease in airflow and pitch, and this is followed by laryngeal muscle activity and increased pitch. This can be cycled throughout the expiration to produce complex vocalizations with oscillations in pitch. A basal breath is hardwired for the laryngeal muscle activity to follow the diaphragm, so the re-cycling of this pattern nested within an expiration (a ‘mini-breath’ in a ‘breath’) demonstrates that the vocalization patterning system engages the entire breathing CPG. This contrasts with the canonical model that activity of the laryngeal premotor neurons control all aspects of producing / patterning vocalizations. Furthermore, this mechanism is exactly how the iRO produces and patterns neonatal vocalizations (Wei et al. 2022) and motivates the likely use of the iRO in adult vocalizations.

Response to recommendations for the authors:

Reviewer #1:

(1) The authors should note in the Discussion that the cellular and circuit mechanisms by which the vocalization pattern generator integrates with the respiratory pattern generator to control expiratory airflow have not been fully worked out, requiring future studies.

This was noted in the discussion section “The iRO likely patterns intonation for endogenous phonation”.

(2) Please change the labeling of the last supplemental figure to Figure Supplemental 5.

Thank you for identifying this.

Reviewer #2:

Major concerns

(1) While it is true that modulation of activity in RAm modulates the laryngeal opening, this statement is an incomplete summary of prior work. Previous studies (Hartmann et al., 2020; Zhang et al., 1992, 1995) found that activation of RAm elicits not just laryngeal adduction but also the production of vocal sounds, albeit vocal sounds that were spectrally dissimilar from speciestypical vocalizations. Moreover, a recent study/preprint that used an activity-dependent labeling approach in mice to optogenetically activate RAm neurons that were active during USV production found that re-activation of these neurons elicits USVs that are acoustically similar to natural USVs (Park et al., 2023). While the authors might not be required to cite that recent preprint (as it is not yet peer-reviewed), the fact that activation of RAm elicits vocal sounds is clear evidence that its effects go beyond modulating the size of the laryngeal opening, as this alone would not result in sound production (i.e., RAm activation must also recruit expiratory airflow). The authors should include these relevant studies in their Introduction. Moreover, the rationale for the model proposed by the authors (that RAm controls laryngeal opening whereas iRO controls expiratory airflow) is unclear with regard to these prior studies. The authors should include a discussion of how these prior findings are consistent with their model (as presented in the Introduction, as well as in Figure 4 and relevant Discussion) that RAm modulates the size of laryngeal opening but not expiratory airflow.

An introduction and discussion of the Veerakumar et. al. 2023 and Park et. al. 2024 manuscripts describing RAm in mice has now been included.

The iRO serves to coordinate the breath airflow and laryngeal adduction to produce sound and the intonation within it that mirrors the breath airflow. This occurs because the iRO can control the breathing CPG (synaptic input to the preBötC inspiratory pacemaker) and is premotor to multiple laryngeal muscles (Wei et. al. 2022). The modulation of the expiratory airflow is by inducing momentary contraction of the diaphragm (via excitation of the preBötC) which opposes (a.k.a. slows) expiration. This change in flow results in a decrease in pitch (Fig. 3 in the revised manuscript, Wei et. al. 2022).

It is our understanding that the basic model for RAm evoked USVs is that RAm evokes laryngeal adduction (and presumed abdominal expiratory muscle activation) and this activity is momentarily stopped during the breath inspiration by inhibition from the preBötC (Park et. al. 2024). So, in this basic model, any change in pitch and expiratory airflow would be controlled by tuning RAm activity (i.e., extent of laryngeal adduction). In this case, the iRO induced inspiratory muscle activity should not occur during expiration, which is not so (Fig. 3). Note, the activity of abdominal expiratory muscles during endogenous and RAm evoked USVs has not been characterized, so the contribution of active expiration remains uncertain. This is an important next step.

We have now included a discussion of this topic which emphasizes that iRO and RAm likely have reciprocal interactions (supported by the evidence of this anatomical structure). These interactions would explain why excitation of either group can evoke USVs and, perhaps, the extent that either group contributes to a USV explains how the pitch / airflow changes. An important future experiment will be to determine the sufficiency of each site in the absence of the other.

(2) The authors provide evidence that the relationship between expiratory airflow and USV pitch is variable (sometimes positive, sometimes negative, and sometimes not related). While the representative spectrograms clearly show examples of all three relationship types, no statistical analyses are included to evaluate whether the relationship between expiratory airflow and USV pitch is different than what one would expect by chance. For example, if USV pitch were actually unrelated to expiratory airflow, one might nonetheless expect spurious periods of positive and negative relationships. The lack of statistical analyses to explicitly compare the observed data to a null model makes it difficult to fully evaluate to what extent the evidence provided by the authors supports their claims.

We have now included two null distributions and compared our observed correlation values to these. The two distributions were created by taking each USV / airflow pair and randomly shuffling either the normalized USV pitch values (pitch shuffled) or the normalized airflow values (airflow shuffled) to simulate the distribution of data should no relationship exist between the USV pitch and airflow.

(3) The relationship between expiratory airflow and USV pitch comes with two important caveats that should be described in the manuscript. First, even in USV types with an overall positive relationship between expiratory airflow and pitch contour, the relationship appears to be relative rather than absolute. For example, in Fig. 2E, both the second and third portions of the illustrated two-step USV have a positive relationship (pitch goes down as expiratory airflow goes down). Nonetheless, the absolute pitch of the third portion of that USV is higher than the second portion, and yet the absolute expiratory airflow is lower. The authors should include an analysis or description of whether the relationship between expiratory airflow and USV pitch is relative vs.

absolute during periods of 'positive intonation'.

The relationship between pitch and airflow is relative and this in now clarified in the text. To determine this, we visualized the relationship between the two variables by scatterplot for each of the USVs syllables and, as the reviewer notes, a given airflow cannot predict the resulting frequency and vice versa.

(4) A second important caveat of the relationship between expiratory airflow and USV pitch is  that changes in expiratory airflow do not appear to account for the pitch jumps that characterize mouse USVs (this lack of relationship also seems clear from the example shown in Fig. 2E). This caveat should also be stated explicitly.

The pitch jumps do not have a corresponding fluctuation in airflow, and this is now stated in the results and discussion.

(5) The authors report that the mode of relationship between expiratory airflow and USV pitch (positive intonation, negative intonation, or no relationship) can change within a single USV. Have the authors considered/analyzed whether the timing of such changes in the mode of relationship coincides with pitch jumps? Perhaps this isn’t the case, but consideration of the question would be a valuable addition to the manuscript.

We analyzed a subset of USVs with pitch jumps that were defined by a change >10 kHz, at least 5ms long, and had one or two jumps. The intonation relationships between the sub-syllables within a USV type were not stereotyped as evidenced by the same syllable being composed of combinations of both modes.

(6) The authors incorrectly state that PAG neurons important for USV production have been localized to the ventrolateral PAG. Tschida et al., 2019 report that PAG-USV neurons are located predominantly in the lateral PAG and to a lesser extent in the ventrolateral PAG (see Fig. 5A from that paper). The finding that iRO neurons receive input from VGlut2+ ventrolateral PAG neurons represents somewhat weak evidence that these neurons reside downstream of PAG-USV neurons. This claim would be strengthened by the inclusion of FOS staining (following USV production), to assess whether the Vglut+ ventrolateral PAG neurons that provide input to iRO are active in association with USV production.

This comment correctly critiques that our PAG à iRO tracing does not demonstrate that the labeled PAG neurons are sufficient nor necessary for vocalization. Directly demonstrating that activation and inhibition the PAG-iRO labeled neurons ectopically drives or prevents endogenous USVs is an important next step. While FOS implies this connectivity, it does not definitely establish it and so this experiment is impacted by some of the caveats of our tracing (e.g. PAG neurons that drive sniffing might be erroneously attributed to vocalization).

Our reading of the literature could not identify an exact anatomical location within the mouse PAG and this site appears to vary within a study and between independent studies (like within and between Tschida et. al. 2019 and Chen et. al. 2021). The labeling we observed aligns with some examples provided in these manuscripts and with the data reported for the retrograde tracing from RAm (Tschida et al 2019).

(7) In Figure S5A, the authors show that USVs are elicited by optogenetic activation of iRO neurons during periods of expiration. In that spectrogram, it also appears that vocalizations were elicited during inspiration. Are these the broadband vocalizations that the authors refer to in the Results? Regardless, if optogenetic activation of iRO neurons in some cases elicits vocalization both during inspiration and during expiration, this should be described and analyzed in the manuscript.

The sound observed on the spectrogram during inspiration is an artefact of laser evoked head movements that resulted in the fiber cable colliding with the plethysmography chamber. In fact, tapping an empty chamber yields the same broad band spectrogram signal. The evoked USV or harmonic band vocalization is distinct from this artefact and highlighted in pink.

(8) Related to the comment above, the authors mention briefly that iRO activation can elicit broadband vocalizations, but no details are provided. The authors should provide a more detailed account of this finding.

The broadband harmonic vocalizations we sometimes observe upon optogenetic stimulation of AAV-ChR2 expressing iRO neurons are akin to those previously described within the mouse vocal repertoire (see Grimsley et. al .2011). We have added this citation and mentioned this within the text. 

(9) The effects of iRO stimulation differ in a couple of interesting ways from the effects of PAGUSV activation. Optogenetic activation of PAG-USV neurons was not found to entrain respiration or to alter the ongoing respiratory rate and instead resulted in the elicitation of USVs at times when laser stimulation overlapped with expiration. In contrast, iRO stimulation increases and entrains respiratory rate, increases expiratory and inspiratory airflow, and elicits USV production (and also potentially vocalization during inspiration, as queried in the comment above). It would be informative for the authors to add some discussion/interpretation of these differences.

We have added a section of discussion to describe the how these different results may be explained by the iRO being a vocal pattern generator versus the PAG as a ‘gating’ signal to turn on the medullary vocalization patterning system (iRO and RAm). See discussion section ‘The iRO likely patterns intonation for endogenous phonation’.

(10) The analysis shown in Fig. 4D is not sufficient to support the author’s conclusion that all USV types elicited by iRO activation are biased to have more positive relationships between pitch and expiratory airflow. The increase in the relative abundance of down fm USVs in the opto condition could account for the average increase in positive relationship when this relationship is considered across all USV types in a pooled fashion. The authors should consider whether each USV type exhibits a positive bias. Although such a comparison is shown visually in Fig. 4G, no statistics are provided. All 7 USV types elicited by optogenetic activation of iRO should be considered collectively in this analysis (rather than only the 5 types currently plotted in Fig. 4G).

In the original submission the statistical analysis of r values between opto and endogenous conditions was included in the figure legend (‘panels E-G, two-way ANOVA with Sidak’s post-hoc test for two-way comparisons was used; all p-values > 0.05), and this has not changed in the revised manuscript. We have now provided the suggested comparison of opto vs endogenous USVs without down fm (Fig. 5D). This positive shift in r is statistically significant (…).

(11) The evidence that supports the author’s model that iRO preferentially regulates airflow and that RAm preferentially regulates laryngeal adduction is unclear. The current study finds that activation of iRO increases expiratory (and inspiratory) airflow and also elicits USVs, which means that iRO activation must also recruit laryngeal adduction to some extent. As the authors hypothesize, this could be achieved by recruitment of RAm through iRO’s axonal projections to that region.

Note, it is more likely that iRO is directly recruiting laryngeal adduction as they are premotor to multiple laryngeal muscles like the thyroarytenoid and cricothyroid (Wei et. al. 2022). The ‘Discussion’ now includes our ideas for how the iRO and RAm likely interact to produce vocalizations.

In the recent preprint from Fan Wang’s group (Park et al., 2023), those authors report that RAm is required for USV production in adults, and that activation of RAm elicits USVs that appear species-typical in their acoustic features and elicits laryngeal adduction (assessed directly via camera). Because RAm activation elicits USVs, though, it must by definition also recruits expiratory airflow. Can the authors add additional clarification of how the evidence at hand supports this distinction in function for iRO vs RAm?

See response to ‘Major Concern #1”.

Minor concerns 

(1) The authors might consider modifying the manuscript title. At present, it primarily reflects the experiments in Figure 2.

We have provided a title that we feel best reflects the major point of the manuscript. We hope that this simplicity enables it to be recognized by a broad audience of neuroscientists as well as specialists in vocalization and language.

(2) The statement in the abstract that "patterns of pitch are used to create distinct 'words' is somewhat unclear. Distinct words are by and large defined by combinations of distinct phonemes. Are the authors referring to the use of "tonemes" in tonal languages? If so, a bit more explanation could be added to clarify this idea. This minor concern includes both the Abstract, as well as the first paragraph of the Introduction.

We have clarified this line in the abstract to avoid the confusing comparison between mouse vocalizations and human speech. In the introduction we have expanded our explanation to clarify that variations in pitch are a component of spoken language that add additional meaning and depth to the underlying, phonemic structure. 

(3) Multiple terms are used throughout the manuscript to refer to expiratory airflow: breath shape (in the title), breath pattern, deviations in exhalation, power of exhalation, exhalation strength, etc. Some of these terms are vague in meaning, and a consolidation of the language would improve the readability of the abstract and introduction.

We have chosen a smaller selection of descriptive words to use when describing these breath features.

(4) Similarly, "exhalation" and "expiration" are both used, and a consistent use of one term would help readability.

See point 3.

(5) In a couple of places in the manuscript, the authors seem to state that RAm contains both laryngeal premotor neurons as well as laryngeal motor neurons. This is not correct to our knowledge., but if we are mistaken, we would ask that the authors add the relevant references that report this finding.

It is our understanding that the RAm is defined as the anatomical region consistent with the murine rostral and caudal ventral respiratory groups composed of multiple premotor neuron pools to inspiratory, expiratory, laryngeal, and other orofacial muscles. This is supported by neurons within RAm that reflect multiple phases of the inspiratory and expiratory cycle (Subramanian et. al. 2018) and excitation of sub-regions within RAm modulating multiple parts of the breathing control system (Subramanian et. al. 2018 and Subramanian 2009). Rabies tracing of the various premotor neurons which define the anatomical region of RAm in the mouse shows that they surround the motor neurons in the loose region of the nucleus ambiguus (the anatomical location of RAm) for multiple muscles of the upper airway system, such as the thyroarytenoid (Wu et. al. 2017, Dempsey et. al. 2021 and Wei et. al. 2022). Given that the name RAm reflects a broad anatomical location, we have used it to describe both the premotor and motor neurons embedded within it. We have now clarified this in the text.

(6) The statistical analysis applied in Figure 1C is somewhat confusing. The authors show two distributions that appear different but report a p-value of 0.98. Was the analysis performed on the mean value of the distributions for each animal, the median, etc.? If each animal has two values (one for USV+ breaths and one for USV- breaths), why not instead compare those with a paired t-test (or Wilcoxon rank sign)? Additional information is needed to understand how this analysis was performed.

The original manuscript version used a two-way anova to compare the normalized histogram of instantaneous frequency for breaths with (USV+) or without (USV-) for each animal (first factor: USV+/-, second factor: Frequency). The p-value for the first factor (USV) was 0.98 showing no statistically significant effect of USV on the distribution of the histogram.

For simplicity, we have instead performed the analysis as suggested and include a bar graph. This analysis shows that the instantaneous frequency of USV breaths is, in fact, statistically significantly lower than those without USVs. We have updated the figure legend and text to reflect this.

(7) The use of the word "syllable" to describe parts of a USV that are produced on a single breath may be confusing to some scientists working on rodent USVs. The term 'syllable' is typically used to describe the entirety of a USV, and the authors appear to use the term to describe parts of a USV that are separated by pitch jumps. The authors might consider calling these parts of USVs "sub-syllables".

We have clarified these descriptions throughout the text. We now refer to the categories as ‘syllable types’, define ‘syllables’ as ‘a continuous USV event’ with no more than 20ms of silence within and finally ‘sub-syllables’ to refer to components of the syllable separated by jumps in frequency (but not gaps in time).

(8) In Figure S3, final row, the authors show a USV produced on a single breath that contains two components separated by a silent period. This type of bi-syllabic USV may be rare in adults and is similar to what the authors showed in their previous work in pups (multiple USVs produced on a single expiration, separated by mini-inspirations). One might assume that the appearance of such USVs in pups and their later reduction in frequency represents a maturation of vocalrespiratory coordination. Nonetheless, the appearance of bi-syllabic USVs has not been reported in adult mice to our knowledge, and the authors might consider further highlighting this finding.

We were also struck by the similarity of these USVs to our study in neonates and such types of similarities sparked an interest in the role of the iRO in patterning adult USVs. We now include a description of the presence and abundance of bi- and tri-syllablic calls observed in our recordings to highlight this finding.

(9) Figure 4 is referenced at the end of the second Results section, but it would seem that the authors intended to reference Figure 2. 

For simplicity we included some of the referenced data within Fig. S5. We appreciate the recommendation.

(10) In the optogenetic stimulation experiments, the authors should clarify why bilateral stimulation was applied. Was unilateral stimulation ineffective or less effective? The rationale provided for the use of bilateral stimulation (to further localize neural activation) is unclear.

The iRO is bilateral and, we presume, functions similarly. So, we attempted to maximally stimulate the system. We have clarified this in the methods.

(11) Figure Supplemental '6' should be '5'.

Thanks!

(12) Last sentence of the Introduction: "Lasty" should be "lastly".

Thanks!

(13) There are two references for Hage et al., 2009. These should be distinguished as 2009a and 2009b for clarity.

Thanks!

please bold prompt

Author response:

The following is the authors’ response to the current reviews.

We thank the reviewers and editor for their careful review of our work. We believe the resulting manuscript is much stronger. We agree with the comments made by Reviewer #2 regarding additional histology and neuronal data analysis, which will be presented in subsequent work.

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The following is the authors’ response to the original reviews.

Reviewer 1 (Public Weaknesses):

It was not always clear what the lesion size was. This information is important for future applica- tions, for example, in the visual cortex, where neurons are organized in retinotopy patterns.

We thank the reviewer for this feedback. While there is some variation in lesion volume for a given parameter set, we have added more details of the volumes of lesions created in our testing (Fig. 4 and Fig. 5).

It would be helpful if the author could add some discussion about whether and how this method could be used in other types of array/multi-contact electrodes, such as passive neuropixels, S- probes, and so on. In addition, though an op-amp was used in the design, it would still be helpful if the author could provide a recommended range for the impedance of the electrodes.

We thank the reviewer for this suggestion. We have both added a demonstration of use in a differ- ent multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

“We demonstrated that our electrolytic lesioning technique works with a linear multicontact probe by testing with a U-Probe in ex vivo rabbit cortex. There are no particular limitations that would prevent our specific electrolytic lesioning technique and device from working with any passive multielectrode probe. The main requirements for use are that the probe has two electrodes that can directly (via whatever necessary adapters) connect to the lesioning device, such that arbitrary current can be passed into them as the anode and cathode. This would limit use of probes, like Neuropixels, where the on-chip acquisition and digitization circuitry generally precludes direct connection to electrodes [1], [2]. The impedance of the multielectrode probe should not be an issue, due to the use of an op amp. We showed use  with a Utah array (20-800 kΩ) and a U-Probe (1-1.5 MΩ). The specific op amp used here has a voltage range of ± 450 V, which assuming a desired output of 150 µA of current would limit electrode impedance to 6 MΩ. Though a different op amp could easily be used to accommodate a higher electrode impedance, it is unlikely that this would be necessary, since most electrodes have impedances between 100 kΩ to 1 MΩ [3].”

Reviewer 2 (Public Weaknesses):

In many of the figures, it is not clear what is shown and the analysis techniques are not well described.

We thank the reviewer for this feedback. We hope that our edits to both the figures and the text have improved clarity for readers.

The flexibility of lesioning/termination location is limited to the implantation site of the multielec- trode array, and thus less flexible compared to some of the other termination methods outlined in Appendix 2.

We thank the reviewer for this point. You are right that the lesioning location is limited to the multielectrode array’s implantation site, while other methods in Appendix 2 do not require prox- imity of the lesion location and the electrophysiology recording site. However, we believe that the closeness of the lesioning location to the microelectrode array is a strength - guaranteeing record- ings from the perilesional area - even with the small negative of reduced flexibility. Multielectrode arrays can be implanted in many areas of cortex. If one wanted to study distal effects of a lesion, additional electrophysiology probes could be implanted to record from those areas. We have noted this on Page 3, Line 117.

“While the link between the lesion location and the multielectrode location technically con- strains the lesion to an area of cortex in which a multielectrode array could be implanted, we see the connection as a positive, because it ensures recording some neuroelectrophysiology from the perilesional area in which recovery is hypothesized to occur (see Appendix 1Data Availabilityappendix.41).”

Although the extent of the damage created through the Utah array will vary based on anatomical structures, it is unclear what is the range of lesion volumes that can be created with this method, given a parameter set. It was also mentioned that they performed a non-exhaustive parameter search for the applied current amplitude and duration (Table S1/S2) to generate the most suitable lesion size but did not present the resulting lesion sizes from these parameter sets listed. Moreover, there’s a lack of histological data suggesting that the lesion size is precise and repeatable given the same current duration/amplitude, at the same location.

We thank the reviewer for this thoughtful feedback. We have added figures (Figs. 4 and 5), where we show the relationship between estimated lesion volume and the current amplitude and duration parameters. These figures include more data from the tests in Supplementary File 1 and Supplementary File 2. While there is some variation in lesion volume for a given current amplitude and duration, there is still a clear relationship between the parameters and lesion volume.

It is unclear what type of behavioral deficits can result from an electrolytic lesion this size and type (∼3 mm in diameter) in rhesus macaques, as the extent of the neuronal loss within the damaged parenchyma can be different from past lesioning studies.

While we appreciate the reviewer’s interest in the behavioral deficits associated with our lesions in rhesus macaques, reporting these falls beyond the scope of this manuscript. Future work will explore the behavioral deficits associated with these lesions

The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study.

We apologize for not being more explicit about the parameters used for the lesion in Monkey F. We have added this in Results on Page 5, Line 209 and in Methods on Page 19, Line 586.

“After this validation and refinement, one proof-of-concept lesion (150 µA direct current passed through adjacent electrodes for 45 seconds) was performed in an in vivo sedated rhe- sus macaque (Monkey F) in order to validate the safety of the procedure.”

“This lesion was created by applying 150 µA of direct current to two adjacent electrodes in the microelectrode array for 45 seconds.”

We also clarified the parameters used for the other lesions in Monkeys H and U in Results on Page 7, Line 233 and in Methods on Page 19, Line 586.

“In all of the fourteen lesions across two awake-behaving rhesus macaques (150 µA direct current passed through adjacent electrodes for 30 or 45 seconds (30s for Monkey U and 45s for Monkey H, except lesion H200120 which was for 50 seconds)), the current source worked as expected, providing a constant current throughout the duration of the procedure.”

“In these lesions, 150 µA of direct current was applied to two adjacent electrodes in the mi- croelectrode array for 30 or 45 seconds (30s for Monkey U, 45s for Monkey H), except in lesion H200120 where current was applied for 50 seconds.”

Monkey F was euthanized shortly after the lesion, so we now mention this on Page 19, Line 583.

“Based on this, and a lack of physiological signs of pain from the anaesthetized pig studies, a lesion was performed on a sedated rhesus macaque who was subsequently euthanized due to unrelated health complications (Monkey F; 16 year-old adult, male rhesus macaque) in order to further verify safety before use in awake-behaving rhesus.”

Because Monkey F was sedated and then euthanized shortly after, there is no behavioral data. As the lesion in sedated Monkey F was used to validate the safety of the procedure, any further data and analysis fall beyond the scope of this manuscript.

As an inactivation method, the electrophysiology recording in Figure 5 only showed a change in pairwise comparisons of clustered action potential waveforms at each electrode (%match) but not a direct measure of neuronal pre and post-lesioning. More evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning. Some exam- ples of this can be showing the number of spike clusters identified each day, as well as analyzing local field potential and multi-unit activity.

The reviewer has pointed out some short comings of the original analysis, which we believe have since been addressed with the revised analysis. LFP and spiking activity are functional measures that are more ambiguous in terms of loss and are also the subject of another manuscript currently under revision.

The advantages over recently developed lesioning techniques are not clear and are not discussed.

We thank the reviewer for noting this. We have added a section, also responding to their later request for us to compare our work to Khateeb et al. 2022, by adding a section to the Discussion on Page 16, Line 434.

“Perhaps the most unique advantage of our technique in comparison with other existing inactivation methods lies in Design Consideration #1: stable electrophysiology pre- and post-inactivation (Appendix 1Data Availabilityappendix.41). While several methods exist that allow for localization and size control of the inactivation (Design Consideration #2) and cross compatibility across regions and species (Design Consideration #3), few have achieved compatibility with stable electrophysiology. For example, some studies record electrophysiology only after the creation of the lesion, preventing comparison with baseline neuronal activity [4]. One recent study, Khateeb, et al., 2022, developed an inactivation method that is effectively combined with stable electrophysiology by creating photothrombotic lesions through a chronic cranial window integrated with an electrocorticography (ECoG) array [5], which may be appropriate for applications where local field potential (LFP) recording is sufficient. This approach has trade-offs with regards to the three design considerations presented in Appendix 1Data Availabilityappendix.41.

While Khateeb, et al., present a toolbox with integrated, stable electrophysiology from an ECoG array pre- and post- inactivation (Design Consideration #1), it demonstrated recordings from an ECoG array with limited spatial resolution. While a higher density ECoG array that would provide higher spatial resolution could be used, increasing the density of opaque electrodes might occlude optical penetration and constrain photothrombotic lesions. Further, ECoG arrays are limited to recording LFP, not electrophysiology at single neuron resolution, potentially missing meaningful changes in the neuronal population activity after lesioning. Khateeb, et al., demonstrated localization and control the size of inactivation (Design Consideration #2). In this manuscript, we have shown that the amount and duration of direct current are significant determinants of lesion size and shape, while with photothrombotic lesions, light intensity and aperture diameter are the significantly relevant parameters. One potential advantage of photothrombotic approaches is the use of optical tools to monitor anatomical and physiological changes after lesioning through the cranial window, though the research utility of this monitoring remains to be demonstrated.

Although the method presented by Khateeb, et al., shows some cross-compatibility (Design Consideration #3), it has greater limitations in comparison with the method presented here. For example, while Khateeb, et al., notes that the approach could be adapted for use in smaller organisms, no modification is needed for use in other species with this work’s approach–so long as a multielectrode probe is implantable. In this manuscript we demon- strate electrolytic lesioning spanning two multielectrode probes across rabbits, pigs, sheep, and rhesus macaques, and our same device could be easily used with other smaller species, like rats, in which multielectrode probes have been successfully implanted [6]. Further, the approach in Khateeb, et al., is limited to superficial brain structures, due to the need for opti- cal accessibility. As noted, fiber optics could allow access to deeper structures, which would bring associated additional tissue damage, but deeper structure lesioning was not demon- strated. In contrast, the approach presented here can be used in any region of cortex in which a multielectrode probe can be implanted, which, depending on the probe used, does not limit it to surface structures. For example, we demonstrated use of our lesioning tech- nique with a linear U-probe (Fig. 8figure.caption.25), which could be used to reach deeper layers of cortex or specific deep cortical structures. In both techniques, the location of the lesion is tied to the location of the electrophysiology (for Khateeb et al., wherever the cra- nial window and ECoG array are; for this technique, wherever the multielectrode probe has been implanted), which ensures that the electrophysiology will include recordings from the perilesional area. Neither work addresses the potential of their technique to induce chronic post-lesion behavioral effects, which is a key goal for future work.”

There is a lack of quantitative histological analysis of the change in neuronal morphology and loss.

We appreciate the reviewer’s desire for a quantitative histological analysis, however this falls out- side of the scope of this manuscript. We are not attempting to make strong claims about the number of neurons lost through lesioning or thoroughly characterize morphological changes in the neurons. The histology is intended to show that lesioning did lead to a loss of neurons, but the precise num- ber of neurons lost is neither in scope nor is likely to be highly conserved across lesions.

There is a lack of histology data across animals and on the reliability of their lesioning techniques across animals and experiments.

We thank the reviewer for this point. As stated above, we have now added Fig. 4 and Fig. 5, which includes volume estimates based on the histology from more of our ex vivo and in vivo testing across animals.

There is a lack of data on changes in cortical layers and structures across the lesioning and non- lesioning electrodes.

We acknowledge that the histology does not have the level of detail that is expected from many modern studies. However, the goal here was dramatically different: we sought to calibrate a novel lesion device, ensure it’s safe use in large mammals (specifically, non-human primates) and pro- vide estimates of the lesion size to compare with the literature. The extent of histology that could be performed and the tools available to us prevent such an in depth analysis. We can say based on shank length of the Utah arrays used and known anatomy that we have affected layer 2/3 and maybe a bit of layer 4.

Reviewer 1 (Recommendations For The Authors):

Figure 5b. It would be helpful if the author could plot the delta match separately for the lesion elec- trodes, near neighbor electrodes, and far neighbors. This would help understand the lesion effect, specifically whether the effect is selective (e.g., more potent for the lesion and adjacent electrodes.)

The fact that neuron loss is not particularly selective can already be seen in the spike waveform plots, arranged spatially on the array. Plenty of clear change is observed far from the lesion elec- trodes (marked with black dots) as well as nearby. We have made mention of this localized non- specificity in the main text and have ensured to remphasize in the figure legened. While a nice suggestion, we currently don’t feel this result rises to the level of a figure given it is not highly specific spatially.

Reviewer 2 (Recommendations For The Authors):

Overall the quality of the paper, the figures and the analysis used could be significantly improved. There is a lack of scientific rigor in the presentation of figures and analysis techniques. It is not clear what the authors are trying to communicate through the figures and their choice of figures to show is confusing (see below).

We thank the reviewer for their pointed critiques and believe we have addressed their concerns with many changes to the text, a revamped waveforms analysis, and both the expansion and addition of results.

The neurophysiology data shown doesn’t suggest neuronal loss, it only shows change which needs strong control data to show it is due to a lesion.

As detailed below, we have presented a revised analysis that provides this control. While the reviewer is right to point out we can distinguish actual neuron loss from neuron silencing, we be- lieve the new analysis rigorously indicates new rates of sample turnover beyond those expected from healthy state.

The histology figure should be replaced with a high-quality representation without folds.

We understand the reviewer’s suggestion. While ideally we would have many histology slices from each lesion, due to cost, we were only able to collect one histology slice per lesion. The folds were introduced by the company that performed the H&E staining, and we unfortunately cannot remove the folds. Therefore, despite the folds, this is the best and only image from this lesion. We hope that the markings on the figure and the comment in the caption is sufficient to explain to readers that the folds are not a result of the lesion but instead a result of the histology process.

The authors suggest that this lesioning method will be compatible with any available multielec- trode probe theoretically. Since all testing was done with a Utah array, it will be helpful to add an explanation about potential constraints that will make a given array compatible with this method.

We thank the reviewer for this suggestion. As stated above, we have both added a demonstration of use in a different multielectrode probe type (with a U-probe) in Fig. 8, and we have added a discussion about which types of multielectrode probes would be suitable on Page 15, Line 420.

The authors should cite and discuss previous studies using electrolytic lesioning in awake-behaving animals to study the causal connection between the brain and behavior. (One example study: Morissette MC, Boye SM. Electrolytic lesions of the habenula attenuate brain stimulation reward. Behavioural brain research. 2008 Feb 11;187(1):17-26.)

We thank the reviewers for this suggestion. We have added a mention of existing electrolytic le- sioning studies on Page 2, Line 88.

“Prior termination studies mostly measure behavioral output, with no simultaneous measures of neuronal activity during the behavior, impairing their ability to provide insight into the causal connection between the brain and behavior [7]–[11], or with no baseline (i.e., pre- lesion) measures of neuronal activity [4].”

The authors should compare their technique with other recent lesioning studies in primates (e.g. Khateeb et al, 2022)

We again thank the reviewer for this point. Specifically not mentioning Khateeb et al. 2022 was a submission error on our part; we cited the paper in Appendix 2 in the version uploaded to the eLife submission portal, but we had uploaded the version prior to citing it to bioRxiv. We have combined addressing this with addressing a previous comment, as mentioned above, with a section in the Discussion on Page 16, Line 434.

In Appendix 2, the authors suggest that a major limitation of optogenetics and chemogenetic in- activation methods is the lack of rhesus-compatible constructs. However, several viral constructs have successful implementation in rhesus monkeys so far (e.g. Galvan A, Stauffer WR, Acker L, El-Shamayleh Y, Inoue KI, Ohayon S, Schmid MC. Nonhuman primate optogenetics: recent advances and future directions. Journal of Neuroscience. 2017 Nov 8;37(45):10894-903; Tremblay et al, Neuron 2020)

We thank the reviewer for pointing us to these papers. We have added a more thorough description of what we meant by lack of rhesus-compatible constructs in that Appendix.

“However, other challenges exist with using optogenetics as an inactivation method in nonhu- man primates, including difficulty reliably affecting behavior [12]. While several constructs for rhesus macaques have been developed [13], [14], reports of successfully inducing be- havioral effects have a small effect size and are less numerous than might be expected [12], and several null results have been published [15]–[17]. Other remaining challenges include the need to develop a head-mounted, battery powered light delivery system for multi-day delivery of light and difficulty integrating illumination with simultaneous chronic neuro- electrophysiology.”

For Figure 5b, only pairwise comparison results from monkey U (L11-14) are shown. It is unclear why such results from monkey H were shown in Figure 5a but not in 5b.

We thank the reviewer for pointing out this unconventional one monkey result. As described in the original submission, we previously omitted Monkey H from the analysis in Figure 5b (now Figure 7) since some of the lesions were closely spaced together, preventing well defined pre- and post- lesion rates of turnover. Never-the-less we have included Monkey H in all the revised analysis and believe even the less cleanly separated data shows useful indications of neuron loss or silencing evoked by the lesion.

Behavioral data (during a motor task) from the awake behaving monkeys (U and H) would greatly strengthen the claim that this lesioning method is capable of creating a behavioral effect and can be adopted to study the relationship between neural function and behavior outcomes.

While we are grateful for the reviewer’s interest in the application of our lesioning technique to studies involving behavior, a behavioral analysis of the effects of our electrolytic lesions falls be- yond the scope of this Tools and Resources manuscript. We would also like to point out that we do not claim that we have achieved a behavioral deficit in this manuscript.

Figure 2 would benefit from an illustration of the Utah array placement and the location of the sites used for lesioning. The authors can either overlay the illustrations on the current ex-vivo and histology images or create a separate schematic to demonstrate that for the readers. Also, Figure 2B needs to be replaced with one without the folds to avoid confusion for the readers.

We have added Figure 2 - figure supplement 1, which shows both the location within the Utah array of the two electrodes used to create the lesions as well as the relative size of the surface area of the lesion and the array. Unfortunately, as the lesion was created under the array, the exact location of the array relative to the lesion is unknown.

As mentioned above, Figure 2B is the only histological image from that lesion. We hope that the markings in the image as well as the caption sufficiently explain that the folds are unrelated to the lesion itself.

Figure 3, the conical region is not well delineated. Data across animals and lesion volume with respect to different parameters should be included.

We have included a supplemental figure, Figure 3 - figure supplement 1, where we have used a dashed white line to clearly indicate the area of damaged parenchyma, in case it was not clear in Figure 3a. We have also added volume estimates from lesions across animals and different param- eters. The ex vivo estimates are shown in Figure 4 and the in vivo estimates are shown in Figure 5.

Figure 4: it is not clear what is being communicated, and where the voltage traces are from.

We thank the reviewer for noting this confusion. We have added some lines in the text to explain what the voltage traces show, both in the caption to Fig. 6 and in the text on Page 7, Line 238.

“Traces only capture the values while the lesioning device was turned on (45 seconds for most lesions and 50 seconds for lesion H200120). A) Voltage traces. Discontinuity at the beginning of the traces indicates transient voltages that were too rapid to be captured by the voltmeter, lasting between 0.13 and 0.33 s. The fluctuating voltages, especially the rapid in- crease in voltage at the beginning of lesioning, emphasize the importance of using a current source to deliver consistent amounts of current into the brain.”

“The voltage across the microelectrode array fluctuated much more than the current did, em- phasizing that we made the correct choice in using a current source to ensure delivery of consistent amounts of current into the brain (Fig. 6figure.caption.19).”

Figure 5: why did the authors choose to use matching units as a measure of the lesion? It is surprising that there are still units on the location that the authors claim to be a lesion. To clarify that it would be helpful to show the location of the lesion in Figure 4a. Also, what can we conclude about the lesion induction when we see units on the lesion electrode? The change in unit match shows that there is a change in the network (although the authors need to show control for that so we know those changes don’t happen due to natural dynamics). It is not clear what is the time duration for pre-pre and post-post (i.e. minutes, seconds, hours). Do these comparisons come from the same time frame or are they coming from two fragments of time for both pre and post- conditions?

Aside from post-mortem histology and tissue assays, there is no good way to confirm neuron loss with chronically implanted electrode arrays in nonhuman primates. Waveforms were chosen as they are the one readily isolated physical measure of the system we are injuring. Although functional measures of activity could indicate neuron loss (topic of following papers), there are many conceivable changes in firing rate patterns that could manifest spuriously as loss, making the estimation of loss even more ambiguous and challenging this way.

We believe the new Figure 7 will make the procedure much more clear, while also providing the control requested by the reviewer, illustrating that new statistical categories of altered waveforms emerge during a lesion, beyond those associated with typical changes in waveform composition within multi-unit recordings seen during recording sample turnover fom healthy animals. We further note that by confining this analysis to four day spans at most, we have limited the impact of daily sample turnover described in the literature (Gallego, 2020).

The time duration for pre-session versus pre-session (pre-post and post-post), is some multiple of the approximate 24 hours between each daily recording session. Therefore, since restricting our- selves to four days separation, between 24 and 96 hours. Spikes are sampled from successful trial periods (so on the order of seconds, compiled into minutes across the whole recording session). Although already described in the main text, these points have been reemphasized in the figure legend.

CNO (line 931) needs to be explained.

We thank the reviewer for this point. We have defined CNO and its relevance in Appendix 2.

“Additionally, chronic inactivation over days may be logistically challenging, as the half life of clozapine N-oxide (CNO, a ligand used to activate DREADD receptors) is on the order of hours.”

eLife assessment

This paper reports a valuable new method for creating localized damage to candidate brain regions for functional and behavioral studies. The authors present solid support for their ability to create long-term local lesions with mm spatial resolution. The paper is likely to be of broad interest to brain researchers working to establish causal links between neural circuits and behavior.

Reviewer #1 (Public Review):

In the paper, the authors illustrated a novel method for Electrolytic Lesioning through a microelectronics array. This novel lesioning technique is able to perform long-term micro-scale local lesions with a fine spatial resolution (mm). In addition, it allows a direct comparison of population neural activity patterns before and after the lesions using electrophysiology. This new technique addresses a recent challenge in the field and provides a precious opportunity to study the natural reorganization/recovery at the neuronal population level after long-term lesions. It will help discover new causal insights investigating the neural circuits controlling behavior.

Comments on revised version:

We appreciate the revisions made by the authors in response to our comments on the previous version of their manuscript. They carefully addressed the majority of the concerns and performed additional experiments. The new figure illustrating the lesion volume as a function of electrolytic lesioning parameters provides a valuable reference for future experiments. In addition, the latest results on different versions of passive multielectrode probes, U-probe, demonstrate that the technique is applicable beyond the specific technical setup they employ. Overall, we believe that the revised manuscript is significantly improved.

Reviewer #2 (Public Review):

This work by Bray et al. presented a customized way to induce small electrolytic lesions in the brain using chronically implanted intracortical multielectrode arrays. This type of lesioning technique has the benefit of high spatial precision and low surgical complexity while allowing simultaneous electrophysiology recording before, during, and after the lesion induction. The authors have validated this lesioning method with a Utah array, both ex vivo and in vivo using pig models and awake-behaving rhesus macaques. Given its precision in controlling the lesion size, location, and compatibility with multiple animal models and cortical areas, the authors believe this method can be used to study cortical circuits in the presence of targeted neuronal inactivation or injury and to establish causal relationships before behavior and cortical activity.

Strengths:

- Overall the techniques, parameters, and data analysis methods are better described in the revised version.

- The authors added the section "Relationship Between Applied Current and Lesion Volume" as well as Figure 4 and 5 to address our comments regarding parameter testing. Multiple combinations of current amplitude and duration were tested and the induced lesion volumes were estimated, providing a better picture of why certain parameters were chosen for in vivo studies.

- The authors added Figure 7 which addressed our comment "more evidence is needed to suggest robust neuronal inactivation or termination in rhesus macaques after electrolytic lesioning." They went into more details to explain the observed changes in pairwise comparisons of spike waveforms (difference in projected radii). Particularly in Fig 7C, they identified a new cluster from the pre-post lesioning group, which effectively represented neuronal loss from the<br /> recorded population.

- The authors discussed their method in the context of other literature and stating its strength and limitation.

Major comments:

-The lack of histology limits the validation of lesion induction, ideally cell loss and neuronal loss in vivo needs to be quantified. In addition based on the lack of access to histology, it is not clear how the lesion volumes are calculated which also impacts the scientific rigor of the work. The authors mention that layers 2/3 and maybe 4 have been impacted. The lack of information on the extent of the lesion severely limits the use of their technique for neuroscience experiments.

-The lack of histology in combination with behavioral measures still limits the impact of the paper in the context of NHP research.

- Figure 5 involves fitting an exponential model to the generated lesion volume given the applied current amplitude and duration. However, the data from ex vivo sheep and pig cortex with the same current amplitude & three durations showed very large variability in lesion volume at Time = 2min (larger than the difference from 2 to ~2.2min). Very limited data points exist for the other two parameter combinations. These may suggest that the exponential fit is not the best model in this scenario.

- Regarding the comment on neuronal inactivation, the authors still did not show any evidence of single unit activity loss or changes in local field potential/multi-unit activity from the region being lesioned.

- Regarding this comment "The lesioning procedure was performed in Monkey F while sedated, but no data was presented for Monkey F in terms of lesioning parameters, lesion size, recorded electrophysiology, histological, or behavioral outcomes. It is also unclear if Monkey F was in a terminal study" the authors explained that "a lesion was performed on a sedated rhesus macaque (monkey F) who was subsequently euthanized due to unrelated health complications, in order to further verify safety before use in awake-behaving rhesus" but still no histology data is shown regarding monkey F to demonstrate this verification. Given that NHPs are highly valuable resources, it's important to make use of all collected data and to show that the induced lesion is comparable to those in the pig cortex.

Reviewer #1 (Public Review):

This paper describes "Ais", a new software tool for machine-learning-based segmentation and particle picking of electron tomograms. The software can visualise tomograms as slices and allows manual annotation for the training of a provided set of various types of neural networks. New networks can be added, provided they adhere to a Python file with an (undescribed) format. Once networks have been trained on manually annotated tomograms, they can be used to segment new tomograms within the same software. The authors also set up an online repository to which users can upload their models, so they might be re-used by others with similar needs. By logically combining the results from different types of segmentations, they further improve the detection of distinct features. The authors demonstrate the usefulness of their software on various data sets. Thus, the software appears to be a valuable tool for the cryo-ET community that will lower the boundaries of using a variety of machine-learning methods to help interpret tomograms.

eLife assessment

This work describes a new software platform for machine-learning-based segmentation of and particle-picking in cryo-electron tomograms. The program and its corresponding online database of trained models will allow experimentalists to conveniently test different models and share their results with others. The paper provides solid evidence that the software will be valuable to the community.

Reviewer #2 (Public Review):

Summary:

Last et al. present Ais, a new deep learning-based software package for the segmentation of cryo-electron tomography data sets. The distinguishing factor of this package is its orientation to the joint use of different models, rather than the implementation of a given approach. Notably, the software is supported by an online repository of segmentation models, open to contributions from the community.

The usefulness of handling different models in one single environment is showcased with a comparative study on how different models perform on a given data set; then with an explanation of how the results of several models can be manually merged by the interactive tools inside Ais.

The manuscripts present two applications of Ais on real data sets; one is oriented to showcase its particle-picking capacities on a study previously completed by the authors; the second one refers to a complex segmentation problem on two different data sets (representing different geometries as bacterial cilia and mitochondria in a mouse neuron), both from public databases.

The software described in the paper is compactly documented on its website, additionally providing links to some YouTube videos (less than an hour in total) where the authors videocapture and comment on major workflows.

In short, the manuscript describes a valuable resource for the community of tomography practitioners.

Strengths:

A public repository of segmentation models; easiness of working with several models and comparing/merging the results.

Weaknesses:

A certain lack of concretion when describing the overall features of the software that differentiate it from others.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Last and colleagues describe Ais, an open-source software package for the semi-automated segmentation of cryo-electron tomography (cryo-ET) maps. Specifically, Ais provides a graphical user interface (GUI) for the manual segmentation and annotation of specific features of interest. These manual annotations are then used as input ground-truth data for training a convolutional neural network (CNN) model, which can then be used for automatic segmentation. Ais provides the option of several CNNs so that users can compare their performance on their structures of interest in order to determine the CNN that best suits their needs. Additionally, pre-trained models can be uploaded and shared to an online database.

Algorithms are also provided to characterize "model interactions" which allows users to define heuristic rules on how the different segmentations interact. For instance, a membrane-adjacent protein can have rules where it must colocalize a certain distance away from a membrane segmentation. Such rules can help reduce false positives; as in the case above, false negatives predicted away from membranes are eliminated.

The authors then show how Ais can be used for particle picking and subsequent subtomogram averaging and for the segmentation of cellular tomograms for visual analysis. For subtomogram averaging, they used a previously published dataset and compared the averages of their automated picking with the published manual picking. Analysis of cellular tomogram segmentation was primarily visual.

Strengths:

CNN-based segmentation of cryo-ET data is a rapidly developing area of research, as it promises substantially faster results than manual segmentation as well as the possibility for higher accuracy. However, this field is still very much in the development and the overall performance of these approaches, even across different algorithms, still leaves much to be desired. In this context, I think Ais is an interesting package, as it aims to provide both new and experienced users with streamlined approaches for manual annotation, access to a number of CNNs, and methods to refine the outputs of CNN models against each other. I think this can be quite useful for users, particularly as these methods develop.

Weaknesses:

Whilst overall I am enthusiastic about this manuscript, I still have a number of comments:

On page 5, paragraph 1, there is a discussion on human judgement of these results. I think a more detailed discussion is required here, as from looking at the figures, I don't know that I agree with the authors' statement that Pix2pix is better. I acknowledge that this is extremely subjective, which is the problem. I think that a manual segmentation should also be shown in a figure so that the reader has a better way to gauge the performance of the automated segmentation.

On page 7, the authors mention terms such as "emit" and "absorb" but never properly define them, such that I feel like I'm guessing at their meaning. Precise definitions of these terms should be provided.

For Figure 3, it's unclear if the parent models shown (particularly the carbon model) are binary or not. The figure looks to be grey values, which would imply that it's the visualization of some prediction score. If so, how is this thresholded? This can also be made clearer in the text.

Figure 3D was produced in ChimeraX using the hide dust function. I think some discussion on the nature of this "dust" is in order, e.g. how much is there and how large does it need to be to be considered dust? Given that these segmentations can be used for particle picking, this seems like it may be a major contributor to false positives.

Page 9 contains the following sentence: "After selecting these values, we then launched a batch particle picking process to determine lists of particle coordinates based on the segmented volumes." Given how important this is, I feel like this requires significant description, e.g. how are densities thresholded, how are centers determined, and what if there are overlapping segmentations?

The FSC shown in Figure S6 for the auto-picked maps is concerning. First, a horizontal line at FSC = 0 should be added. It seems that starting at a frequency of ~0.045, the FSC of the autopicked map increases above zero and stays there. Since this is not present in the FSC of the manually picked averages, this suggests the automatic approach is also finding some sort of consistent features. This needs to be discussed.

Page 11 contains the statement "the segmented volumes found no immediately apparent false positive predictions of these pores". This is quite subjective and I don't know that I agree with this assessment. Unless the authors decide to quantify this through subtomogram classification, I don't think this statement is appropriate.

In the methods, the authors note that particle picking is explained in detail in the online documentation. Given that this is a key feature of this software, such an explanation should be in the manuscript.

eLife assessment

In this study, camera trapping and species distribution models are used to show that human disturbance in mountain forests in the eastern Himalayas pushes medium-sized and large mammal species into narrower habitat space, thus increasing their co-occurrence. While the collected data provide a useful basis for further work, the study presents incomplete evidence to support the claim that increased co-occurrence may indicate positive interactions between species.

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing. Also, the authors do not clearly distinguish between significant (true) non-random associations and random associations.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we would need to better understand what the individual species do and "like" (their environmental niche).

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the study is not set up in a way to test this directly.

The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one could then add the layer of interspecific associations.

The change in activity and space use could be analysed by looking at the activity times and spatial distribution directly. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

The per-species models are simplistic: the predictors are only linear, and there are no statistical interactions. It is unclear how spatial autocorrelations of residuals were treated, although they form the basis for the association analysis. Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. What is the advance in ecological understanding at the community level?

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.

Strengths:

The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.

Weaknesses:

The authors use the terms associations and interactions interchangeably.

This is not the case. In fact, we state specifically that "... interspecific associations should not be directly interpreted as a signal of biotic interactions between pairs of species…" However, co-occurrence can be an important predictor of likely interactions, such as competition and predation. We stand by our original text.

It is not clear what the authors mean by "associations". A brief clarification would be helpful.

Our specific definition of what is meant here by spatial association can be found in the Methods section. To clarify, the calculation of the index of associations is based on the covariance for the two species of the residuals (epsilon) after consideration of all species-specific response to known environmental covariates. These covariances are modelled to allow them to vary with the level of human disturbance, measured as human presence and human modification. After normalization, the final index of association is a correlation value that varies between -1 (complete disassociation) and +1 (complete positive association).

Also, the authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing.

Suggesting the ecological underpinnings of the associations observed here would mainly be speculation at this point, but the associations demonstrated in this analysis do suggest promising areas for the more detailed research suggested.

Also, the authors do not distinguish between significant (true) non-random associations and random associations. In my opinion, associations are those in which two species co-occur more or less than expected by chance. This is not well addressed in the present version of the manuscript.

Results were considered to be non-random if correlation coefficients (for spatial association) or overlap (for temporal association) fell outside of 95% Confidence Intervals. This is now stated clearly in the Methods section.  In Figure 3—figure supplement 1-3 and Figure 4—figure supplement 1-3, p<0.01 levels are also presented.

The obtained results support the conclusions of the study.

Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.

Reviewer #2 (Public Review):

Summary:

This study analyses camera trapping information on the occurrence of forest mammals along a gradient of human modification of the environment. The key hypotheses are that human disturbance squeezes wildlife into a smaller area or their activity into only part of the day, leading to increased co-occurrence under modification. The method used is joint species distribution modelling (JSDM).

Strengths:

The data source seems to be very nice, although since very little information is presented, this is hard to be sure of. Also, the JSDM approach is, in principle, a nice way of simultaneously analysing the data.

Weaknesses:

The manuscript suffers from a mismatch of hypotheses and methods at two different levels.

(1) At the lower level, we first need to understand what the individual species do and "like" (their environmental niche). That information is not presented, and the methods suggest that the representation of each species in the JSDM is likely to be extremely poor.

The response of each species to the environmental covariates provides a window into their environmental niche, encapsulated in the beta coefficients for each environmental covariate. This information is presented in Figure 2.

(2) The hypothesis clearly asks for an analysis of the statistical interaction between human disturbance and co-occurrence. Yet, the model is not set up this way, and the authors thus do a lot of indirect exploration, rather than direct hypothesis testing.

Our JSDM model is set up specifically to examine the effect of human disturbance on co-occurrence, after controlling for shared responses to environmental variables.  It directly tests the first hypothesis, since, if increase in indices of human disturbance had not tended to increase the measured spatial correlations between species as detected by the model, we would have rejected our stated hypothesis that human modification of habitats results in increased positive spatial associations between species.

Even when the focus is not the individual species, but rather their association, we need to formulate what the expectation is. The hypotheses point towards presenting the spatial and the temporal niche, and how it changes, species for species, under human disturbance. To this, one can then add the layer of interspecific associations.

Examining each species one by one and how each one responds to human disturbance would miss the effects of any meaningful interactions between species.  The analysis presented provides a means to highlight associations that would have been overlooked.  Future research could go on to analyze the strongest associations in the community and the strongest effects of human disturbance so as to uncover the underlying interactions that give rise to them and the mechanisms of human impact.  We believe that this will prove to be a much more productive approach than trying to tackle this problem species by species and pair by pair.

The change in activity and space use can be analysed much simpler, by looking at the activity times and spatial distribution directly. It remains unclear what the contribution of the JSDM is, unless it is able to represent this activity and spatial information, and put it in a testable interaction with human disturbance.

The topic is actually rather complicated. If biotic interactions change along the disturbance gradient, then observed data are already the outcome of such changed interactions. We thus cannot use the data to infer them! But we can show, for each species, that the habitat preferences change along the disturbance gradient - or not, as the case may be.

Then, in the next step, one would have to formulate specific hypotheses about which species are likely to change their associations more, and which less (based e.g. on predator-prey or competitive interactions). The data and analyses presented do not answer any of these issues.

We suggest that the so-called “simpler” approach described above is anything but simple, and this is precisely what the Joint Species Distribution Model improves upon.  As pointed out in the Introduction, simply examining spatial overlap is not enough to detect a signal of meaningful biotic interaction, since overlap could be the result of similar responses to environmental variables.  With the JSDM approach, this would not be considered a positive association and would then not imply the possible existence of meaningful interaction.

Another more substantial point is that, according to my understanding of the methods, the per-species models are very inappropriate: the predictors are only linear, and there are no statistical interactions (L374). There is no conceivable species in the world whose niche would be described by such an oversimplified model.

While interaction terms can be included in the JSDM, this would considerably increase the complexity of the models.  In previous work, we have found no strong evidence for the importance of interaction terms and they do not improve the performance of the models.

We have no idea of even the most basic characteristics of the per-species models: prevalences, coefficient estimates, D2 of the model, and analysis of the temporal and spatial autocorrelation of the residuals, although they form the basis for the association analysis!

The coefficient estimates for response to environmental variables used in the JSDM are provided in Figure 2 and Figure 2—source data 1.

Why are times of day and day of the year not included as predictors IN INTERACTION with niche predictors and human disturbance, since they represent the temporal dimension on which niches are hypothesised to change?

Also, all correlations among species should be shown for the raw data and for the model residuals: how much does that actually change and can thus be explained by the niche models?

The discussion has little to add to the results. The complexity of the challenge (understanding a community-level response after accounting for species-level responses) is not met, and instead substantial room is given to general statements of how important this line of research is. I failed to see any advance in ecological understanding at the community level.

We agree that the community-level response to human disturbance is a complex topic, and we believe it is also a very important one.  This research and its support of the spatial compression hypothesis, while not providing definitive answers to detailed mechanisms, opens up new lines of inquiry that makes it an important advance.  For example, the strong effects of human disturbance on certain associations that were detected here could now be examined with the kind of detailed species by species and pair by pair analysis that this reviewer appears to demand.

Reviewer #1 (Recommendations For The Authors):

L27 indicates instead of "idicates".

We thank the reviewer for catching that error.

L64 I would refer to potential interactions or just associations. It is always hard to provide evidence for the existence of true interactions.

We have revised to “potential interactions” to qualify this statement.

L69 Suggestion: distort instead of upset.

We thank the reviewer for catching that error.

L70-71 Here, authors use the term associations. Please, be consistent with the terminology throughout the manuscript.

We thank the reviewer for raising this important point.  The term “co-occurrence” appears to be used inconsistently in the literature, so we have tried to refer to it only when referencing the work of us. For us, co-occurrence means “spatial overlap” without qualification as to whether it is caused by interaction or simply by similar responses to environmental factors (see Blanchet et al. 2020, Argument 1). In our view, interactions refer to biotic effects like predation, competition, commensalism, etc., while associations are the statistical footprint of these processes.   In keeping with this understanding, in Line 73, we changed "association" to the stronger word "interaction," but in Line 76, we keep the words "spatiotemporal association", which is presumed to be the result of those interactions. In Line 91, we have changed “interactions” to “associations,” as we do not believe interactions were demonstrated in that study. 

L76 "Species associations are not necessarily fixed as positive or negative..." This sentence is misleading. I would say that species associations can vary across time and space, for instance along an environmental gradient.

We thank the reviewer for pointing out the potential for confusion.  In Line 79, we have changed as suggested.

L78 "Associations between free-ranging species are especially context-dependent" Loose sentence. Please, explain a bit further.

We have changed the sentence to be more specific; ”Interactions are known to be context-dependent; for example, gradients in stress are associated with variation in the outcomes of pairwise species interactions.”

L83-85 This would be a good place to introduce the 'stress gradient' hypothesis, which has also been applied to faunal communities in a few studies. According to this hypothesis, the incidence of positive associations should increase as environmental conditions harden.

In our review of the literature, we find that the stress gradient hypothesis is somewhat controversial and does not receive strong support in vertebrates.  We have added the phrase “…the controversial stress-gradient hypothesis predicts that positive associations should increase as environmental conditions become more severe…”

L86-88 Well, overall, the number of studies examining spatiotemporal associations in vertebrates is relatively small. That is, bird associations have not received much more attention than those of mammals. I find this introductory/appealing paragraph a bit rough. I think the authors can do better and find a better justification for their work.

We thank the reviewer for the comments.  We have rewritten the paragraph extensively to make it clearer and to provide a stronger justification for the study.

L106 "[...] resulting in increased positive spatial associations between species" I'd say that habitat shrinking would increase the level of species clustering or co-occurrence, but in my opinion, not necessarily the incidence of positive associations. It is not clear to me if the authors use positive associations as a term analogous to co-occurrence.

We thank the reviewer for raising this very important distinction.  Habitat shrinking would increase levels of species co-occurrence, but this is not particularly interested.  We wanted to test whether there were effects on species interactions, as revealed by associations.  We find that the terms association and co-occurrence are used somewhat loosely in the literature and so have made some new effort to clarify and systematize this in the manuscript.  For example, there appear to be a differences in the way “co-occurrence” is used in Boron 2023 and in Blanchet 2020. We do not use the term "positive spatial association" as analogous to "spatial co-occurrence.". Spatial co-occurrence, which for us has the meaning of spatial overlap, could simply be the result of similar reactions to environmental co-variates, not reflecting any biotic interaction. Joint Species Distribution Models enable the partitioning of spatial overlap and segregation into that which can be explained by responses to known environmental factors, and that which cannot be explained and thus might be the result of biotic interactions.  It is only the latter that we are calling spatial association, which can be positive or negative.   These associations may be the statistical footprint of biotic interactions.

Results:

Difference between random and non-random association patterns. It is not clear to me if the reported associations are significant or not. The authors only report the sign of the association (either positive or negative) but do not clarify if these associations indicate that two species coexist more or less than expected by chance. In my opinion, that is the difference between true ecological associations (e.g., via facilitation or competition effects) and random co-existence patterns. This is paramount and should be addressed in a new version of the manuscript.

This information is provided in Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3.  This is referenced in the text as follows, “… correlation coefficients for 18 species pairs were positive and had a 95 % CI that did not overlap zero, and the number increased to 65 in moderate modifications but dropped to 29 at higher modifications" and so on. This criterion for significance (ie., greater than expected by chance) is now stated at the end of the Materials and methods.  In Figure 3—figure supplement 1,2,3 and Figure 4—figure supplement 1,2,3, those correlations that were significant at p<0.01 are also shown.

I am also missing a more ecological explanation for the observed findings. For instance, the top-ranked species in terms of negative associations is the red fox, whereas the muntjac seems to be the species whose presence can be used as an indicator for that of other species. What are the mechanisms underlying these patterns? Do red foxes compete for food with other species? Do the species that show positive associations (red goral, muntjac) have traits or a diet that are more different from those of other species? More discussion on these aspects (role of traits and the trophic niche) would be necessary to better understand the obtained results.

The purpose of this paper was to test the compression hypotheses, and we have tried to keep that as the focus.  However, the analysis does open up interesting lines of inquiry for future research to decipher the details of the interactions between species and the mechanisms by which human disturbance facilitates or disrupts these interactions. The reviewer raises some interesting possibilities, but at this point, any discussion along these lines would be largely speculation and could lengthen the paper without great benefit. 

Reviewer #2 (Recommendations For The Authors):

The manuscript should be accompanied by all data and code of analysis.

All data and RScripts have been made available in Science Data Bank: https://doi.org/10.57760/sciencedb.11804.

The sentence "not much is known" is weak: it suggests the authors did not bother to quantify what IS known, and simply waved any previous knowledge aside. Surely we have some ideas about who preys on whom, and which species have overlapping resource requirements (e.g., due to jaw width). For those, we would expect a particularly strong signal, if the association is indeed indicative of interactions.

We believe that the reviewer is referring to the statement in Line 90-92 about the lack of understanding of the resilience of terrestrial mammal associations to human disturbance.  We have added a reference to one very recent publication that addresses the issue (Boron et al., 2023), but otherwise we stand by our statement. We have, however, added a qualifier to make it clear that we did indeed look for previous knowledge; "However, a review of the literature indicates that ...."

Figures:

Fig. 1. This reviewer considers that this is too trivial and should be deleted.

This is a graphical statement of the hypotheses and may be helpful to some readers.

Fig. 2. Using points with error bars hides any potential information.

Done as suggested.

That only 4 predictors are presented is unacceptably oversimplified.

Only 4 predictors are included because, in previous work, we found that adding additional predictors or interactions did little to improve the model’s performance (Li et al. 2018, 2021 and 2022) and could lead to over-fitting.

Fig. 5. and 6. aggregate extremely strongly over species; it remains unclear which species contribute to the signal, and I guess most do not.

The number of detection events presented in Table 1 should help to clarify the relative contribution of each species to the data presented in Figures 5 and 6.

This reviewer considers that the introduction 'oversells' the paper.

L55: can you give any such "unique ecological information"

L60: Lyons et al. (Kathleen is the first name) has been challenged by Telford et al. (2016 Nature) as methodologically flawed.

The first name has been deleted.  The methodological flaw has to do with interpretation of the fossil record and choice of samples, not with the need to partition shared environmental preferences and interactions.

L61 contradicts line 64: Blanchet et al. (2022, specifying some arguments from Dormann et al. 2018 GEB) correctly point out that logically one cannot infer the existence or strength from co-occurrence data. It is thus wrong to then claim (citing Boron et al.) that such data "convey key information about interactions". The latter statement is incorrect. A tree and a beetle can have extremely high association and nothing to do with each other. Association does not mean anything in itself. When two species are spatially and temporally non-overlapping, they can exhibit perfect "anti-association", yet, by the authors' own definition, cannot interact.

We believe that the reviewer’s concerns arise from a misunderstanding of how we use the term association.  In our usage, an association is not the same as co-occurrence or overlap, which may simply be the result of shared responses to environmental variables.  The co-occurring tree and beetle would not be found to have any association in our analysis, only shared environmental sensitivities.  In contrast, associations can be the statistical footprint of interactions, and would be overlaid onto any overlap due to similar responses to the environment.  In the case of negative associations, such as might be the result of competitive exclusion or avoidance of predators, the two species would share environmental responses but show lower than expected spatial overlap.  Even though they might be only rarely found in the same vicinity, they would indeed be interacting when they were together.

Joint Species Distribution Models "allow the partitioning of the observed correlation into that which can be explained by species responses to environmental factors... and that which remains unexplained after controlling for environmental effects and which may reflect biotic interactions." (Garcia Navas et al. 2021). It is the latter that we are calling “associations.”

L63: Gilbert reference: Good to have a reference for this statement.

This point is important, but the reviewer’s comments below have made it clear that it is even more important to point out that strong interactions should be expected to lead to significant associations.  We have added a statement to clarify this.

L70-72: Incorrect, interactions play a role, not associations (which are merely statistical).

In this, we agree, and we have revised the statement to refer to interactions, not associations. In our view, an interaction is a biological phenomenon, while an association is the resulting statistical signal that we can detect.

L75: Associations tell us nothing, only interactions do. Since these can not be reliably inferred, this statement and this claim are wrong.

We thank the reviewer for raising this point, but we beg to disagree. Strong interactions should be expected to lead to significant associations that can be detected in the data. Associations, which can be measured reliably, are the evidence of potential interactions, and hence associations can tell us a great deal.  We have added a note to this effect after the Gilbert reference above to clarify this point.

However, we do accept that associations must be interpreted with caution. As Blanchet et al. 2020 explain, " …the co-occurrence signals (e.g. a significant positive or negative correlation value) estimated from these models could originate from any abiotic factors that impact species differently. Therefore, this correlation cannot be systematically interpreted as a signal of biotic interactions, as it could instead express potential non-measured environmental drivers (or combinations of them) that influence species distribution and co-distribution.”  Or alternatively an association could be the result of interaction with a 3rd species. 

L87: Regarding your claim, how would you know you DO understand? For that, you need to formulate an expectation before looking at the data and then show you cannot show what you actually measure. (Jaynes called this the "mind-projection fallacy".)

We are not sure if the reviewer is criticizing our paper or the entire field of community ecology.  Perhaps it is the statement that “….resilience of interspecific spatiotemporal associations of terrestrial mammals to human activity remains poorly understood….”  Since we are confident that the reviewer believes that mammals do interact, we guess that it is the term “association” that is questioned.  We have revised this to “…the impacts of human activity on interspecific interactions of terrestrial mammals remains poorly understood…” 

In this particular case, we did formulate an expectation before looking at the data, in the form of the two formal hypotheses that are clearly stated in the Introduction and illustrated in Figure 1. If the hypotheses had not been supported, then we would have accepted that we do not understand. But as the data are consistent with the hypotheses, we submit that we do understand a bit more now.

eLife assessment

This study provides a valuable resource by thoroughly benchmarking multiple sequencing-based tRNA quantification methods. The suggested best practice is supported by solid evidence from in silico experiments in multiple scenarios. The major weakness of the manuscript is the incomplete validation of newly generated experimental datasets.

Reviewer #1 (Public Review):

Summary:

In the manuscript titled "Benchmarking tRNA-Seq quantification approaches by realistic tRNA-Seq data simulation identifies two novel approaches with higher accuracy," Tom Smith and colleagues conducted a comparative evaluation of various sequencing-based tRNA quantification methods. The inherent challenges in accurately quantifying tRNA transcriptional levels, stemming from their short sequences (70-100nt), extensive redundancy (~600 copies in human genomes with numerous isoacceptors and isodecoders), and potential for over 100 post-transcriptional chemical modifications, necessitate sophisticated approaches. Several wet-experimental methods (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA) combined with bioinformatics tools (bowtie2-based, SHRiMP, and mimseq) have been proposed for this purpose. However, their practical strengths and weaknesses have not been comprehensively explored to date. In this study, the authors systematically assessed and compared these methods, considering factors such as incorrect alignments, multiple alignments, misincorporated bases (experimental errors), truncated reads, and correct assignments. Additionally, the authors introduced their own bioinformatic approaches (referred to as Decision and Salmon), which, while not without flaws (as perfection is unattainable), exhibit significant improvements over existing methods.

Strengths:

The manuscript meticulously compares tRNA quantification methods, offering a comprehensive exploration of each method's relative performance using standardized evaluation criteria. Recognizing the absence of "ground-truth" data, the authors generated in silico datasets mirroring common error profiles observed in real tRNA-seq data. Through the utilization of these datasets, the authors gained insights into prevalent sources of tRNA read misalignment and their implications for accurate quantification. Lastly, the authors proposed their downstream analysis pipelines (Salmon and Decision), enhancing the manuscript's utility.

Weaknesses:

As discussed in the manuscript, the error profiles derived from real-world tRNA-seq datasets may still harbor biases, as reads that failed to "align" in the analysis pipelines were not considered. Additionally, the authors did not validate the efficacy of their "best practice" pipelines on new real-world datasets, preferably those generated by the authors themselves. Such validation would not only confirm the improvements but also demonstrate how these pipelines could alter biological interpretations.<br /> Because tRNA-sequencing methods have not been widely used (compared to mRNA-seq), many readers would not be familiar with the characteristics of different methods introduced in this study (QuantM-tRNA, mim-tRNA, YAMAT, DM-tRNA, and ALL-tRNA; bowtie2-based, SHRiMP, and mimseq; what are the main features of "Salmon?"). The manuscript will read better when the basic features of these methods are described in the manuscript, however brief.

Reviewer #2 (Public Review):

Summary:

The authors provided benchmarking study results on tRNA-seq in terms of read alignment and quantification software with optimal parameterization. This result can be a useful guideline for choosing optimal parameters for tRNA-seq read alignment and quantification.

Strengths:

Benchmarking results for read alignment can be a useful guideline to choose optimal parameters and mapping strategy (mapping to amino acid) for various tRNAseq.

Weaknesses:

The topic is highly specific, and the novelty of the analysis might not be widely useful for general readers.

Some details of the sequencing data analysis pipeline are not clear for general readers:

(1) The explanation of the parameter D for bowtie2 sounds ambiguous. "How much effort to expend" needs to be explained in more detail.

(2) Please provide optimal parameters (L and D) for tRNA-seq alignment.

(3) I think the authors chose L=10 and D=100 based on Figure 1A. Which dataset did you choose for this parameterization among ALL-tRNAseq, DM-tRNAseq, mim-tRNAseq, QuantM-tRNA-seq, and YAMAT-seq?

(4) Salmon does not need a read alignment process such as Bowtie2. Hence, it is not clear "Only results from alignment with bowtie2" in Figure legend for Figure 4a.

Author response:

We thank the reviewers for their critical appraisal of our manuscript. We will address the points of confusion and/or lack of clarity in a revised manuscript. We agree with reviewer 1 that applying the best practice pipeline(s) on new experimental data and comparing this approach with current practices would be a useful demonstration of how this alters the biological interpretation. This is something we are in the process of completing but believe this is best addressed in a separate manuscript where we can focus on the associated biological findings, allowing this manuscript to remain focused on the accurate quantification of tRNA-Seq data.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This manuscript provides useful information about the lipid metabolite 15d-PGJ2 as a potential regulator of myoblast senescence. The authors provide experimental evidence that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas. However, the manuscript is incomplete in its current form, as it lacks robust support from the data regarding the main conclusions related to senescence and technical concerns related to the senescence models used in this study.

We are grateful to the editors and the reviewers for their time and comments in sharpening the science and the writing of the manuscript. We have attached a detailed response to emphasize that the manuscript does include robust evidence regarding the claims, which could have been missed during the review process. We have provided a better context for these points now.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.

Strengths:

The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.

Weaknesses:

The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.

As pointed out Doxo induces systemic inflammation along with inducing DNA damage-mediated senescence. Therefore, along with the inflammatory markers of the SASP (CXCL1/2, TNF1α, IL6, PTGS1/2, PTGDS) we also observed an increase in the mRNA levels of canonical markers of DNA damage-mediated senescence. We observed an increase in the mRNA levels of cell cycle and senescence associated proteins p16 and p21 (Fig. 1C). We also observed an increased nuclear accumulation of p21 (Fig. 1A) and increased levels of phosphorylated H2A.X in the nucleus (Fig. 1B).

In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment.

We measured the viability of C2C12 cells after 24 hours of treatment with 15d-PGJ2 using the MTT assay and observed that the viability of cells was decreased after treatment with 15d-PGJ2 (10 µM) but not with 15d-PGJ2 (1 µM, 2 µM, 4 µM, or 5 µM) (see Fig. S2A of the updated manuscript). The results and figures of the manuscript have been updated accordingly.

Also, in the myoblast experiments, are the effects of treatment with Dox reversible?

The treatment with Doxorubicin is irreversible as the senescent phenotype was not reversed after withdrawal of Doxorubicin, even after 20 days.

In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours.

Figure 3 is an acute experiment for only 1 hour, at which time no cell death was observed. Specifically, we measured the phosphorylation of Erk and Akt proteins after 1 hour of treatment with 15d-PGJ2 (10 µM) during which we did not observe any cell death. 

Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.

We observe a ~30% increase in the phosphorylation of Erk proteins after treatment with 15d-PGJ2 in 0.2% serum medium compared to treatment with vehicle (DMSO). This is reproducible and significant.

The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2.

Our data does not suggest higher levels of C184S mutant in the Golgi compared with WT (Fig. S4A). We observed that the ratio of HRas levels in the Golgi to the HRas levels in the plasma membrane were similar in C2C12 cells expressing HRas C184S and HRas WT (Fig. S4A graph columns 1 and 5).

Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.

Palmitoylation of HRas C181S is required for the localization of HRas at the plasma membrane. The inhibition of palmitoylation of C181, either by mutation (C181S) or treatment with protein palmitoyl transferase inhibitor (2-Bromopalmitate), results in the accumulation of HRas at Golgi(Rocks et al., 2005) (Fig. S4A). Modification of HRas at C184 by 15d-PGJ2 (Fig. 3A) could inhibit the palmitoylation of HRas at C181. However, our data does not support this hypothesis as modification of HRas WT by 15d-PGJ2 does not increase the level of HRas at the Golgi, like in the case of inhibition of cysteine palmitoylation due to C181S mutation.

To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears. 

Endogenous HRas (wild type) is present in the C2C12 cells overexpressing the EGFP-tagged HRas constructs. Therefore, we only observe a partial rescue in the differentiation after 15d-PGJ2 treatment in C2C12 cells expressing the C184S mutant (Fig. 4D and E). However, since HRas is expressed under high expression CMV promoter and in the absence of other regulatory elements, the overexpressed constructs do show a dominant effect over the endogenous HRas, showing cysteine mutant dependent inhibition of differentiation of myoblasts after treatment with 15dPGJ2 (Fig. 4D and E).

Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.

The mRNA levels of MyoD, MyoG, and MHC in C2C12 cells expressing HRas constructs after treatment with 15d-PGJ2 were normalized to the mRNA levels in C2C12 cells expressing corresponding constructs and were treated with vehicle (DMSO). mRNA levels in C2C12 cells treated with vehicle were not shown as they were normalized to 1. MHC protein levels in C2C12 cells expressing HRas constructs after 15d-PGJ2 treatment were normalized to that in C2C12 cells treated with vehicle (DMSO). Since the hypothesis to study the effect of HRas cysteine mutations on the differentiation of myoblasts after treatment with 15d-PGJ2, C2C12 cells expressing HRas WT serve as adequate control. Fig. 2 shows the effect of 15dPGJ2 on muscle differentiation when HRas was not overexpressed.

Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.

The inhibition of differentiation in C212 cells after treatment with 15d-PGJ2 cannot be attributed to the general toxicity of 15d-PGJ2 in cells. We show that the inhibition of differentiation of myoblasts after 15d-PGJ2 depends on modification of HRas at C184 i.e. failure to modify HRas at C184 (Fig. 3A) and resultant activation (Fig. 3B) by 15d-PGJ2 rescues this inhibition of differentiation of C2C12 cells (Fig. 4D and E), dissecting the inhibition of differentiation of myoblasts by 15d-PGJ2 from general toxic effects of 15d-PGJ2 on cell physiology.

Please note that the effect of 15d-PGJ2 on cell physiology is context-specific. On one hand, 15d-PGJ2 has been shown to exert tumor-suppressor effects by inhibiting the proliferation of ovarian cancer cells and lung adenocarcinoma cells (de Jong et al., 2011; Slanovc et al., 2024), 15d-PGJ2 also exerts pro-carcinogenic effects by induction of epithelial to mesenchymal transition in breast cancer cells MCF7 and inhibition of tumor-suppressor protein p53 in MCF7 and PC-3 cells (Choi et al., 2020; Kim et al., 2010).

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

The novelty of the study is compromised as the activation of PGD and 15d-PGJ2, as well as the regulation of HRas and cell proliferation, have been previously reported. 

Literature does not support this statement, and it is important to clarify this misimpression for the field as a whole. 

Let us clarify- 

Covalent modification of HRas by 15d-PGJ2 has been reported only twice in the literature(Luis Oliva et al., 2003; Yamamoto et al., 2011) in fibroblasts and neurons respectively. 

Interaction between Hras and 15d-PGJ2 in skeletal muscles has not been shown before, even though both Hras and 15d-PGJ2 are shown to be key regulators of muscle homeostasis. 

Activation of Hras by 15d-PGJ2 was reported first by Luis Oliva et al (Luis Oliva et al., 2003). However, this study does not comment on the functional implications of activation of Hras signaling. 

Recently, our lab contributed to a study where the functional implication of activation of Hras signaling due to covalent modification by 15d-PGJ2 was shown in the maintenance of senescence phenotype (Wiley et al., 2021). 

15d-PGJ2 was shown to inhibit the differentiation of myoblasts by Hunter et al (Hunter et al., 2001). This study hypothesized that the inhibition of myoblast differentiation is via 15d-PGJ2 mediated activation of the PPARγ signaling, the study also showed inhibition of myoblast differentiation independent of PPARγ activity, suggesting the presence of other mechanisms.

This is the first study to show a molecular mechanism where activation of Hras signaling in skeletal myoblasts due to covalent modification by 15d-PGJ2 at C184 of Hras inhibits the differentiation of skeletal myoblasts.

Additionally, there are major technical concerns related to the senescence models, limiting data interpretation regarding the relevance to senescent cells.

Major concerns:

(1) The C2C12 cell line is not an ideal model for senescence study due to its immortalized nature and lack of normal p16 expression. A more suitable myoblasts model is recommended, with a more comprehensive characterization of senescence features.

C2C12 is a good model for DNA damage-based senescence that is used in this manuscript. Several reports in the literature have shown the induction of senescence in C2C12 cells. Moiseeva et al 2023 show induction of senescence in C2C12 cells after etoposide-mediated DNA damage. Moustogiannis et al 2021 show the induction of replicative senescence in C2C12 cells. In this study, we show that C2C12 cells undergo DNA damage-mediated senescence after treatment with Doxo. We measured the induction of senescence in C2C12 cells upon DNA damage using several physiological (Nuclear Size, Cell Size, and SA β-gal) and molecular markers (mRNA levels of p21 and SASP factors (IL6 and TGFβ), protein levels of p21) of senescence (see Fig. 1 of the updated manuscript). The results and the figures in the manuscript have been updated accordingly.

(2) The source of increased PGD or its metabolites in the conditioned medium is unclear. Including other senescence models, such as replicative or oncogeneinduced senescence, would strengthen the study.

Fig. 1E shows time-dependent increase in the expression of PGD2 biosynthetic enzymes in senescent C2C12 cells. Fig. 1F shows an increase in the levels of 15dPGJ2 secreted by senescent C2C12 cells in the conditioned medium. This data shows that senescent C2C12 cells are the source of PGD and its metabolites in the conditioned medium.

Again, C2C12 is not suitable for replicative senescence due to its immortalized status.

We and others have shown that C2C12 cells undergo senescence, and this manuscript only used DNA damage induced senescence.

(3) In the in vivo part, it is unclear whether the increased expression of PTGS1, PTGS2, and PTGDS is due to senescence or other side effects of DOXO.

We concur that this is a limitation of this study and the subsequent work will demonstrate the origin of prostaglandin biosynthesis after treatment with Doxo in vivo.

(4) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of a conditioned medium.

Figure 2A tests the effect of prostaglandin PGD2 and its metabolites secreted by the senescent cells on the differentiation of myoblasts. Therefore, we inhibited the synthesis of PGD2 in senescent cells by treatment with AT-56, and then collected the conditioned medium. Conditioned medium collected from senescent C2C12 cells treated with vehicle (DMSO) served as a control for the experiment, whereas differentiation of C2C12 cells without any treatment serves as a positive control.

There is no explanation of how differentiation was quantified or how the fusion index was calculated.

The fusion index was calculated using a published myotube analyzer software (Noë et al., 2022). Appropriate information has been added to the materials and methods section of the manuscript.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Line 3: Expand SA in "SA β-gal".

The manuscript has been updated accordingly (See line 3).

Line 68: HRas is highly regulated by lipid modifications.

The manuscript has been updated accordingly (See line 67).

Figures

Figure S1A seemed incomplete (maybe some processing issue).

The Figure has been updated in the revised manuscript (See Fig. S1A).

Figure S1B-H are mislabeled.

The figure has been updated in the revised manuscript (See Fig. S1C, D, E, and F).

Figures S1E-H are not mentioned in the manuscript.

The manuscript has been updated accordingly (See line 120).

Many supplementary figures are not cited in the article.

The manuscript has been updated accordingly. (See lines 85, 120, 123, 166, 225, 356, 364, 412, and 413)

Reviewer #2 (Recommendations For The Authors):

(1) Clarify the injection method for Doxorubicin in B6J mice on line 83 (IP or IM).

Mice were injected intraperitoneally with Doxorubicin (as mentioned in the materials and methods, see lines 83 and 794)

(2) Address missing information in figures or figure legends.

There is missing piece in Sup Fig 1A.

The figure has been updated in the revised manuscript (See Fig. S1A).

Correct labels in Sup Fig 1C and 1D.

The figure has been updated in the revised manuscript (See Fig. S1C, D, E, and F).

How would the authors explain the dramatic differences in the morphology of C2C12 cells treated with DOXO between bright field and SA-beta-gal staining images in Sup Fig 1B and 1C.

The SA β-gal image after treatment with Doxo does show a flattened cell morphology. Another field of view from the same experiment has been added in the figure to show the difference in the cell morphology more prominently in the revised manuscript (See Fig. 1H).

Provide explanations for Sup Fig 1E-1G, including the meaning of the y-axis and the blue dots and red lines.

We have provided an explanation for the multiple reaction monitoring mass spectrometry used to measure the concentration of 15d-PGJ2 in the conditioned medium in the revised manuscript (see lines 119-130 and the legends of Fig. S1C, D, and E)

(3) Please review the calculation of qPCR data in Figure 1C for correctness, ensuring reference samples with an average expression level of 1.

The data in Fig. 1C was plotted using 2-ΔCT instead of 2-ΔΔCT to show the variability in the expression of mRNAs isolated from animals treated with Saline.

(4) Please explain the calculation of 15d-PGJ2/cell concentration in Figure 1F and provide raw data for review, considering the substantial changes and small error bars. The method or result section lacks an explanation of how this calculation was performed. Additionally, there is no mention of the cell number count.

All the raw values (concentration of 15d-PGJ2 measured using mass spec and cell numbers counted at the time of collection of conditioned medium) are provided in the supplementary table 1. The standard curve to calculate the concentration of 15dPGJ2 in the conditioned medium is shown in Fig. S1F. The cell number was counted after trypsinization using a hemocytometer on the day of collection of the conditioned medium.

(5) Please clarify how cell number normalization and doubling time calculation were done in Fig 2B. Consider replacing the figure with a growth curve showing confluence on the y-axis for easier interpretation.

Cells were counted every 24 hours and the normalization was done to the number of cells counted on day 0 of the treatment (to consider attaching efficiency and other cell culture parameters). Doubling time was calculated as the reciprocal of the slope of the graph of log2(normalized cell number) vs time.

eLife assessment

This manuscript outlines an interaction between senescence-related 15d-PGJ2 and the proliferation and differentiation of myoblasts, with potential implications for muscle health. This manuscript is useful in understanding the role of lipid metabolite 15d-PGJ2 in myoblast proliferation and differentiation. However, in its current form, the manuscript is incomplete as there are several concerns in the statistical analysis, lack of clarity on the mechanistic details, and concerns about the use of an immortalized C2C12 myoblasts cell line to draw major conclusions related to senescence-associated secreted phenotype.

Reviewer #1 (Public Review):

Summary:

The authors show that upon treatment with Doxorubicin (Doxo), there is an increase in senescence and inflammatory markers in the muscles. They also show these genes get upregulated in C2C12 myoblasts when treated with conditioned media or 15d-PGJ2. 15dPGJ2 induces cell death in the myoblasts, decreases proliferation (measured by cell numbers), and decreases differentiation and fusion. 15d-PGJ2 modified Cys184 of HRas, which is required for its activation as indicated by the FRET analysis with RAF RBD. They also showed that 15d-PGJ2 activates ERK signaling, but not Akt signaling, through the electrophilic center. 15d-PGJ2 inhibits Golgi localization of HRAS (only WT, not C181 or C184 mutant). They also showed that expressing the WT HRas followed by 15d-PGJ2 treatment led to a decrease in the levels of MHC mRNA and protein, and this defect is dependent on C184. This is a well-written manuscript with interesting insights into the mechanism of action of 15d-PGJ2. However, some clarification and experiments will help the paper advance the field significantly.

Strengths:

The data clearly shows that 15d-PGJ2 has a negative role in the myoblast cells and that it leads to modification of HRas protein. Moreover, the induction of biosynthetic enzymes in the PGD2 pathway also supports the induction of 15d-PGJ2 in Doxorubicin-treated cells. Both conditioned media experiments and the 15d-PGJ2 experiments show that 15d-PGJ2 could be the active component secreted by the senescent myoblasts.

Weaknesses:

The genes that are upregulated in the muscles upon injection with Doxo are also markers for inflammation. Since Doxo is also known to induce systemic inflammation, it is important to delineate these two effects (Inflammatory cells vs senescent cells). The expression of beta Gal and other markers of senescence in the tissue sections will help to delineate these.

In Figure 2, where the defect in the differentiation of myoblasts upon treatment with 15d-PGJ2 is shown, most of the cells die within 48 hours at higher concentrations, making it difficult to perform the experiments. This also shows that 15d-PGJ2 was toxic to these cells. Lower concentrations show a decrease in the differentiation based on the lower number of nuclei in fibers and low expression of MyoD, MyoG, and MHC. However, it is unclear if this is due to increased cell death or defective differentiation. It would be a lot more informative if the cell count, cell division, and cell death could be plotted for these concentrations of the drug during the experiment. Also, in the myoblast experiments, are the effects of treatment with Dox reversible?

In Figure 3, most of the experiments are done at a high concentration, which induces almost complete cell death within 48 hours. Even at such a high concentration of 15dPGJ2, the increase in ERK phosphorylation is minimal.

The experiment Figure 4C shows that C181 and C84 mutants of the HRas show higher levels in Golgi compared with WT. However, this could very well be due to the defect in palmitoylation rather than the modification with 15d-PGJ2. Though the authors allude to the possibility that intracellular redistribution of HRas by 15d-PGJ2 requires C181 palmitoylation, the direct influence of C184 modification on C181 palmitoylation is not shown. To have a meaningful conclusion, the authors need to compare the palmitoylation and modification with 15d-PGJ2.

To test if the inhibition of myoblast differentiation depends on HRas, they overexpressed the HRas and mutants in the C2C12 lines. However, this experiment does not take the endogenous HRAs into consideration, especially when interpreting the C184 mutant. An appropriate experiment to test this would be to knock down or knock out HRas (or make knock-in mutations of C184) and show that the effect of 15d-PGJ2 disappears. Moreover, in this specific experiment, it is difficult to interpret without a control with no HRas construct and another without the 15d-PGJ2 treatment.

Moreover, the overall study does not delineate the toxic effects of 15d-PGJ2 from its effect on the differentiation.

Reviewer #2 (Public Review):

Summary:

In this study, Swarang and colleagues identified the lipid metabolite 15d-PGJ2 as a potential component of senescent myoblasts. They proposed that 15d-PGJ2 inhibits myoblast proliferation and differentiation by binding and regulating HRas, suggesting its potential as a target for restoring muscle homeostasis post-chemotherapy.

Strengths:

The regulation of HRas by 15d-PGJ2 is well controlled.

Weaknesses:

(1) I still think the novelty is limited by previous published findings. The authors themselves noted that the accumulation of 15d-PGJ2 in senescent cells has been reported in various cell types, including human fibroblasts, HEPG2 hepatocellular carcinoma cells, and HUVEC endothelial cells (PMCID: PMC8501892). Although the current study observed similar activation of 15d-PGJ2 in myoblasts, it appears to be additive rather than fundamentally novel. The covalent adduct of 15d-PGJ2 with Cys-184 of H-Ras was reported over 20 years ago (PMID: 12684535), and the biochemical principles of this interaction are likely universal across different cell types. The regulation of myogenesis by both HRas and 15d-PGJ2 has also been previously extensively reported (PMID: 2654809, 1714463, 17412879, 20109525, 11477074). The main conceptual novelty may lie in the connection between these points in myoblasts. But as discussed in another comment, the use of C2C12 cells as a model for senescence study is questionable due to the lack of the key regulator p16. The findings in C2C12 cells may not accurately represent physiological-relevant myoblasts. It is recommended that these findings be validated in primary myoblasts to strengthen the study's conclusions.

(2) The C2C12 cell line is not an ideal model for senescence study.<br /> C2C12 cells are a well-established model for studying myogenesis. However, their suitability as a model for senescence studies is questionable. C2C12 cells are immortalized and do not undergo normal senescence like primary cells as C2C12 cells are known to have a deleted p16/p19 locus, a crucial regulator of senescence (PMID: 20682446). The use of C2C12 cells in published studies does not inherently validate them as a suitable senescence model. These studies may have limitations, and the appropriateness of the C2C12 model depends on the specific research goals.<br /> In the study by Moustogiannis et al. (PMID: 33918414), they claimed to have aged C2C12 cells through multiple population doublings. However, the SA-β-gal staining in their data, which is often used to confirm senescence, showed almost fully confluent "aged" C2C12 cells. This confluent state could artificially increase SA-β-gal positivity, suggesting that these cells may not truly represent senescence. Moreover, the "aged" C2C12 cells exhibited normal proliferation, which contradicts the definition of senescence. Similar findings were reported in another study of C2C12 cells subjected to 58 population doublings (PMID: 21826704), where even at this late stage, the cells were still dividing every 2 or 3 days, similar to younger cells at early passages. More importantly, I do know how the p16 was detected in that paper since the locus was already mutated. In terms of p21, there was no difference in the proliferative C2C12 cells at day 0.<br /> In the study by Moiseeva et al. in 2023 (PMID: 36544018), C2C12 cells were used for senescence modeling for siRNA transfection. However, the most significant findings were obtained using primary satellite cells or confirmed with complementary data.<br /> In conclusion, while molecular changes observed in studies using C2C12 cells may be valid, the use of primary myoblasts is highly recommended for senescence studies due to the limitations and questionable senescence characteristics of the C2C12 cell line.

(3) Regarding source of increased PGD in the conditioned medium, I want to emphasize that it's unclear whether the PGD or its metabolites increase in response to DNA damage or the senescence state. Thus, using a different senescent model to exclude the possibility of DNA damage-induced increase will be crucial.

(4) Similarly for the in vivo Doxorubicin (Doxo) injection, both reviewers have raised concerns about the potential side effects of Doxo, including inflammation, DNA damage, and ROS generation. These effects could potentially confound the results of the study. The physiological significance of this study will heavily rely on the in vivo data. However, the in vivo senescence component is confounded by the side effects of Doxo.

(5) Figure 2A lacks an important control from non-senescent cells during the measurement of C2C12 differentiation in the presence of conditioned medium. The author took it for granted that the conditioned medium from senescent cells would inhibit myogenesis, relying on previous publications (PMID: 37468473). However, that study was conducted in the context of myotonic dystrophy type 1. To support the inhibitory effect in the current experimental settings, direct evidence is required. It would be necessary to include another control with conditioned medium from normal, proliferative C2C12 cells.

(6) Statistical analyses problems.<br /> Only t-test was used throughout the study even when there are more than two groups. Please have a statistician to evaluate the replicates and statistical analyses used.<br /> For the 15d-PGJ2/cell concentration measurements in Figure 1F, there were only two replicates, which was provided in the supplementary table after required. Was that experiment repeated with more biological replicates?<br /> For figure 1C, Fig 1F, 1G, 1J, 2C, 2E, 3A, 3E, 3F, 4D, 4E, please include each data points in bar graphs as used in Fig 1D, or at least provide how many biological replicates were used for each experiment?<br /> There is no error bar in a lot of control groups (Fig 2C, 2E, 3EF, 4E, S4B).<br /> For qPCR data in Figure 1C, the author responded in that the data in was plotted using 2-ΔCT instead of 2-ΔΔCT to show the variability in the expression of mRNAs isolated from animals treated with Saline. This statement does not align with the method section. Please revise.

(7) For Figure 1, the title may not be appropriate as there is insufficient data to support the inhibition of myoblast differentiation.

Two term that are completly related, lietrature and art, but kno we have the posibility to connect thus art to the digital world.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Eaton et al. examine the regulation of transcription directionality using a powerful genomic approach (more about the methodology below). Their data challenge the notion that the polyadenylation signal-reading Cleavage and Polyadenylation (CPA) complex is responsible for controlling promoter directionality by terminating antisense transcription. Namely, depletion of the required CPA factor RBBP6 has little effect on antisense transcription measured by POINT. They find instead that initiation is intrinsically preferential in the sense direction and additionally maintained by the activities of an alternative processing complex called Integrator, together with the kinase CDK9. In the presence of CDK9 activity, depletion of Integrator endoribonuclease INTS11 leads to globally increased transcription in the antisense direction, and minor effects in the sense direction. However, CDK9 inhibition reveals that sense transcription is also sensitive to INS11 depletion. The authors suggest that CDK9 activity is stronger in the sense direction, preventing INTS11-mediated premature termination of sense transcrpts.

Strengths:

The combination of acute depletion of the studied factors using degron approaches (important to limit possible secondary effects), together with novel and very sensitive nascent transcriptomics methods POINT and sPOINT is very powerful. The applied spike-in normalization means the analysis is more rigorous than most. Using this methodology allowed the authors to revisit the interesting question of how promoter/transcription directionality is determined.

The data quality appears very good and the fact that both global analysis as well as numerous gene-specific examples are shown makes it convincing.

The manuscript is well written and hence a pleasure to read.

We appreciate this positive assessment.

Weaknesses:

I am slightly worried about the reproducibility of the data - it is unclear to me from the manuscript if and which experiments were performed in replicate (lack of table with genomic experiments and GEO access, mentioned in more detail in below recommendations to authors), and the methods could be more detailed.

All sequencing data was deposited with GEO. Multiple biological replicates were performed for each sequencing experiment.  Bigwig files are presented as a table in the GEO submissions. This data has now been made public.

A separate discussion section would be useful, particularly since the data provided challenge some concepts in the field. How do the authors interpret U1 data from the Dreyfuss lab in light of their results? How about the known PAS-density directionality bias (more PAS present in antisense direction than in sense) - could the differential PAS density be still relevant to transcription directionality?

As suggested, we have expanded our discussion to relate our findings to existing data. We think the results from the Dreyfuss lab are very important and highlight the role of U1 snRNA in enforcing transcriptional elongation.  It does this in part by shielding PAS sequences.  Recent work from our lab also shows that U1 snRNA opposes the Restrictor complex and PNUTS, which otherwise suppress transcription (Estell et al., Mol Cell 2023).  Most recently, the Adelman lab has demonstrated that U1 snRNA generally enhances transcription elongation (Mimoso and Adelman., Mol Cell 2023).  Our work does not challenge and is not inconsistent with these studies.

The role of U1 in opposing PAS-dependent termination inspired the idea that antisense transcriptional termination may utilise PASs.  This was because such regions are rich in AAUAAA and comparatively poor in U1 binding sites. However, our RBBP6 depletion and POINT-seq data suggest that PAS-dependent termination is uncommon in the antisense direction. As such, other mechanisms suppress antisense transcription and influence promoter directionality. In our paper, we propose a major role for the Integrator complex.

We do not completely rule out antisense PAS activity and discuss the prior work that identified polyadenylated antisense transcripts. Nevertheless, this was detected by oligo-dT primed RT-PCR/Northern blotting, which cannot determine the fraction of non-polyadenylated RNA that could result from PAS-independent termination (e.g. by Integrator).  To do that requires an analysis of total nascent transcription as achieved by our POINT-seq.  Based on these experiments, Integrator depletion has a greater impact on antisense transcription than RBBP6 depletion. 

I find that the provided evidence for promoter directionality to be for the most part due to preferential initiation in the sense direction should be stressed more. This is in my eyes the strongest effect and is somehow brushed under the rug.

We agree that this is an important finding and incorporated it into the title and abstract.  As the reviewer recommends, we now highlight it further in the new discussion.

References 12-17 report an effect of Integrator on 5' of protein-coding genes, while data in Figure 2 appears contradictory. Then, experiments in Figure 4 show a global effect of INST11 depletion on promoter-proximal sense transcription. In my opinion, data from the 2.5h time-point of depletion should be shown alongside 1.5h in Figure 2 so that it is clear that the authors found an effect similar to the above references. I find the current presentation somehow misleading.

We are grateful for this suggestion and present new analyses demonstrating that our experiment in Figure 2 concurs with previous findings (Supplemental Figures 2A and B). Our original heatmap (Figure 2E) shows a very strong and general antisense effect of INTS11 loss. On the same scale, the effects in the sense direction are not as apparent, which is also the case using metaplots.  New supplemental figure 2A now shows sense transcription from this experiment in isolation and on a lower scale, demonstrating that a subset of genes shows promoter-proximal increases in transcription following INTS11 depletion.  This is smaller and less general than the antisense effect but consistent with previous findings.  Indeed, our new analysis in supplemental figure 2B shows that affected protein-coding genes are lowly expressed, in line with Hu et al., Mol Cell 2023. This explains why a sense effect is not as apparent by metaplot, for which highly expressed genes contribute the most signal.

As a result of our analyses, we are confident that the apparently larger effect at the 2.5hr timepoint (Figure 4) that we initially reported is due to experimental variability and not greater effects of extended INTS11 depletion. Overlaying the 1.5h and 2.5h datasets (Supplemental Figure 4B) revealed a similar number of affected protein-coding genes with a strong (83%) overlap between the affected genes.  To support this, we performed qPCR on four affected protein-coding transcripts which revealed no significant difference in the level of INTS11 effect after 2.5h vs 1.5h (Supplemental Figure 4C).

We now present data for merged replicates in Figures 2 and 4 which reveal very similar average profiles for -INTS11 vs +INTS11 at both timepoints. Overall, we believe that we have resolved this discrepancy by showing that it amounts to experimental variability and because the most acutely affected protein-coding genes are lowly expressed. As detailed above, we show this in multiple ways (and validate by qPCR) We have revised the text accordingly and removed our original speculation that differences reflected the timeframe of INTS11 loss.

Conclusion/assessment:

This important work substantially advances our understanding of the mechanisms governing the directionality of human promoters. The evidence supporting the claims of the authors is compelling, with among others the use of advanced nascent transcriptomics including spike-in normalization controls and acute protein depletion using degron approaches.

In my opinion, the authors' conclusions are in general well supported.

Not only the manuscript but also the data generated will be useful to the wide community of researchers studying transcriptional regulation. Also, the POINT-derived novel sPOINT method described here is very valuable and can positively impact work in the field.

We are grateful for the reviewers' positive assessment of our study.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

We appreciate this positive assessment.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

We agree that other (so far, unknown) factors promote sense transcription over antisense, which was demonstrated by our short POINT.  We have provided an expanded discussion on this in the revision. In our opinion, demonstrating that sense transcription is driven by preferential initiation in that direction is a key finding and we agree that the identification of the underlying mechanism constitutes an interesting avenue for future study.

Reviewer #3 (Public Review):

Summary:

Using a protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in the sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:

The strength of the paper is the acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

We appreciate this positive assessment.

Weaknesses:

While the manuscript is well written, the details on the panel are not sufficient. The methods could be elaborated to aid understanding. Additional discussion on how the authors' findings contradict the existing model of anti-sense transcription termination should be added.

We have added more detail to the figure panels, which we hope will help readers to navigate the paper more easily. Specifically, the assay employed for each experiment is indicated in each figure panel. As requested, we provide a new and separate discussion section in the revision.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Congratulations on this important piece of work!

Some specific suggestions.

MAJOR

-The data are not available (Accession "GSE243266" is currently private and is scheduled to be released on Sep 01, 2026.) This should be corrected and as a minimum, the raw sequencing files as well as the spike-in scaled bigwig files should be provided in GEO.

We have made the data public. Raw and bigwig files are provided as part of the GEO upload.

MINOR

- It would be useful for readers if you could include catalog numbers of the reagents used in the study.

We have included this information in our revision.

- A table in experimental procedures summarizing the genomic experiments performed in this study as well as published ones reanalyzed here would be helpful.

This is now provided as part of the resources table.

- It would be easier for reviewers to evaluate the manuscript if the figure legends were included together with the figures on one page. This is now allowed by most journals.

We have used this formatting in the revision.

- Providing some captions for the results sections would be helpful.

We have included subheadings as suggested.

Reviewer #2 (Recommendations For The Authors):

Generally, I would suggest writing the experiment-type above panels where it is not immediately obvious what they are so a reader can appreciate the figures without referencing the legend. E.g. write POINT-seq on Figure 1B just to make it obvious to someone looking at the figures what methodology they are looking at. Likewise, you could write RNAPII ChIP-seq for Supplementary Figures 3D and 3E.

We have carried out this recommendation.

Can a y-axis be indicated on POINT-seq genome browser tracks? This could make them easier to interpret.

Y-axis scales are provided as RPKM as stated in the figure legends.

The authors could address/speculate in the text why there is less POINT-seq signal for the antisense transcript in the treatment condition in Figure 1B? Or could consider including a different example locus where this is not the case for clarity.

Acute depletion of poly(A) factors (like RBBP6) results in a strong read-through beyond the poly(A) signal of protein-coding genes as Figure 1 shows.  However, it also causes a reduction in transcription levels, which can be seen in the figure and is correctly noted by the reviewer in this comment.  We see this with other poly(A) factor depletions (e.g. CPSF73 and CPSF30 – Eaton et al., 2020 and Estell et al., 2021) and other labs have observed this too (e.g for CPSF73-dTAG depletion (Cugusi et al., Mol Cell 2022)).  Plausible reasons include a limited pool of free RNAPII due to impaired transcriptional termination or limited nucleotide availability due to their incorporation within long read-through transcripts. For these reasons, we have retained the example in Figure 1B as a typical representation of the effect. Moreover, the heatmap in Figure 1D fairly represents the spectrum of effects following RBBP6 loss – highlighting the strong read-through beyond poly(A) signals and the marginal antisense effects.

"The established effect of INTS11 at snRNAs was detected in our POINT-seq data and demonstrates the efficacy of this approach (Figure 2B)." The authors could explain this point more clearly in the text and describe the data - e.g. As expected, depletion of INTS11 leads to increased POINT-seq signal at the 3' end of snRNAs, consistent with defects in transcriptional termination. This is highlighted by the RNU5A-1 and RNU5B-1 loci (Figure 2B).

We agree and have added more context to clarify this.

I would suggest adjusting the scale of the heatmap in Figure 2E - I think it would be easier to interpret if the value of 0 was white - with >0 a gradient of orange and <0 a gradient of blue (as is done in Figure 1C). I think making this change would make the point as written in the text clearer i.e. "heatmap analysis demonstrates the dominant impact of INTS11 on antisense versus sense transcription at most promoters (Figure 2E)." I'm assuming most of the sense transcription would be white (more clearly unchanging) when the scale is adjusted.

We agree and have done this. The reviewer is correct that most sense transcription is unchanged by INTS11 loss.  However, as we alluded to in the original submission, a subset of transcripts shows a promoter-proximal increase after INTS11 depletion. We have expanded the analyses of this effect (see responses to other comments) but stress that it is neither as general nor as large as the antisense effect.

The authors make the point that there is mildly increased transcription over the 5' end of some genes upon INST11 depletion and show a track (Supplementary Fig 2A). It is not immediately obvious from the presentation of the meta-analysis in Figure 2D how generalisable this statement is. Perhaps the size of the panel or thickness of the lines in Figure 2D could be adjusted so that the peak of the control (in blue) could be seen. Perhaps an arrow indicating the peak could be added? I'm assuming the peak at the TSS is slightly lower in the control compared to INST11 depletion based on the authors' statement.

We have provided multiple new analyses of this data to highlight where there are promoter-proximal effects of INTS11 loss in the sense direction.  Please see our response to the public review of reviewer 1 and new supplemental figures 2A, 2B, 4A and 4B which highlight the sense transcription increased in the absence of INTS11.

The authors label Figure 4 "Promoters lose their directionality when CDK9 is inhibited" - but in INST11 depleted cells treated with CDK9i they find that there still is a bias towards sense transcription. Suggested edit "Some promoter directionality is lost when CDK9 is inhibited" or similar.

We agree and have made this change.

The authors conclude that INTS11-mediated effects are the result of perturbation of the catalytic activities of Integrator, the authors should perform rescue experiments with the catalytically dead E203Q-INTS11 mutant.

This is a very good suggestion and something we had intended to pursue.  However, as we will describe below (and shown in Supplemental Figure 4G), there were confounding issues with this experiment.

The E203Q mutant of INTS11 is widely used in the literature to test for catalytic functions of INTS11.  However, we have found that this mutation impairs the ability of INTS11 to bind other Integrator modules in cells. Based on co-immunoprecipitation of flag-tagged WT and E203Q derivatives, INTS1 (backbone module), 10 (tail module), and 8 (phosphatase module) all show reduced binding to E203Q vs. WT. Because E203Q INTS11 is defective in forming Integrator complexes, rescue experiments might not fully distinguish the effects of INTS11 activity from those caused by defects in complex assembly. While this may at first seem unexpected, in the analogous 3’ end processing complex, catalytic mutants of CPSF73 (which is highly related to INTS11) negatively affect its interaction with other complex members (Kolev and Steitz, EMBO Reports 2005).

We hypothesise that INTS11 activity is most likely involved in attenuating promoter-proximal transcription, but we cannot formally rule out other explanations and discuss this in our revision. Regardless of how INTS11 attenuates transcription, our main conclusion is on its requirement to terminate antisense transcription whether this involves its cleavage activity or not.

The authors suggest that CDK9 modulates INTS11 activity/assembly and suggest this may be related to SPT5. Is there an effect of CDK9 inhibition on the snRNA's highlighted in Figure 2B?

We believe that snRNAs are different from protein-coding genes concerning CDK9 function. Shona Murphy’s lab previously showed that, unlike protein-coding genes, snRNA transcription is insensitive to CDK9 inhibition, and that snRNA processing is impaired by CDK9 inhibition (Medlin et al., EMBO 2003 and EMBO 2005).  We reproduce these findings by metaanalysis of 15 highly expressed and well-separated snRNAs and by qRT-PCR of unprocessed RNU1-1, RNU5A-1 and RNU7-1 snRNA following CDK9 inhibition. We observe snRNA read-through by POINT-seq following INTS11 loss whether CDK9 is inhibited or not (left panel, below). Note the higher TES proximal signal in CDK9i conditions, which likely reflects the accumulation of unprocessed snRNA as validated by qPCR for three example snRNAs (right panel, below).

Author response image 1.

For Figure 4, would similar results be observed using inhibitors targeting other transcriptional CDKs such as CDK7,12/13?

In response to this suggestion, we analysed four selected protein-coding transcripts (the same 4 that we used to validate the CDK9i results) by qRT-PCR in a background of CDK7 inhibition using the THZ2 compound (new Supplemental Figure 4E).  THZ2 suppresses transcription from these genes as expected.  Interestingly, expression is restored by co-depleting Integrator, recapitulating our findings with CDK9 inhibition.  As CDK7 is the CDK-activating kinase for CDK9, its inhibition will also inhibit CDK9 so THZ2 may simply hit this pathway upstream of where CDK9 inhibitors.  Second, CDK7 may independently shield transcription from INTS11.  We allude to both interesting possibilities.

What happens to the phosphorylation state of anti-sense engaged RNAPII when INTS11 is acutely depleted and/or CDK9 is inhibited? This could be measured by including Ser5 and Ser2 antibodies in the sPOINT-seq assay and complemented with Western Blot analysis.

We have performed the western blot for Ser5 and Ser2 phosphorylation as suggested.  Both signals are mildly enhanced by INTS11 loss, which is consistent with generally increased transcription.  Ser2p is strongly reduced by CDK9 inhibition, which is consistent with the loss of nascent transcription in this condition.  Interestingly, both modifications are partly recovered when INTS11 is depleted in conjunction with CDK9 inhibition. This is consistent with the effects that we see on POINT-seq and shows that the recovered transcription is associated with some phosphorylation of RNAPII CTD.  This presumably reflects the action(s) of kinases that can act redundantly with CDK9.

We have not performed POINT-seq with Ser5p and Ser2p antibodies under these various conditions.  Our rationale is that our existing data uses an antibody that captures all RNAPII (regardless of its phosphorylation status), which we feel most comprehensively assays transcription in either direction. Moreover, the lab of Fei Chen (Hu et al., Mol Cell 2023) recently published Ser5p and Ser2p ChIP-seq following INTS11 loss. By ChIP-seq, they observe a bigger increase in antisense RNAPII occupancy vs. sense providing independent and orthogonal support for our POINT-seq data.  Interestingly, this antisense increase is not paralleled by proportional increases in Ser5p or Ser2p signals.  This suggests that the unattenuated antisense transcription resulting from INTS11 loss does not have high Ser5p or Ser2p.  Since CDK7 and 9 are major Ser5 and 2 kinases, this supports our model that their activity is less prevalent for antisense transcription.  We now discuss these data in our revision.   

The HIV reporter RNA experiments should be performed with the CDK9 inhibitor added to the experimental conditions. Presumably CDK9 inhibition would result in no upregulation of the reporter upon addition of TAT and/or dTAG. Perhaps the amount of TAT should be reduced to still have a dynamic window in which changes can be detected. It is possible that reporter activation is simply at a maximum. Can anti-sense transcription be measured from the reporter?

We have performed the requested CDK9 inhibitor experiment to confirm that TAT-activated transcription from the HIV promoter is CDK9-dependent (new supplemental figure 4F).  Consistent with previous literature on HIV transcription, CDK9 inhibition attenuates TAT-activated transcription.  Importantly, and in line with our other experiments, depletion of INTS11 results in significant restoration of transcription from the HIV promoter when CDK9 is inhibited. Thus, TAT-activated transcription is CDK9-dependent and, as for endogenous genes, CDK9 prevents attenuation by INTS11.

While TAT-activated transcription is high, we do not think that the plasmid is saturated. When considering this question, we revisited previous experiments using this system to study RNA processing (Dye et al., Mol Cell 1999, Cell 2001, Mol Cell 2006). In these cases, mutations in splice sites or polyadenylation sites have a strong effect on RNA processing and transcription around HIV reporter plasmids. Effects on transcription and RNA processing are; therefore, apparent in the appropriate context. In contrast, we find that the complete elimination of INTS11 has no impact on RNA output from the HIV reporter. Our original experiment assessing the impact of INTS11 loss in +TAT conditions used total RNA.  One possibility is that this allows non-nascent RNA to accumulate which might confound our interpretation of INTS11 effects on ongoing transcription.  However, the new experiment described in the paragraph above was performed on chromatin-associated (nascent) RNA to rule this out.  This again shows no impact of INTS11 loss on HIV promoter-derived transcription in the presence of TAT.

To our knowledge, antisense transcription is not routinely assayed from plasmids. They generally employ very strong promoters (e.g. CMV, HIV) to drive sense transcription.  Crucially, their circular nature means that RNAPII going around the plasmid could interfere with antisense transcription coming the other way which does not happen in a linear genomic context. This is why we restricted our use of plasmids to looking at the effects of stimulated CDK9 recruitment (via TAT) on transcription rather than promoter directionality.   

The authors should clearly state how many replicates were performed for the genomics experiments. Ideally, a signal should be quantified and compared statistically rather than relying on average profiles only.

We have stated the replicate numbers for sequencing experiments in the relevant figure legends. All sequencing experiments were performed in at least two biological replicates, but often three. In addition, we validated their key conclusions by qPCR or with orthogonal sequencing approaches.

Reviewer #3 (Recommendations For The Authors):

The authors provide strong evidence in support of their claims.

ChIP-seq of pol2S5 and S2 upon INST11 and CDK9 inhibition will strengthen the observation that transcription in the sense direction is more efficient.

We view the analysis of total RNAPII as the most unbiased way of establishing how much RNAPII is going one way or the other. Importantly, ChIP-seq was very recently performed for Ser2p and Ser5p RNAPII derivatives in the lab of Fei Chen (Hu et al., Mol Cell 2023). Their data shows that loss of INTS11 increases the occupancy of total RNAPII in the antisense direction more than in the sense direction, which is consistent with our finding. Interestingly, the increased antisense RNAPII was not paralleled with an increase in Ser2p or Ser5p. This suggests that, following INTS11 loss, the unattenuated antisense transcription is not associated with full/normal Ser2p or Ser5p. These modifications are normally established by CDK7 and 9; therefore, this published ChIP-seq suggests that they are not fully active on antisense transcription when INTS11 is lost. This supports our overall model that CDK9 (and potentially CDK7 as suggested for a small number of genes in new Supplemental Figure 4E) is more active in the sense direction to prevent INTS11-dependent attenuation. We now discuss these data in our revision.

In Supplementary Figure 2, the eRNA expression increases upon INST11 degradation, I wonder if the effects of this will be appreciated on cognate promoters? Can the authors test some enhancer:promoter pairs?

We noticed that some genes (e.g. MYC) that are regulated by enhancers show reduced transcription in the absence of INTS11. Whilst this could suggest a correlation, the transcription of other genes (e.g. ACTB and GAPDH) is also reduced by INTS11 loss although they are not regulated by enhancers.  A detailed and extensive analysis would be required to establish any link between INTS11-regulated enhancer transcription and the transcription of genes from their cognate promoters.  We agree that this would be interesting, but it seems beyond the scope of our short report on promoter directionality.

Line 111, meta plot was done of 1316 genes. Details on this number should be provided. Overall, the details of methods and analysis need improvement. The layout of panels and labelling on graphs can be improved.

We have now explained the 1316 gene set.  In essence, these are the genes separated from an expressed neighbour by at least 10kb.  This distance was selected because depletion of RBBP6 induces extensive read-through transcription beyond the polyadenylation site of protein-coding genes.  To avoid including genes affected by transcriptional read-through from nearby transcription units we selected those with a 10kb gap between them. This was the only selection criteria so is unlikely to induce any unintended biases. Finally, we have added more information to the figure panels and their legends, which we hope will make our manuscript more accessible.

eLife assessment

The important study uses a new experimental method to provide compelling evidence on how sense- and anti-sense transcription is differentially regulated. The method described here can generally be used to study the alterations in transcription. This paper will be of interest to scientists working in the gene regulation community.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Eaton et al. examine the regulation of transcription directionality using a powerful genomic approach (more about the methodology below).<br /> Their data challenge the notion that the polyadenylation signal-reading Cleavage and Polyadenylation (CPA) complex is responsible for controlling promoter directionality by terminating antisense transcription. Namely, depletion of the required CPA factor RBBP6 has little effect on antisense transcription measured by POINT. They find instead that initiation is intrinsically preferential in the sense direction and additionally maintained by the activities of an alternative processing complex called Integrator, together with the kinase CDK9. In the presence of CDK9 activity, depletion of Integrator endoribonuclease INTS11 leads to globally increased transcription in the antisense direction, and minor effects in the sense direction. However, CDK9 inhibition reveals that sense transcription is also sensitive to INS11 depletion. The authors suggest that CDK9 activity is stronger in the sense direction, preventing INTS11-mediated premature termination of sense transcripts.

Strengths:

The combination of acute depletion of the studied factors using degron approaches (important to limit possible secondary effects), together with novel and very sensitive nascent transcriptomics methods POINT and sPOINT is very powerful. The applied spike-in normalization means the analysis is more rigorous than most. Using this methodology allowed the authors to revisit the interesting question of how promoter/transcription directionality is determined.

The data quality appears very good and the fact that both global analysis as well as numerous gene-specific examples are shown makes it convincing.

The manuscript is well written and hence a pleasure to read.

Weaknesses:

The bias in transcriptional initiation directionality remains to be elucidated.

Conclusion/assessment:

This important work substantially advances our understanding of the mechanisms governing the directionality of human promoters. The evidence supporting the claims of the authors is compelling, with a.o. the use of advanced nascent transcriptomics including spike-in normalization controls and acute protein depletion using degron approaches.

In my opinion the authors' conclusions are well supported.

Not only the manuscript but also the data generated will be useful to the wide community of researchers studying transcriptional regulation. Also, the POINT-derived novel sPOINT method described here is very valuable and can positively impact work in the field.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper which uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figure 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

Reviewer #3 (Public Review):

Summary:

Using protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:

The strength of paper is acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

Weaknesses:

While manuscript is well written, the details on panel is not sufficient. The methods can be more elaborate for better understanding. Additional discussion on how authors findings contradict the existing model of anti-sense transcription termination should be added.

in the revised manuscript, authors have added details on panels and elaborated method and other sections for better understanding.

トル

eLife assessment

The study presents valuable findings on the molecular mechanisms of glucose-stimulated insulin secretion from pancreatic islets, focusing on the main regulatory elements of the signaling pathway in physiological conditions. While the evidence supporting the conclusions is solid, the study can be strengthened by the use of a beta cell line or knockout mice. The work will be of interest to cell biologists and biochemists working on diabetes.

Reviewer #1 (Public Review):

Summary:

This study investigated the mechanism by which PGE2 inhibits the release of insulin from pancreatic beta cells in response to glucose. The researchers used a combination of cell line experiments and studies in mice with genetic ablation of the Kv2.2 channel. Their findings suggest a novel pathway where PGE2 acts through EP2/EP4 receptors to activate PKA, which directly phosphorylates a specific site (S448) on the Kv2.2 channel, inhibiting its activity and reducing GSIS.

Strengths:

- The study elegantly demonstrates a potential pathway connecting PGE2, EP2/EP4 receptors, PKA, and Kv2.2 channel activity, using embryonic cell line.<br /> - Additional experiments in INS1 and primary mouse beta cells with altered Kv2.2 function partially support the inhibitory role of PGE2 on GSIS through Kv2.2 inhibition.

Weaknesses:

- A critical limitation is the use of HEK293T cells, which are not pancreatic beta cells. Functional aspects can differ significantly between these cell types.<br /> - The study needs to address the apparent contradiction of PKA activating insulin secretion in beta cells, while also inhibiting GSIS through the proposed mechanism.<br /> - A more thorough explanation is needed for the discrepancies observed between the effects of PGE2 versus Kv2.2 knockdown/mutation on the electrical activity of beta cells and GSIS.

Reviewer #2 (Public Review):

The authors identified new target elements for prostaglandin E2 (PGE2) through which insulin release can be regulated in pancreatic beta cells under physiological conditions. In vitro extracellular exposure to PGE2 could directly and dose-dependently inhibit the potassium channel Kv2.2. In vitro pharmacology revealed that this inhibition occurs through the EP2/4 receptors, which activate protein kinase A (PKA). By screening specific sites of the Kv2.2 channel, the target phosphorylation site (S448) for PKA regulation was found. The physiological relevance of the described signaling cascade was investigated and confirmed in vivo, using a Kv2.2 knockdown mouse model.

The strength of this manuscript is the novelty of the (EP2/4-PKA-Kv2.2 channel) molecular pathway described and the comprehensive methodological toolkit the authors have relied upon.

The introduction is detailed and contains all the information necessary to place the claims in context. Although the dataset is comprehensive and a logical lead is consistently built, there is one important point to consider: to clarify that the described signaling pathway is characteristic of normal physiological conditions and thus differs from pathological changes. It would be useful to carry out basic experiments in a diabetes model (regardless of whether this is in mice or rats).

Author response:

We thank the reviewers for their positive evaluation and constructive feedback on our study.

We acknowledge the concern regarding the use of HEK293T cells. In the revised manuscript, we will provide a more detailed explanation of the role of the PKA pathway in the regulation of GSIS by PGE2. To validate this regulation through Kv2.2, we will overexpress the Kv2.2 mutant channel in beta cells and assess its impact. Additionally, we will verify the specificity of the antibodies for EP1-EP4 receptors by knockdown. To confirm the receptors involved in PGE2 function, we will use additional EP receptor blockers or perform receptor knockdown experiments.

We will clarify that the described signaling pathway operates under normal physiological conditions and differs from pathological changes.

We once again thank the reviewers for their positive evaluation and constructive suggestions.

eLife assessment

This work describes a novel affinity interactomics approach that allows investigators to identify networks of protein-protein interactions in cells. The important findings presented here describe the application of this technique to the SH3 domain of the membrane remodeling Bridging Integrator 1 (BIN1), the truncation of which leads to centronuclear myopathy. The authors present solid evidence that BIN1 SH3 engages with an unexpectedly high number of cellular proteins, many of which are linked to skeletal muscle disease, and evidence is presented to suggest that BIN1 may play a role in mitosis creating the potential for new avenues in drug development efforts. Some of the findings, however, remain rather preliminary, lack sufficient replicates and may require additional experiments to definitively support the conclusions.

Reviewer #1 (Public Review):

Summary:

In this paper, Zambo and coworkers use a powerful technique, called native holdup, to measure the affinity of the SH3 domain of BIN1 for cellular partners. Using this assay, they combine data using cellular proteins and proline-containing fragments in these proteins to identify 97 distinct direct binding partners of BIN1. They also compare the binding interactome of the BIN1 SH3 domain to the interactome of several other SH3 domains, showing varying levels of promiscuity among SH3 domains. The authors then use pathway analysis of BIN1 binding partners to show that BIN1 may be involved in mitosis. Finally, the authors examine the impact of clinically relevant mutations of the BIN1 SH3 domain on the cellular interactome. The authors were able to compare the interactome of several different SH3 domains and provide novel insight into the cellular function of BIN1. Generally, the data supports the conclusions, although the reliance on one technique and the low number of replicates in each experiment is a weakness of the study.

Strengths:

The major strength of this paper is the use of holdup and native holdup assays to measure the affinity of SH3 domains to cellular partners. The use of both assays using cell-derived proteins and peptides derived from identified binding partners allows the authors to better identify direct binding partners. This assay has some complexity but does hold the possibility of being used to measure the affinity of the cellular interactome of other proteins and protein domains. Beyond the utility of the technique, this study also provides significant insight into the cellular function of BIN1. The authors have strong evidence that BIN1 might have an undiscovered function in cellular mitosis, which potentially highlights BIN1 as a drug target. Finally, the study provides outstanding data on the cellular binding properties and partners of seven distinct SH3 domains, showing surprising differences in the promiscuity of these proteins.

Weaknesses:

There are several weaknesses of the study. First, the authors rely completely on a single technique to measure the affinity of the cellular interactome. The native holdup is a relatively new technique that is powerful yet relatively unproven. However, it appears to have the capacity to measure the relative affinity of proteins and the authors describe the usefulness of the technique. Second, and most important, the authors use a relatively small number of replicates for the holdup assays. The holdup technique will have biological variation in the cellular lysate or purified protein that could impact the results, so more replicates would enhance the reliability of the results.

Reviewer #2 (Public Review):

Summary:

The authors report here interesting data on the interactions mediated by the SH3 domain of BIN1 that expand our knowledge on the role of the SH3 domain of BIN1 in terms of mediating specific interactions with a potentially high number of proteins and how variants in this region alter or prevent these protein-protein interactions. These data provide useful information that will certainly help to further dissect the networks of proteins that are altered in some human myopathies as well as the mechanisms that govern the correct physiological activity of muscle cells.

Strengths:

The work is mostly based on improved biochemical techniques to measure protein-protein interaction and provide solid evidence that the SH3 domain of BIN1 can establish an unexpectedly high number of interactions with at least a hundred cellular proteins, among which the authors underline the presence of other proteins known to be causative of skeletal muscle diseases and not known to interact with BIN1. This represents an unexpected and interesting finding relevant to better define the network of interactions established among different proteins that, if altered, can lead to muscle disease. An interesting contribution is also the detailed identification of the specific sites, namely the Proline-Rich Motifs (PRMs) that in the interacting proteins mediate binding to the BIN1 SH3 domain.

Weaknesses:

Less convincing, or too preliminary in my opinion, are the data supporting BIN1 co-localization with PRC1. Indeed, the affinity of PRC1 is significantly lower than that of DNM2, an established BIN1 interacting protein. Thus, this does not provide compelling evidence to support PRC1 as a significant interactor of BIN1. Similarly, the localization data appears somewhat preliminary to substantiate a role of BIN1 in mitotic processes. These findings may necessitate additional experimental work to be more convincing.

Author response:

The following is the authors’ response to the previous reviews.

Reviewer #1

We modified the text regarding PRC1 according to the reviewer’s recommendation.

Reviewer #2

Following the reveiwer’s advise, we introduced the holdup assay, as well as the native holdup assay in more details.

This new part now also discusses the question of replicates in more details. We do not agree with the eLife assessment on this matter, but we think that this assessment was made because analyzing holdup data requires a different approach compared to more conventional interactomic approaches and these differences were not introduced in sufficient depth. We hope that the inclusion of more background reasoning, as well as by providing a more detailed comparison of the measured independent BIN1 interactomes, now included on Figure S4, will eliminate all confusion in the reader.

We thank the reviewer for guiding us to a previous work that was done on Grb2. Indeed, the finding of this earlier work aligns perfectly with our finding suggesting general similarities in SH3 domain mediated interactions.

Underlying issues (underlying crisis) need to transform into a thorn in the side of the government in order to create an existential crisis for the government in order to be taken seriously

Governments need to be forced to do something that's radical - they prefer keeping things the same. Why? Because doing more is consumption of more energy, it's politically dangerous, doing anything out of ordinary raises accountability.

What's the greatest crisis for a government? Prospect of not getting elected. How does that crisis correlate with other crisis?

sounds like "opportunity" is correct

Résumé de la vidéo [00:00:00][^1^][1] - [00:20:41][^2^][2]:

Cette vidéo présente une discussion approfondie sur l'éducation, en se concentrant sur les idées fausses courantes concernant le système scolaire et l'impact des stéréotypes de genre. Louise Tourret, journaliste et productrice de l'émission "Être et savoir" sur France Culture, partage ses observations et expériences pour démystifier ces idées reçues et souligner l'importance d'une approche réfléchie de l'éducation.

Points forts: + [00:00:13][^3^][3] Introduction de Louise Tourret * Présentation de son ouvrage sur les idées fausses de l'école * Expérience en tant que journaliste et productrice * Brève expérience en tant qu'enseignante contractuelle + [00:02:08][^4^][4] Impact des stéréotypes de genre dans l'éducation * Influence des stéréotypes sur les élèves et les enseignants * Réflexion sur la mixité et l'égalité de genre à l'école * Importance de repenser les pratiques éducatives + [00:11:02][^5^][5] Déconstruction des mythes éducatifs * Discussion sur l'école et le sexisme * Examen des idées reçues sur les élèves à haut potentiel intellectuel (HPI) * Importance de valoriser l'expérience des enseignants + [00:16:17][^6^][6] Rôle de la formation continue des enseignants * Interrogation sur le développement professionnel des enseignants * Impact des idées reçues sur les pratiques pédagogiques * Nécessité d'une réflexion collective sur l'éthique de l'enseignement

Résumé de la vidéo [00:20:43][^1^][1] - [00:41:03][^2^][2] : La vidéo aborde la question de l'éducation en France, en examinant les défis et les perspectives d'avenir du système éducatif. Elle soulève des préoccupations sur la manière dont l'école peut créer une distance sociale plutôt que de rapprocher les gens, et comment cela peut influencer les attitudes politiques et sociales. La vidéo explore également les différences entre les pédagogies françaises et nordiques, l'importance de la culture générale dans l'éducation française, et la nécessité d'adapter l'école aux besoins individuels des élèves tout en préservant l'héritage culturel.

Points saillants: + [00:20:43][^3^][3] Les défis de l'éducation en France * Discussion sur l'impact de l'éducation sur les choix politiques * Analyse du ressentiment développé à l'école envers les diplômés * Réflexion sur l'humiliation et le sentiment de perte dans le système scolaire + [00:23:00][^4^][4] Comparaison avec les pédagogies nordiques * Évaluation des idées fausses sur l'adoption des pédagogies nordiques * Importance des maîtres spécialisés et de la formation continue en Finlande * Différences culturelles dans la perception de l'enfant et de l'élève + [00:26:01][^5^][5] La culture générale et l'éducation * La valeur de la culture générale dans l'éducation française * Transmission du patrimoine culturel et révérence pour celui-ci * Tension entre l'héritage culturel et un projet d'école pour tous + [00:31:01][^6^][6] L'évaluation et la sélection dans l'éducation * Débat sur l'efficacité de l'évaluation de 0 à 20 * Impact de Parcoursup et de la sélection basée sur les notes * Importance de diversifier les notions de réussite scolaire + [00:34:33][^7^][7] La forme scolaire et les méthodes d'enseignement * Discussion sur l'enseignement mutuel et les alternatives à la forme scolaire traditionnelle * Réflexion sur la liberté pédagogique et l'auto-évaluation des enseignants * L'importance de l'adaptation des espaces éducatifs et de la coopération

Résumé de la vidéo [00:41:06][^1^][1] - [00:55:48][^2^][2]:

Cette partie de la vidéo aborde les stéréotypes de genre dans l'éducation, la réussite scolaire des filles par rapport aux garçons, et les défis rencontrés par les filles dans les domaines des mathématiques et des sciences. L'intervenant discute également de l'impact des neurosciences sur l'éducation, les mythes qui les entourent, et la manière dont les enseignants peuvent intégrer ces connaissances dans leur pratique pédagogique.

Points forts: + [00:41:06][^3^][3] Stéréotypes de genre et éducation * Discussion sur la réussite des filles à l'école * Les garçons s'orientent vers des filières plus prestigieuses * Questionnement sur la représentation des femmes dans les sciences + [00:44:01][^4^][4] Impact des neurosciences sur l'éducation * Les neurosciences apportent des connaissances mais pas de solutions toutes faites * Prudence face aux neuromythes et à l'application pratique * Importance de la formation des enseignants et de l'adaptation pédagogique + [00:49:01][^5^][5] La coopération dans l'éducation * La coopération est essentielle mais souvent limitée par le système * Les contradictions dans les pratiques éducatives * L'importance de la réflexion sur la pratique professionnelle

eLife assessment

This useful manuscript extends prior work to identify OVO as a major transcriptional activator of the female germline gene expression program. Using a combination of solid genomic strategies, the authors demonstrate that OVO binds to the promoters of hundreds of genes in the female germline and promotes their expression.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Benner et al. identify OVO as a transcriptional factor instrumental in promoting expression of hundreds of genes essential for female germline identity and early embryo development. Prior data had identified both ovo and otu as genes activated by OVO binding to the promoters. By combining ChIP-seq, RNA-seq and analysis of prior datasets, the authors extend these data to hundreds of genes and therefore propose that OVO is a master transcriptional regulator of oocyte development. They further speculate that OVO may function to promote chromatin accessibility to facilitate germline gene expression. Overall, the data compellingly demonstrate a much broader role for OVO in activation of genes in the female germline than previously recognized. By contrast, the relationship between OVO, chromatin accessibility and the timing of gene expression is only correlative, and more work will be needed to determine the mechanisms by which OVO promotes transcription.

Strengths

Here Benner at al. convincingly show that OVO is a transcriptional activator that promotes expression of hundreds of genes in the female germline. The ChIP-seq and RNA-seq data included in the manuscript are robust and the analysis is compelling.

Importantly, the set of genes identified are essential for maternal processes, including egg production and patterning of the early embryo. Together, these data identify OVO as a major transcriptional activator of the numerous genes expressed in the female germline, deposited into the oocyte and required for early gene expression. This is an important finding as this is an essential process for development and prior to this study the major drivers of this gene expression program were unknown.

Weaknesses

The novelty of the manuscript is somewhat limited as the authors show that, like two prior, well-studied OVO target genes, OVO binds to promoters of germline genes and activates transcription. The fact that OVO performs this function more broadly is not particularly surprising.

A major challenge to understanding the impact of this manuscript is the fact that the experimental system for the RNA-seq, the tagged constructs, and the expression analysis that provides the rationale for the proposed pioneering function of OVO are all included in a separate manuscript.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Benner et al. identify OVO as a transcriptional factor instrumental in promoting the expression of hundreds of genes essential for female germline identity and early embryo development. Prior data had identified both ovo and otu as genes activated by OVO binding to the promoters. By combining ChIP-seq, RNA-seq, and analysis of prior datasets, the authors extend these data to hundreds of genes and therefore propose that OVO is a master transcriptional regulator of oocyte development. They further speculate that OVO may function to promote chromatin accessibility to facilitate germline gene expression. Overall, the data compellingly demonstrate a much broader role for OVO in the activation of genes in the female germline than previously recognized. By contrast, the relationship between OVO, chromatin accessibility, and the timing of gene expression is only correlative, and more work will be needed to determine the mechanisms by which OVO promotes transcription.

We fully agree with this summary.  

Strengths:

Here Benner et al. convincingly show that OVO is a transcriptional activator that promotes expression of hundreds of genes in the female germline. The ChIP-seq and RNA-seq data included in the manuscript are robust and the analysis is compelling.

Importantly, the set of genes identified is essential for maternal processes, including egg production and patterning of the early embryo. Together, these data identify OVO as a major transcriptional activator of the numerous genes expressed in the female germline, deposited into the oocyte and required for early gene expression. This is an important finding as this is an essential process for development and prior to this study, the major drivers of this gene expression program were unknown.

We are delighted that this aspect of the work came across clearly. Understanding the regulation of maternal effect genes has been something of a black-box, despite the importance of this class of genes in the history of developmental genetics. The repertoire of essential oogenesis/embryonic development genes that are bound by and respond to OVO are well characterized in the literature, but nothing is known about how they are transcriptionally regulated. We feel the manuscript will be of great interest to readers working on these genes.

Weaknesses:

The novelty of the manuscript is somewhat limited as the authors show that, like two prior, well-studied OVO target genes, OVO binds to promoters of germline genes and activates transcription. The fact that OVO performs this function more broadly is not particularly surprising.

Clearly, transcription factors regulate more than one or two genes. Never-the-less we were surprised at how many of the aspects of oogenesis per se and maternal effect genes were OVO targets. It was our hypothesis that OVO would have a transcriptional effect genome-wide, however, it was less clear whether OVO would always bind at the core promoter, as is with the case of ovo and otu. Our results strongly support the idea that core promoter proximal binding is essential for OVO function; a conclusion of work done decades ago, which has not been revisited using modern techniques. 

A major challenge to understanding the impact of this manuscript is the fact that the experimental system for the RNA-seq, the tagged constructs, and the expression analysis that provides the rationale for the proposed pioneering function of OVO are all included in a separate manuscript.

This is a case where we ended up with a very, very long manuscript which included a lot of revisiting of legacy data. It was a tough decision on how to break up all the work we had completed on ovo to date. In our opinion, it was too much to put everything into a single manuscript unless we wanted a manuscript length supplement (we were also worried that supplemental data is often overlooked and sometimes poorly reviewed). We therefore decided to split the work into a developmental localization/characterization paper and a functional genomics paper. As it stands both papers are long. Certainly, readers of this manuscript will benefit from reading our previous OVO paper, which we submitted before this one. The earlier manuscript is under revision at another journal and we hope that this improved manuscript will be published and accessible shortly.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Benner et al. interrogate the transcriptional regulator OVO to identify its targets in the Drosophila germline. The authors perform ChIP-seq in the adult ovary and identify established as well as novel OVO binding motifs in potential transcriptional targets of OVO. Through additional bioinformatic analysis of existing ATAC-seq, CAGE-seq, and histone methylation data, the authors confirm previous reports that OVO is enriched at transcription start sites and suggest that OVO does not act as part of the core RNA polymerase complex. Benner et al. then perform bulk RNA-seq in OVO mutant and "wildtype" (GAL4 mediated expression of OVO under the control of the ovo promoter in OVO mutants) ovaries to identify genes that are differentially expressed in the presence of OVO. This analysis supports previous reports that OVO likely acts at transcription start sites as a transcriptional activator. While the authors propose that OVO activates the expression of genes that are important for egg integrity, maturation, and for embryonic development (nanos, gcl, pgc, bicoid), this hypothesis is based on correlation and is not supported by in vivo analysis of the respective OVO binding sites in some of the key genes. A temporal resolution for OVO's role during germline development and egg chamber maturation in the ovary is also missing. Together, this manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis but lacks important in vivo experimental evidence that would validate the high-quality datasets.

We thank reviewer 2 for the appreciation of the genomics data and analysis. Some of the suggested in vivo experiments are clear next steps, which are well underway. These are beyond the scope of the current manuscript. 

Temporal analysis of ovo function in egg chamber development is not easy, as only the weakest ovo alleles have any egg chambers to examine. However, we will also point out the long-known phenotypes of some of those weak alleles in the text (e.g. ventralized chambers in ovoD3/+). We will need better tools for precise rescue/degradation during egg chamber maturation.     

Strengths:

The manuscript contains relevant ChIP-seq and RNA-seq datasets of OVO targets in the Drosophila ovary alongside thorough bioinformatic analysis

Thank you. We went to great lengths to do our highly replicated experiments in multiple ways (e.g. independent pull-down tags) and spent considerable time coming up with an optimized and robust informatic analysis.

Weaknesses:

(1) The authors propose that OVO acts as a positive regulator of essential germline genes, such as those necessary for egg integrity/maturation and embryonic/germline development. Much of this hypothesis is based on GO term analysis (and supported by the authors' ChIP-seq data). However accurate interpretation of GO term enrichment is highly dependent on using the correct background gene set. What control gene set did the authors use to perform GO term analysis (the information was not in the materials and methods)? If a background gene set was not previously specified, it is essential to perform the analysis with the appropriate background gene set. For this analysis, the total set of genes that were identified in the authors' RNA-seq of OVO-positive ovaries would be an ideal control gene set for which to perform GO term analysis. Alternatively, the total set of genes identified in previous scRNA-seq analysis of ovaries (see Rust et al., 2020, Slaidina et al., 2021 among others) would also be an appropriate control gene set for which to perform GO term analysis. If indeed GO term analysis of the genes bound by OVO compared to all genes expressed in the ovary still produces an enrichment of genes essential for embryonic development and egg integrity, then this hypothesis can be considered.

We feel that this work on OVO as a positive regulator of genes like bcd, osk, nos, png, gnu, plu, etc., is closer to a demonstration than a proposition. These are textbook examples of genes required for egg and early embryonic development. Hopefully, this is not lost on the readers by an over-reliance on GO term analysis, which is required but not always useful in genome-wide studies. 

We used GO term enrichment analysis as a tool to help focus the story on some major pathways that OVO is regulating. To the specific criticism of the reference gene-set, GO term enrichment analysis in this work is robust to gene background set. We will update the GO term enrichment analysis text to indicate this fact and add a table using expressed genes in our RNA-seq dataset to the manuscript and clarify gene set robustness in greater detail in the methods of the revision. We will also try to focus the reader’s attention on the actual target genes rather than the GO terms in the revised text.

We have updated the GO term analysis by including all the expressed genes in our RNA-seq datasets as a background control. Figure 6 has been updated to include the significant GO terms. We have outlined changes in the methods section below.

Lines 794-801:

“Gene ontology enrichment analysis was completed with g:Profiler’s g:GOSt software (Raudvere et al. 2019) on the set of genes overlapping OVO ChIP peaks over the TSS and significantly upregulated in the presence of ectopic OVO (525 genes in total). All genes that were considered to be expressed in our RNA-seq datasets were used as a background control (10,801 genes in total). Default parameters were used for the enrichment analysis except for ‘statistical domain scope’ was set to ‘custom’ (our control background genes were uploaded here), ‘significance threshold’ was set to ‘Bonferroni correction’, and only GO biological process terms were searched for enrichment with the gene list. The GO terms listed in Figure 6 represent the 24 smallest GO term sizes according to Table S5.”

(2) The authors provide important bioinformatic analysis of new and existing datasets that suggest OVO binds to specific motifs in the promoter regions of certain germline genes. While the bioinformatic analysis of these data is thorough and appropriate, the authors do not perform any in vivo validation of these datasets to support their hypotheses. The authors should choose a few important potential OVO targets based on their analysis, such as gcl, nanos, or bicoid (as these genes have well-studied phenotypes in embryogenesis), and perform functional analysis of the OVO binding site in their promoter regions. This may include creating CRISPR lines that do not contain the OVO binding site in the target gene promoter, or reporter lines with and without the OVO binding site, to test if OVO binding is essential for the transcription/function of the candidate genes.

Exploring mechanism using in vivo phenotypic assays is awesome, so this is a very good suggestion. But, it is not essential for this work -- as has been pointed out in the reviews, in vivo validation of OVO binding sites has been comprehensively done for two target genes, ovo and otu. The “rules” appear similar for both genes. That said, we are already following up specific OVO target genes and the detailed mechanism of OVO function at the core promoter. We removed some of our preliminary in vivo figures from the already long current manuscript. We continue to work on OVO and expect to include this type of analysis in a new manuscript.

(3) The authors perform de novo motif analysis to identify novel OVO binding motifs in their ChIP-seq dataset. Motif analysis can be significantly strengthened by comparing DNA sequences within peaks, to sequences that are just outside of peak regions, thereby generating motifs that are specific to peak regions compared to other regions of the promoter/genome. For example, taking the 200 nt sequence on either side of an OVO peak could be used as a negative control sequence set. What control sequence set did the authors use as for their de novo motif analysis? More detail on this is necessary in the materials and methods section. Re-analysis with an appropriate negative control sequence set is suggested if not previously performed.

We apologize for being unclear on negative sequence controls in the methods. We used shuffled OVO ChIP-seq peak sequences as the background for the de novo motif analysis, which we will better outline in the methods of the revision. This is a superior background set of sequences as it exactly balances GC content in the query and background sequences. We are not fond of the idea of using adjacent DNA that won’t be controlled for GC content and shadow motifs. Furthermore, the de novo OVO DNA binding motifs are clear, statistically significant variants of the characterized in vitro OVO DNA binding motifs previously identified (Lu et al., 1998; Lee and Garfinkel, 2000; Bielinska et al., 2005), which lends considerable confidence. We also show that the OVO ChIP-seq read density are highly enriched for all our identified motifs, as well as the in vitro motifs. We provide multiple lines of evidence, through multiple methods, that the core OVO DNA binding motif is 5’-TAACNGT-3’. We have high confidence in the motif data.

We have added the below text to the methods section for further clarity on motif analysis parameters.

Lines 808-812

“The default parameters were used for de novo motif enrichment analysis, including the use of shuffled input sequences as a control. After identifying ‘OVO Motif One’, OVO ChIP peaks that contained that sequence were removed and the resulting ChIP peaks were resubmitted for STREME analysis deriving derivative OVO DNA binding motifs like above.”

(4) The authors mention that OVO binding (based on their ChIP-seq data) is highly associated with increased gene expression (lines 433-434). How many of the 3,094 peaks (conservative OVO binding sites), and what percentage of those peaks, are associated with a significant increase in gene expression from the RNA-seq data? How many are associated with a decrease in gene expression? This information should be added to the results section.

Not including the numbers of the overlapping ChIP peaks and expression changes in the text was an oversight on our part. The numbers that relate to this (666 peaks overlapping genes that significantly increased in expression, significant enrichment according to Fishers exact test, 564 peaks overlapping genes that significantly decreased in expression, significant depletion according to Fishers exact test) are found in figure 4C and will be added to the text.

We have modified the results section to include the overlap between the RNA-seq and ChIP-seq data.

Lines 463-468

“We found that 2,298 genes that were expressed in our RNA-seq data overlapped an OVO ChIP peak. 666 genes significantly increased in expression and were bound by OVO, which is a significant enrichment according to a Fisher’s exact test (Figure 4C, cyan dots, p < 0.01, odds ratio = 2.21). While conversely, 564 genes decreased in expression and were bound by OVO, indicating a significant depletion according to a Fisher’s exact test (Figure 4C, blue dots, p < 0.01, odds ratio = 0.85).”

(5) The authors mention that a change in endogenous OVO expression cannot be determined from the RNA-seq data due to the expression of the OVO-B cDNA rescue construct. Can the authors see a change in endogenous OVO expression based on the presence/absence of OVO introns in their RNA-seq dataset? While intronic sequences are relatively rare in RNA-seq, even a 0.1% capture rate of intronic sequence is likely to be enough to determine the change in endogenous OVO expression in the rescue construct compared to the OVO null.

This is a good point. The GAL4 transcript is downstream of ovo expression in the hypomorphic ovoovo-GAL4 allele. We state in the text that there is a nonsignificant increase in GAL4 expression with ectopic rescue OVO, although the trend is positive. We calculated the RPKM of RNA-seq reads mapping to the intron spanning exon 3 and exon 4 in ovo-RA and found that there is also a nonsignificant increase in intronic RPKM with ectopic rescue OVO (we will add to the results in the revision). We would expect OVO to be autoregulatory and potentially increase the expression of GAL4 and/or intronic reads, but the ovoovoGAL4>UASp-OVOB is not directly autoregulatory like the endogenous locus. It is not clear to us how the intervening GAL4 activity would affect OVOB activity in the artificial circuit. Dampening? Feed-forward? Is there an effect on OVOA activity? Regardless, this result does not change our interpretation of the other OVO target genes.

We have added the analysis of intronic ovo RNA-seq to the results as outlined below.

Lines 512-520

“Transcriptionally, ovo RNA-seq reads are likely derived from the UASp-3xFHA-OVO-B cDNA rescue or are indistinguishable between the genomic locus and rescuing cDNA transgene. We found a nonsignificant increase in exon 3 to exon 4 intronic ovo reads with the expression of ectopic rescue OVO (log2 fold change = 0.76, p-adj = 0.26). These intronic reads would be derived from the endogenous ovo locus, but it is difficult to conclusively determine if the endogenous ovo locus would respond transcriptionally to ectopic OVO downstream of UASp (for example, the pathway for ovo is no longer autoregulatory in ovoovo-GAL4/ovoΔBP; UASp-3xFHA-OVO-B germ cells, there is an additional GAL4>UASp activation step). So, we could not confidently assess whether ovo responded transcriptionally to ectopic rescue OVO.”

(6) The authors conclude with a model of how OVO may participate in the activation of transcription in embryonic pole cells. However, the authors did not carry out any experiments with pole cells that would support/test such a model. It may be more useful to end with a model that describes OVO's role in oogenesis, which is the experimental focus of the manuscript.

We did not complete any experiments in embryonic pole cells in this manuscript and base our discussion on the potential dynamics of OVO transcriptional control and our previous work showing maternal and zygotic OVO protein localization in the developing embryonic germline. Obviously, we are highly interested in this question and continue to work on the role of maternal OVO. We agree that we are extended too far and will remove the embryonic germ cell model in the figure. We will instead focus on the possible mechanisms of OVO gene regulation in light of the evidence we have shown in the adult ovary, as suggested.

We have removed figure 7 and have re-written the last two paragraphs of the discussion as below.

Lines 645-663

“The requirement for OVO at the TSS of target genes has been well characterized at its own locus as well as its downstream target otu. Our OVO ChIP and expression data confirm findings from previous work that OVO is binding to these target promoters, and in the case of otu, strongly responds transcriptionally to the presence of OVO. Although we did not test the requirement for OVO DNA binding motifs at other OVO bound genes in this work, this has been extensively explored before, showing that removal of OVO

DNA binding sites overlapping the TSS results in a strong decrease in reporter expression (Lü et al. 1998; Bielinska et al. 2005; Lü and Oliver 2001). Removal of more distal upstream OVO DNA binding sites also reduces reporter expression to a lesser degree. However, for most cases tested, removal of OVO DNA binding sites while leaving the rest of the enhancer regions intact, never totally abolished reporter expression. These dynamics are highly similar to work that has been completed on the pioneer factor zelda (zld). Adding zld DNA binding motifs to a stochastically expressed transcriptional reporter increases the activity and response of the reporter (Dufourt et al. 2018). Distally located zld DNA binding motifs influenced reporter expression to a lesser degree than proximal sites. A single zld DNA binding site adjacent to the TSS produced the strongest reporter activity. Importantly, just like the activity of OVO transgenic reporters, there is not an absolute requirement for zld DNA binding to activate reporter expression, however, the addition of TSS adjacent zld DNA binding motifs does strongly influence reporter response. We know that zld achieves this reporter response through its pioneering activity (Xu et al. 2014; Harrison et al. 2011), whether OVO achieves this similar effect on gene expression through a shared mechanism, or in cooperation with other transcription factors needs to be further explored.”

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The Results section could be streamlined by limiting the discussion of analysis to only those details that are unusual or essential for understanding the science. For example, the fact that MACS3 was used to call peaks seems most suitable for the Methods section.

We have removed the below excerpts from the results section to streamline the text.

‘We compared immuno-purified OVO associated DNA with input DNA as a control, for a total of 12 ChIPseq libraries, which we sequenced using the Illumina system. After quality control and alignment to the Drosophila r6.46 genome (Gramates et al. 2022), we used MACS3 (Zhang et al. 2008)’

The Supplemental Tables are referred to out of order. Table S2 is referred to on line 143 while Table S1 is not referred to until the Methods section.

We have reorganized the order of the tables in the manuscript text.

In the analysis of CAGE-seq data, it is unclear whether there is anything distinctive about the ~2000 regions bound by OVO but that is not near TSS in the ovary dataset. Are these TSS that are not active in the ovary or are these non-promoter bound OVO sites? If they are TSS of genes not in the CAGE-seq data set, are these genes expressed in other tissues or just expressed at lower levels in the ovary?

This was a good point that prompted us to take a closer look at the characteristics of OVO binding and its relationships to promoters and other gene elements. 45% of OVO ChIP peaks overlapped the TSS while 55% were either non-overlapping downstream or upstream of the TSS. When plotting OVO ChIP read density, there was still a striking enrichment of OVO binding over the TSS, even though the ChIP peak was not overlapping the TSS (new figure 1K). This is possibly due to weaker direct OVO binding at the TSS that was not considered significant in the peak calling software or were indirect interactions of the distal OVO binding and the TSS. We outline this in the below text added to the results section on the OVO ChIP. To showcase these results, we have included a new panel in figure 1K. We removed the panel showing the enrichment over the cage-seq TSS, but this same data remains in the heatmap shown in figure 1L, so no information is lost. To directly answer the Cage-seq questions considering the OVO bound over the annotated TSS results, we found that 1,047 chip peaks overlapped CAGE-seq TSS, which is only 347 fewer than the annotated TSS overlap (1,394). Of the 1,394 genes that were bound by over the TSS, all of them were considered to be expressed in our RNA-seq dataset, indicating that these might just be more lowly expressed genes that for whatever reason were not considered to be enriched TSSs in the CAGE-seq data. This difference is likely not significant.

Lines 235-251

“Although OVO ChIP peaks overlapping genes showed a strong read density enrichment over the TSS, we found that only 45% (1,394/3,094) of OVO ChIP peaks directly overlapped a TSS. 43% (1,339/3,094) of OVO ChIP peaks were found to overlap the gene body downstream of the TSS (intronic and exonic sequences) and 12% (366/3,094) did not overlap any gene elements, indicating that they were intergenic.

We were interested in the differences between OVO binding directly over the TSS or at more distal upstream and downstream sites. We decided to plot the OVO ChIP read density of these different classes of OVO binding patterns and found that OVO bound over the TSS produced a sharp read density enrichment over the TSS which was consistent with what was found for all OVO bound genes (Figure 1K). OVO binding along the gene body surprisingly also showed a read density enrichment over the TSS, although the magnitude of read density enrichment was notably less than TSS OVO binding. Intergenic OVO binding also showed these same characteristics with a notable upstream read density enrichment possibly indicative of enhancer binding. This indicates that although the significantly called OVO ChIP peaks did not overlap the TSS, there was still a propensity for TSS sequences to be enriched with OVO ChIP over the input control. This could be due to weaker direct in vivo binding of OVO to these TSSs or indirect interactions between the upstream/downstream OVO bound sequences and the TSS, possibly through a looping enhancer-promoter interaction. However, regardless of the location of the OVO ChIP peak, OVO seemed to always be enriched at or in close proximity to TSSs.”

It would be helpful for the authors to provide a bit more detailed analysis of chromatin states of OVObound regions in GSC, 8c NC, and 32c NC (or some more clarity in the current analysis). Are the regions that are bound by OVO accessible in all these cell types or specifically enriched for accessibility in a subset? The authors state that OVO binding is correlated with open chromatin, but whether these are regions that are open in all cell types analyzed or a subset is not clear from the data presented. Promoters are often accessible regardless of cell type, so it is unclear what exactly is to be concluded from this association. Also, is the proximity to open chromatin features for OVO-bound promoters (as shown in Figure 2C) different than non-OVO-bound promoters (the two classes shown Figure 1L, for example)?

We utilized previously published datasets of staged germ cell chromatin status to look at the association of chromatin status and OVO binding. Unfortunately, not all the same germ cell stages were profiled for each chromatin mark from the datasets derived for these two papers. For example, only H3K4me3 data exists for GSCs, and only gsc and 8c data exists for H3K9me3, while the other chromatin marks had more profiles, even including later stages. We focused specifically on gsc and 32c (essentially stage 5 egg chambers) for the other chromatin marks since that is when the ovo hypomorphic egg chambers arrest. A nice control would have been chromatin states in somatic follicle cells of the ovary, since we know germ cell genes such as ovo and otu are not expressed and presumably the chromatin states in somatic cell types would be different than germ cells. However, chromatin states for somatic follicle cells were not published in these two papers and we are not aware of any other existing datasets to compare too. Essentially, we need to determine the changes in chromatin states with and without OVO, which we are currently working on. 

We did further analyze chromatin states and differential OVO binding in respect to gene elements, and found that OVO binding, regardless of the relationship to the gene element, is always open (gsc and 32c ATAC). OVO binding over the gene body shows the same enrichment for open chromatin and transcriptionally active histone marks. We compared the profiles of these chromatin marks and the promoters of OVO bound and not bound genes and consistent with the suggestion that promoters are generally open, we found that this was the case. However, there is an enrichment for open chromatin and transcriptionally active histone marks for OVO bound genes compared to non-OVO bound genes. This could be a consequence of OVO binding or indirect consequence of a downstream OVO target. Regardless, as has been suggested, future experiments directly measuring chromatin status and OVO needs to be performed. The below excerpts have been added to the text to supplement the comments provided above.

Lines 328-343

“The association of OVO binding with active histone marks and open chromatin was striking, but open chromatin is likely a general phenomenon of promoters (Haines and Eisen, 2008). Indeed, when measuring the read density for GSC and 32C ATAC-seq for OVO bound and OVO non-bound promoters, there is an enrichment for open chromatin at the TSS regardless of OVO binding. However, we did notice an increase in enrichment for OVO bound promoters compared to OVO non-bound promoters (Figure S1G), possibly suggesting that OVO bound promoters are more open or have an increase in accessibility when compared to non-OVO bound promoters. This same relationship held true for the transcriptionally active histone mark H3K27ac in GSCs (Figure S1H). Since only 45% of OVO ChIP peaks overlapped TSSs, we plotted the read density of the above chromatin marks over OVO ChIP peak maximums for OVO bound over the TSS, gene body, or intergenic regions (Figure S2A-D). We found that OVO bound regions that were not overlapping the TSS still showed the same propensity for enrichment of open chromatin and active histone marks. Intergenic regions were especially enriched for open chromatin measured through ATAC-seq. Altogether suggesting that OVO binding genome-wide is tightly associated with open chromatin regardless of germ cell stage, and active transcription in GSCs. In other words, chromatin state data suggests OVO is acting positively on its target genes and raises the possibility that OVO-binding and open chromatin are related.”

For clarity, it would help the reader if the authors mentioned the male-specific TATA-associated factors as a rationale for testing the role of OVO binding in core promoter function. This is currently mentioned in the Discussion on lines 575-577, but would help in understanding the motivation behind the detailed analysis of the promoter binding of OVO in the Results and make the negative result more clearly impactful.

We have introduced the male specific tata factors as suggested and have condensed the two intro paragraphs in this section into one, as shown below.

Lines 347-363

“Our data thus far clearly indicates that OVO binding occurs at or very near the core promoter, a region recognized by an enormous collection of factors that associate with RNA polymerase to initiate transcription (Aoyagi and Wassarman 2000; Vo Ngoc, Kassavetis, and Kadonaga 2019). The highly organized polymerase complex has sequence-specific DNA recognition sites with incredibly precise spacing between them, with an overall DNA footprint of a little less than 100bp (Rice, Chamberlin, and Kane 1993; FitzGerald et al. 2006; Ohler et al. 2002). There are upstream binding sites such as TATA, sites at transcription start, such as the initiator (INR), and downstream promoter elements (DPE) (Vo Ngoc, Kassavetis, and Kadonaga 2019). The combinations of these DNA motifs is not random in mammals and Drosophila (FitzGerald et al. 2006), and distinct combinations of different motifs at the TSS of genes expressed in Drosophila are conserved over tens of millions of years of evolution (Chen et al. 2014). The male germline expresses a number of TATA-associated factors that have been implicated in male-specific promoter usage for gene expression (M. Hiller et al. 2004; M. A. Hiller et al. 2001; Lu et al. 2020; V. C. Li et al. 2009). It is possible that OVO is a female germline specific TATA-associated factor, and if so, OVO binding sites at core promoters should share precise spacing with other core promoter elements, suggesting it is likely part of the complex. If not, then OVO is more likely to facilitate binding of the basal transcriptional machinery. Because of the extended footprint of engaged RNA polymerase, OVO and the basal machinery would not be likely to occupy the same region at the same time.”

The description of the system used for the RNA-seq would benefit from additional clarity. It is not clear as written why it is "Lucky" that there is an mRNA isoform with extended exon 2 required for egg chamber development beyond stage 5. How does this requirement compare to the global requirement for OVO, which seems to be required for germ cell development even before stage 5? Understanding this system is essential for interpreting the RNA-seq results. Indeed, the authors have a separate manuscript (currently on bioRxiv) that explains the details of this system. As such, the current description requires that the reader refer to this additional pre-print. Could the authors include a diagram to better illustrate this system? Furthermore, since this RNA-seq is being performed on tissue that includes nurse cells, follicle cells, and germ cells from multiple stages of development, it is important for the authors to clearly state in which cell types OVO is expressed and likely functional. (While this is well beyond this manuscript, this analysis is the type that might benefit from the use of single-cell sequencing as a means to deconvolute the phenotypic effects of OVO loss.)

We have rewritten the text to better describe the system for RNA-seq. We have also included a figure (Figure S1A) showing the alleles used that should help provide clarity for the readers. We agree that moving forward single cell experiments will be critical to have a better understanding of the transcriptional changes and chromatin dynamics with and without OVO. We have included the below changes to the text.

Lines 409-423

“Previous work from our lab has identified a transheterozygous ovo allelic combination (ovoovo-GAL4/ovoΔBP) that greatly reduces OVO activity resulting in sterility, however, female germ cells are able to survive up until at least stage 5 of oogenesis (Benner et al. 2023). ovoovo-GAL4 is a CRISPR/Cas9 derived T2A-GAL43xSTOP insertion upstream of the splice junction of exon 3 in the ovo-RA transcript (Figure S1A).

Importantly, this insertion in the extended exon 3 would disrupt roughly 90% of the ovo-B transcripts. However, since about 10% of ovo-B transcripts utilize an upstream splice junction in exon 3, these transcripts would not be disrupted with the T2A-GAL4-3xSTOP insertion and thus allow for enough OVO activity for germ cell survival (Benner et al. 2023). Since ovoovo-GAL4 expresses GAL4 in place of full length OVO due to the T2A sequences, we can drive expression of a rescuing OVO-B construct downstream of UASp to generate OVO+ female germ cells, which in fact does rescue the arrested germ cell phenotype of ovoovo-GAL4/ovoΔBP ovaries. Therefore, in order to determine genes that are transcriptionally responsive to OVO, we compared the gene expression profiles in sets of ovaries that had the ovo hypomorphic phenotype with a negative control rescue construct (ovoovo-GAL4/ovoΔBP; UASp-GFP)(Figure 4A) versus those that drive expression of the rescue construct expressing OVO-B (ovoovo-GAL4/ovoΔBP; UASp-3xFHAOVO-B)(Figure 4B).”

Lines 427-432

“The adult female ovary contains somatic cells, germline stem cells, and germline derived nurse cells that would be profiled in a bulk ovary tissue RNA-seq experiment. Although OVO is only required and expressed in germline derived cell types, we chose to dissect one day old post-eclosion ovoovoGAL4/ovoΔBP; UASp-3xFHA-OVO-B female ovaries to enrich for early stages of oogenesis and collected only ovarioles containing the germarium through previtellogenic egg chambers.”

On lines 526-532, it is unclear why the genes fs(1)N, fs(1)M3, and closca are particularly sensitive to the ovoD3 allele. What is this allele trans heterozygous with in the assay that allows development through egg laying? Why might these genes be unique in their sensitivity?

These genes are not particularly sensitive, the transheterozygous hypomorphic ovo ovaries are weak enough to reveal the role of OVO for these genes. We rewrote this paragraph to try and provide more clarity to the relationship between OVO+ binding at these vitelline membrane genes and the phenotype of OVOD3 expressing females.

Lines 562-577

“We also found that the genes fs(1)N, fs(1)M3, and closca, were all bound by OVO and responded transcriptionally to the presence of ectopic rescue OVO. These genes are significant because they constitute a set of genes that are expressed in the germline and the encoded proteins are eventually incorporated into the vitelline membrane providing the structural integrity and impermeability of the egg (Mineo, Furriols, and Casanova 2017; Ventura et al. 2010). Loss-of-function of these three genes results in flaccid eggs that are permeable to dye and fail to develop. The loss-of-function phenotype of fs(1)N, fs(1)M3, and closca closely resembles the dominant antimorph ovoD3 phenotype. The ovoD3 allele is the weakest of the original dominant-negative ovo alleles and produces defective eggs allowing us to explore the role of OVO in late stages (Busson et al. 1983; Komitopoulou et al. 1983). ovoD3/ovo+ transheterozygous females express a repressive form of OVO that results in dominant sterility, and importantly, these females lay flaccid eggs with compromised vitelline membranes that are permeable to the dye neutral red (Oliver, Pauli, and Mahowald 1990). Since OVO+ is bound at the TSS of fs(1)N, fs(1)M3, and closca, and these three genes respond transcriptionally to OVO+, then it is plausible that the repressive OVOD3 is negatively regulating these three genes that are required for vitelline membrane formation. This is evidence that OVO is not only involved in regulating the expression of numerous essential maternal pathways for embryonic development, but it is also essential for regulating genes that are required for egg integrity and maturation.”

The Discussion of OVO as a pioneer factor is highly speculative and based only on correlative data. In fact, the expression data in the embryonic germline is not included in this manuscript, but rather in a separate bioRxiv preprint. This makes it challenging to understand, why this is extensively discussed here. However, there are experiments that could begin to test this proposal. OVO could be expressed in an exogenous tissue and test whether it promotes accessibility. Also, mutations could be made (using gene editing) to identify previously known OVO binding sites in the otu and/or other promoters and these could be assayed for accessibility. By selecting promoters of genes that are not essential for germline development, the authors could directly test the role of OVO in promoting chromatin accessibility. Alternatively, are there reasons that the system used for RNA-seq couldn't be similarly used for ATACseq? It is imperfect but could provide insights into chromatin accessibility in the absence of OVO.

We have largely removed the speculation on pioneering activity, reference to embryonic germline OVO dynamics included in the previous work, and Figure 7. These are excellent suggestions for experiments and ones we are currently pursuing. Below is the modified discussion. 

Lines 645-663

“The requirement for OVO at the TSS of target genes has been well characterized at its own locus as well as its downstream target otu. Our OVO ChIP and expression data confirm findings from previous work that OVO is binding to these target promoters, and in the case of otu, strongly responds transcriptionally to the presence of OVO. Although we did not test the requirement for OVO DNA binding motifs at other OVO bound genes in this work, this has been extensively explored before, showing that removal of OVO

DNA binding sites overlapping the TSS results in a strong decrease in reporter expression (Lü et al. 1998; Bielinska et al. 2005; Lü and Oliver 2001). Removal of more distal upstream OVO DNA binding sites also reduces reporter expression to a lesser degree. However, for most cases tested, removal of OVO DNA binding sites while leaving the rest of the enhancer regions intact, never totally abolished reporter expression. These dynamics are highly similar to work that has been completed on the pioneer factor zelda (zld). Adding zld DNA binding motifs to a stochastically expressed transcriptional reporter increases the activity and response of the reporter (Dufourt et al. 2018). Distally located zld DNA binding motifs influenced reporter expression to a lesser degree than proximal sites. A single zld DNA binding site adjacent to the TSS produced the strongest reporter activity. Importantly, just like the activity of OVO transgenic reporters, there is not an absolute requirement for zld DNA binding to activate reporter expression, however, the addition of TSS adjacent zld DNA binding motifs does strongly influence reporter response. We know that zld achieves this reporter response through its pioneering activity (Xu et al. 2014; Harrison et al. 2011), whether OVO achieves this similar effect on gene expression through a shared mechanism, or in cooperation with other transcription factors needs to be further explored.”

The authors suggest that OVO binding is essential for transcriptional activation, but that this may be indirect and that expression of other transcription factors might be necessary for activating gene expression. Did the motif analysis of the OVO-bound regions suggest additional transcription factors that might provide this function?

We did find other motifs significantly enriched in OVO ChIP peaks. We performed XSTREME analysis on the same set of OVO ChIP peaks which allowed us to determine if any of these motifs were significant matches to DNA binding motifs of known transcription factors. Notably, the DNA binding motifs of GAF and CLAMP were enriched in OVO ChIP peaks. GAF is required in germline clones and the potentially for co-regulation of genes is possible. Other enriched motifs did not match any known binding motifs of other transcription factors but we reported some of the most significantly enriched motifs that were alongside of OVO in Figure S1C-F. The below text outlines changes made to the text incorporating these findings.

Lines 170-182

“Along with the OVO DNA binding motif, other motifs were also significantly enriched in OVO ChIP peaks. The motif 5’-GWGMGAGMGAGABRG-3’ (Figure S1C) was found in 18% of OVO ChIP peaks and is a significant match to the DNA binding motifs of the transcription factors GAF (Trl) (Omelina et al. 2011) and CLAMP (Soruco et al. 2013). Trl germline clones are not viable, indicating that GAF activity is required in the germline during oogenesis (Chen et al. 2009). The possibility that OVO binds with and regulates genes alongside of GAF given the enrichment of both transcription factors DNA binding motifs is intriguing. Other significantly enriched motifs 5’-ACACACACACACACA-3’ (29% of peaks, Figure S1D), 5’RCAACAACAACAACA-3’ (26% of peaks, Figure S1E), and 5’-GAAGAAGAAGAAGAR-3’ (17% of peaks,

Figure S1F) were present in OVO ChIP peaks, however, these motifs did not significantly match known

DNA binding motifs of other transcription factors. Determining the factors that bind to these sequences

will certainly help elucidate our understanding of transcriptional control with relationship to OVO in the female germline.”

The figures would benefit from a bit more detail in the legends (see comments below).

Minor comments:

In multiple places throughout the document, the citations are inadvertently italicized (see lines 57-59, 91, and 327 as examples.)

We have changed this in these locations and other instances in the text.

On line 76, when discussing OVO as a transcription factor this is referencing the protein and not the gene. Thus, should be written OVO and not ovo.

We have made the correction ovo to OVO.

On line 349, "core" promoters is likely what is meant rather than "care" promoters.

We have corrected ‘care’ to ‘core’ in the text.

On line 404, the authors state that they wanted to use a "less conservative log2 fold change" but it is not clear what they are comparing to. This is important to understand the motivation.

We are talking about the gene expression comparison between the ectopic ovo rescue and ovo hypomorphic ovaries. “less conservative” was an unfortunate phrasing. We have rewritten the text to state this directly to the reader.

Lines 435-444

“We then performed RNA-seq in quadruplicate and measured the changes in gene expression between ectopic rescue OVO and hypomorphic OVO ovaries. We used a significance level of p-adj < 0.05 and a log2 fold change cutoff of >|0.5| to call differential expression between these two sets of ovaries. We utilized these log2 fold change cutoffs for two reasons. Our control ovary genotype (ovoovo-GAL4/ovoΔBP; UASp-GFP) has hypomorphic OVO activity, hence germ cells can survive but are arrested. With the addition of ectopic rescue OVO in ovoovo-GAL4/ovoΔBP; UASp-3xFHA-OVO-B ovaries, we predicted that genes that were directly regulated by OVO would transcriptionally respond, however, we were unsure as to what degree the response would be in comparison to hypomorphic OVO. We reasoned that if the changes were not significant between genotypes, then minor changes in gene expression would not matter.”

On line 615, it is unclear what is meant by "showing expression with only 10s of bp of sequence in reporters."

This is in reference to some of the previously studied ovo reporter deletion lines, however, we have decided to remove the below text in the revised discussion.

“, despite being remarkably compact. The OVO-dependent ovo core promoter is very compact; showing expression with only 10s of bp of sequence in reporters.” 

It would be useful to cite and discuss Dufourt et al. Nature Communications 2018 (PMID30518940) regarding the role of Zelda in potentiating transcriptional activation when mentioned on line 624.

We have added this and the relationship to previous similar work on OVO in the discussion.

Lines 645-663

“The requirement for OVO at the TSS of target genes has been well characterized at its own locus as well as its downstream target otu. Our OVO ChIP and expression data confirm findings from previous work that OVO is binding to these target promoters, and in the case of otu, strongly responds transcriptionally to the presence of OVO. Although we did not test the requirement for OVO DNA binding motifs at other OVO bound genes in this work, this has been extensively explored before, showing that removal of OVO

DNA binding sites overlapping the TSS results in a strong decrease in reporter expression (Lü et al. 1998; Bielinska et al. 2005; Lü and Oliver 2001). Removal of more distal upstream OVO DNA binding sites also reduces reporter expression to a lesser degree. However, for most cases tested, removal of OVO DNA binding sites while leaving the rest of the enhancer regions intact, never totally abolished reporter expression. These dynamics are highly similar to work that has been completed on the pioneer factor zelda (zld). Adding zld DNA binding motifs to a stochastically expressed transcriptional reporter increases the activity and response of the reporter (Dufourt et al. 2018). Distally located zld DNA binding motifs influenced reporter expression to a lesser degree than proximal sites. A single zld DNA binding site adjacent to the TSS produced the strongest reporter activity. Importantly, just like the activity of OVO transgenic reporters, there is not an absolute requirement for zld DNA binding to activate reporter expression, however, the addition of TSS adjacent zld DNA binding motifs does strongly influence reporter response. We know that zld achieves this reporter response through its pioneering activity (Xu et al. 2014; Harrison et al. 2011), whether OVO achieves this similar effect on gene expression through a shared mechanism, or in cooperation with other transcription factors needs to be further explored.”

On line 1006 (Figure 1 legend), it is unclear what is meant by "The percentage of OVO ChIP peaks each motif was found". Is a word missing?

This was unclear, we have revised the sentence below.

Lines 1035-1036

“The percentage of OVO ChIP peaks containing each motif and their corresponding p-value are indicated to the right.”

In the Figure 1 legend, please include citations for the Garfinkel motif and Oliver motif.

Included, as below.

Lines 1036-1039

“H) OVO ChIP minus input control ChIP-seq read coverage density centered on the location of the four de novo OVO DNA binding motifs and previously defined in vitro OVO DNA binding motifs (Lü et al. 1998, Bielinska et al. 2005, Lee and Garfinkel 2000).”

In Figure 2 legend, it is unclear if B is all instances of a given motif or the DNA motifs that are bound by ChIP. Please clarify.

We meant only the OVO DNA binding motifs that were within significant OVO ChIP peaks. We have revised the legend below.

Lines 1049-1052

“A, B) OVO ChIP minus input control, GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq read coverage density centered on each OVO peak maximum or OVO DNA binding motif located within a significant OVO ChIP peak.”

The Figure legend for 2D could use more explanation. What do the lines and circles indicate?

These lines and circles indicate the amount of overlapping peaks measured between the two datasets with solid circles. We have included a better description of what these indicate in the figure legend.

Lines 1054-1058

“D) Total number of significant peaks (left) and the total number of overlapping peaks (top) between OVO

ChIP and GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq. Lines connecting solid dots indicates the amount of overlapping peaks between those two corresponding datasets.”

In Figure 4C, bring the 564 blue dots forward so they are not masked by the yellow dots.

We have brought the colored dots forward in both figure 4C and 4D.

In Figure 4E, what is the order of the heatmaps?

The order is genes with the highest to lowest OVO read density enrichment. We have included this in the figure 4 legend.

Lines 1086-1087

“The order of the heatmap is genes with the highest to lowest amount of OVO ChIP read density.”

In Figure 5, the order of the tracks is not immediately obvious. It appears to be those chromatin features most associated with OVO ChIP and those less correlated. Additional clarity could be provided by showing these tracks (and in Supplemental Figure S2) in different colors with a reference to the figure legend about what the colors might indicate.

We have changed the colors and order of the tracks to be more similar and consistent in both figures.

Lines 1090-1093

“ovo gene level read coverage tracks for OVO ChIP minus input (black), GSC and 32c ATAC-seq (light blue), GSC and 32C H3K27ac (green), H3K4me3 (dark blue), GSC and 32c H3K27me3 (orange), and GSC and 8c H3K9me3 (pink) ChIP-seq, and ovoΔBP/ovoovo-GAL4; UASp-3xFHA-OVO-B minus ovoΔBP/ovoovo-GAL4; UASp-GFP RNA-seq (red).”

In Figure S1 legend, what is the reference to the da-GAL4 X UAS transgene in the title?

This was an error on our part and we have removed it.

Reviewer #2 (Recommendations For The Authors):

Overall, the manuscript would benefit from revisions of the writing style. At times it is difficult to distinguish between hypothesis and results. The use of colloquial phrases/prose was distracting while reading, which the authors may consider revising. Some sentences were confusing or extraneous, and the authors may consider revising those. Occasionally sentences within the results sections seem more appropriate for the materials and methods.

(1) The manuscript is generally clear; however, it is at times difficult to distinguish between hypothesis and results. The use of colloquial phrases/prose was distracting while reading, which the authors may consider revising. Examples include:

a)  Lines 48-49 "While thematic elements of this complex orchestration have been well studied, coordinate regulation of the symphony has not."

We have edited this sentence below.

Lines 48-50

“While the complex interactions between maternally supplied mRNAs and proteins have been well studied, transcriptional regulation driving the expression of these pathways are less well understood.“

b)  Lines 232-233 "In other words, where exactly does transcription start at these genes."

We have removed this sentence.

c)  Line 385, the word "sham" could be changed to "negative control" or "GFP control"

We have rewritten this sentence below.

Lines 419-423

“Therefore, in order to determine genes that are transcriptionally responsive to OVO, we compared the gene expression profiles in sets of ovaries that had the ovo hypomorphic phenotype with a negative control rescue construct (ovoovo-GAL4/ovoΔBP; UASp-GFP)(Figure 4A) versus those that drive expression of the rescue construct expressing OVO-B (ovoovo-GAL4/ovoΔBP; UASp-3xFHA-OVO-B)(Figure 4B)”

d)  Line 490 "For the big picture"

We have removed this and revised with the below sentence.

Lines 530-531

“To do this, we performed Gene Ontology enrichment analysis with gProfiler software (Raudvere et al. 2019).”

(2) Some sentences were confusing or extraneous, and the authors may consider revising them. Examples include:

a)  Lines 195-196 "Therefore, we plotted the significant ChIP (minus input) read density peaks centered on the location of the motif itself."

We have removed the word ‘peaks’ and ‘itself’, as below.

Lines 200-201

“Therefore, we plotted the significant ChIP (minus input) read density centered on the location of the motif.”

b)  Lines 201-203 "... over the location of the motifs, strongly reinforces the idea that our dataset contains regions centered on sequence-specifically bound OVO transcription factor in the ovary."

We have edited this sentence to clarify below.

Lines 204-208

“While it is possible that OVO comes into contact with regions of DNA in three-dimensional nuclear space non-specifically, the presence of OVO motifs within a large percentage of significant ChIP peaks in vivo and enrichment of OVO ChIP read density at the location of the motifs, strongly reinforces the idea that our OVO ChIP dataset contains regions centered on sequences specifically bound by OVO in the ovary.”

c)  Lines 326-328 "The combinations of these elements...tens of millions of years of evolution."

We have revised this sentence below.

Lines 354-357

“The combinations of these DNA motifs is not random in mammals and Drosophila (FitzGerald et al. 2006), and distinct combinations of different motifs at the TSS of genes expressed in Drosophila are conserved over tens of millions of years of evolution (Chen et al. 2014).”

d)  Lines 444-446 "To address this directly, we tested the idea that genes with... and thus downstream of OVO."

We have removed this sentence in its entirety.

e)  Line 579-580 "Where OVO binding in close proximity, in any ...activates transcription"

We have removed this sentence in its entirety.

(3)    Occasionally sentences within the results sections seem more appropriate for the materials and methods. For example, lines 213-218.

(4)    At the end of line 375, do the authors mean "only" instead of "also"?

We have modified this sentence below.

Lines 411-414

“ovoovo-GAL4 is a CRISPR/Cas9 derived T2A-GAL4-3xSTOP insertion upstream of the splice junction of exon 3 in the ovo-RA transcript (Figure S1A). Importantly, this insertion in the extended exon 3 would disrupt roughly 90% of the ovo-B transcripts. However, since about 10% of ovo-B transcripts utilize an upstream splice junction in exon 3, these transcripts would not be disrupted with the T2A-GAL4-3xSTOP insertion and thus allow for enough OVO activity for germ cell survival (Benner et al. 2023).”

(5)    In line 392 the authors say that they dissected ovaries "one day post-eclosion" but the methods section says that ovaries were 3-5 days old. Please clarify.

We meant one day old for the RNAseq experiments. We have changed this in the text.

Lines 679-681

“Twenty, one day old post-eclosion ovoΔBP/ovoovo-GAL4; UASp-GFP and ovoΔBP/ovoovo-GAL4; UASp-3xFHAOVO-B ovaries were dissected and germariums through previtellogenic egg chambers were removed with microdissection scissors and placed in ice cold PBS making up one biological replicate.”

(6)    In line 668 the authors mention CRISPR/Cas9 in the methods, but no such experiment was described.

We have removed this from the Methods header.

Interesting link

Reviewer #1 (Public Review):

(1) Significance of findings and strength of evidence.

(a) The work presented in this manuscript is intended to support the authors' novel idea that HIV DNA integration strongly favors "triple-stranded" R-loops in DNA formed either during transcription of many, but not all, genes or by strand invasion of silent DNA by transcripts made elsewhere, and that HIV infection promotes R-loop formation mediated by incoming virions in the absence of reverse transcription. The authors were able to demonstrate a reverse transcription-independent increase in R-loop formation early during HIV infection, while also demonstrating increased integration into sequences that contain R-loop structures. Furthermore, this manuscript also identifies that R-loops are present in both transcriptionally active and silent regions of the genome and that HIV integrase interacts with R-loops. Although the work presented supports a correlation between R-loop formation and HIV DNA integration, it does not prove the authors' hypothesis that R-loops are directly targeted for integration. Direct experimentation, such as in vitro integration into defined DNA targets, will be required. Further, the authors provide no explanation as to how current sophisticated structural models of concerted retroviral DNA integration into both strands of double-stranded DNA targets can accommodate triple-stranded structures. Finally, there are serious technical concerns with the interpretation of the integration site analyses.

(2) Public review with guidance for readers around how to interpret the work, highlighting important findings but also mentioning caveats.

(a) Introduction: The authors provide an excellent introduction to R-loops but they base the rationale for this study on mis-citation of earlier studies regarding integration in transcriptionally silent regions of the genome. E "most favored locus" cited in the very old reference 6 comprises only 5 events and has not been reproduced in more recent, much larger datasets. For example, see the study of over 300.000 sites in freshly infected PBMC cited in https://doi.org/10.1371/journal.ppat.1009141, which shows a 15-fold preference for integration in expressed genes and no evidence of clustering of sites (as seen in expressed genes) in non-expressed DNA. Further, as far as I can tell, they present no examples in the Results section of R-loops in non-expressed DNA serving as integration targets.

(b) Figure 1: Demonstrates models for HIV infections in both cell lines and primary human CD4+ T cells. R-loop formation was determined through a method called DRIPc-seq which utilizes an antibody specific for DNA-RNA hybrid structures and sequences these regions of the genome using RNaseH treatment to show that when RNA-DNA hybrids are absent then no R-loops are detected. In these models of in vitro and ex vivo infection, the authors show that R-Loop formation increases following HIV infection between 6 hour post-infection and 12 hours post-infection, depending on the cell model. However, these figures lack a mock-infected control for each cell model to assess R-loop formation at the same time points. They would also benefit from a control showing that virus entry is necessary, such as omitting the VSV G protein donor.

Additionally, they use intracellular staining to confirm DRIPc-Seq results, by demonstrating an increase in R-loop formation at 6 hours post-infection in HeLa cells. It would have been more relevant to use primary T cells for this assay, but HeLa cells probably provided easier and clearer imaging.

(c) Figure 2: This figure shows that cells infected with HIV show more R-loops as well as longer sequences containing R-loop structures. Panel B shows that these R-loops were distributed throughout different genomic features, such as both genic and intergenic regions of the genome. However, the data are presented in such a way that it is impossible to determine the proportion of R-loops in each type of genomic feature. The reader has no way to tell, for example, the proportion of R-loops in genic vs intergenic DNA and how this value changes with time. Furthermore, increased R-loop formation due to HIV infection showed poor correlation with gene expression, suggesting that R-loops were not forming due to transcriptional activation, although the difference between 0 and the remaining time points is not apparent, nor is the meaning of the absurd p values.

(d) Figure 3: This figure shows the use of cell lines carrying R-loop inducible (mAIRN) or non-inducible (ECFP) genes to model the association of HIV integration with R-loop structures. The authors demonstrate the functional validation of R-loop induction in the cell line model. Additionally, when R-loops are induced there is a significant increase in HIV integration in the R-loop forming vector sequence when R-loops are induced with doxycycline. This result shows a correlation between expression and integration that is much stronger in the R-loop forming gene than in the unreferenced ECFP gene but does not prove that integration directly targets R-loops. It is possible, for example, that some features of the DNA sequence, such as base composition affect both integration and R-loop formation independently. As described more fully below, there is also a serious concern regarding the method used to quantify the integration frequencies.

(e) Figure 4: This figure shows evidence of increased HIV integration within regions of the genome containing R-loops with an additional preference for integration within the R-loop and a decrease in frequency of integration further from the R-loop. Identifying a preference for R-loops is very intriguing but the authors do also demonstrate that integration does occur when R-loops are not present. Also Panel A, which shows that regions of cell DNA that form R-loops have a higher frequency of Integration sites than those that do not, should also be controlled for the level of gene expression of the two types of region.

(f) Figure 5: In this figure, the authors demonstrate that HIV integrase binds to R-loops through a number of protein assays, but does not show that this binding is associated with enzymatic activity. ESMA of integrase identified increased binding to DNA-RNA over dsDNA. Additionally, precipitation of RNA-DNA hybrids pulled down HIV integrase. A proximity ligation assay detecting R-loops and HIV-integrase showed co-localization within the nucleus of HeLa cells. HeLa cells were probably used due to their efficiency of transduction but are not physiologically relevant cell types.

(g) Discussion: In the discussion, the authors address how their work relates to previous evidence of HIV integration by association of LEDGF/p75 and CPSF6. They also cite that LEDGF/p75 has possible R-loop binding capabilities. They also discuss what possible mechanisms are driving increases in R-loop formation during HIV infection, pointing to possible HIV accessory proteins. They also state that how HIV integrates in transcriptionally silent regions is still unknown but do point out that they were able to show R-loops appear in many different regions of the genome but did not show that R-loops in transcriptional inactive regions are integration targets. More seriously, they failed to make a connection between their work and the current understanding of the biochemical and structural mechanism of the integration reaction.

A dação é operada com bem imóvel apenas.

Nesse sentido:

Caso o Código Tributário Nacional tivesse ido além, autorizando a dação em pagamento não apenas em bens imóveis, mas também em bens móveis, o novo dispositivo provavelmente seria declarado inconstitucional nesta parte. Isso porque, o Supremo Tribunal Federal, antes da edição da Lei Complementar nº 104/2001, no julgamento da ADI 1.917, entendeu que a dação em pagamento em bens móveis implicava em ofensa ao princípio da licitação, insculpido no inciso XXI do artigo 37 da Constituição Federal

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Observe que, para maior parte da doutrina, o dispositivo do art. 141 também indica que as hipóteses de suspensão, extinção e exclusão do crédito tributário são taxativas, não podendo ser ampliadas por meio de lei ordinária. Esse foi, inclusive, o entendimento adotado pelo Supremo Tribunal Federal quando enfrentou o tema nas primeiras oportunidades. Posteriormente, no entanto, ao julgar a Medida Cautelar na ADI 2405, a Corte modificou sua posição, entendendo como possível que um Estado criasse uma nova modalidade de extinção – dação em pagamento - até então não prevista no texto do Código Tributário Nacional. O Argumento se fundou na seguinte premissa: se o Estado pode o mais, que é conceder a remissão (perdoar a dívida), também pode o menos, que seria aceitar formas alternativas de pagamento.

Ação direta de inconstitucionalidade: medida cautelar: L. estadual (RS) 11.475, de 28 de abril de 2000, que introduz alterações em leis estaduais (6.537/73 e 9.298/91) que regulam o procedimento fiscal administrativo do Estado e a cobrança judicial de créditos inscritos em dívida ativa da fazenda pública estadual, bem como prevê a dação em pagamento como modalidade de extinção de crédito tributário. I - Extinção de crédito tributário criação de nova modalidade (dação em pagamento) por lei estadual: possibilidade do Estado-membro estabelecer regras específicas de quitação de seus próprios créditos tributários. Alteração do entendimento firmado na ADInMC 1917-DF, 18.12.98, Marco Aurélio, DJ 19.09.2003: conseqüente ausência de plausibilidade da alegação de ofensa ao art. 146, III, b, da Constituição Federal, que reserva à lei complementar o estabelecimento de normas gerais reguladoras dos modos de extinção e suspensão da exigibilidade de crédito tributário. [...] (ADI 2405 MC, Relator(a):  Min. CARLOS BRITTO, Relator(a) p/ Acórdão:  Min. SEPÚLVEDA PERTENCE, Tribunal Pleno, julgado em 06/11/2002, DJ 17-02-2006 PP-00054 EMENT VOL-02221-01 PP-00071 LEXSTF v. 28, n. 327, 2006, p. 14-56)

Comment by reviewer_SorenPold: Both the article in itself and its design in DJMP raise questions about the architecture and materiality of the book and publishing, including academic publishing, through its discussion of artist books and open access. The interesting discussion is of course how ways of publishing, textual formats, ways of writing, editing and reading relate to different kinds of politics, e.g. institutional, economic, ways of ascertaining quality, etc. These are very important questions, both in global politics (e.g. the discussions on ‘fake news’ and its relation to social media), in institutional politics (e.g. the standards and quality assessment of academic publishing) and in art and literature (e.g. whether readers are able and willing to actually read and understand different forms of texts). In general, it is a question of how the text mediates and transforms the reading, how meaning is produced and how/whether it reaches an audience, whether it is productive of e.g. meaning, knowledge and/or action. It is a discussion of the text between mediator and tool.

It is noteworthy how little has happened after several decades of digital publishing and a plethora of death sentences for books and print: Even though some things have changed and are changing e.g. WWW’s ‘non-linear’ and labyrinthine, multi-cursal (Aarseth 1997) hypertext and the collaborative writing tools and platforms like wikis and social media are part of our everyday textual culture, we still have books and journals. Why? Is it because, as Stuart Moulthrop suggested already in 1991, that although hypertext affords new visions about a shared writing space, the responsibility for changes of this magnitude come from a diverse elite (of software developers, literary theorists, legislators, capitalists) who despite their differences remain allegiant to the institutions of intellectual property (the book, the library, the university, the publishing house). In other words, Moulthrop suggests that “it seems equally possible that engagement with interactive media will follow the path of reaction, not revolution.” (Moulthroup 2003 (1991), Andersen and Pold 2014). Is it because of institutional conservatism, because readers are conservative and slowly adapting (as the rather slow development of hypertext seems to suggest), is it a political battle (as the current discussions of the role of digital media, social media versus traditional media might suggest)? And to which extend is it a battle we should go for, if it includes breaking down the kinds of authority that comes with established publication formats and editorial processes (at least the current political climate raises some concerns).

I know that many of these concerns are afterthoughts to an article and a design done before the current situation, and in this sense, they are more reflections that might be relevant for further work. However, the questions remain, whether hypertext and collaborative authoring always leads to more freedom and productive reading/writing? Whether deconstructing the order of the text and its extended argument is always a good thing? We have of course examples of great hypertextual formats that function well as tools and presentation of knowledge, e.g. the encyclopedia, but maybe there are also good reasons to preserve the extended argument of the book and the article? Today it seems simply wrong to assert that "hypertext does not permit a tyrannical, univocal voice” (Landow 1992) faced with Trump and Wilders’ tweets. Consequently, I think, the argument of the article and its design could relate to the history of hypertext and electronic literature, though the discussion of artist books and open access publishing is also relevant.

The implementation in DJMP is in many ways exemplary and manages to present the article in nice ways, including the posters, the ability to comment and follow keywords. It allows its reader to access and use the text in different ways, and gives the valuable possibility of commenting and reading other people’s comments. In this it also follows paths from hypertext and electronic literature/digital culture, e.g. Electronic Book Review of A Peer Reviewed Journal About_. The design in many ways affords that it can do as it ‘preaches’, and in this way experiments with different ways of publishing academic texts. This is needed and current academia is not open enough to these kinds of experiments, that are, as argued, much more than making open access a homogeneous project – there is a need for an ongoing critical struggle that includes the forms of publishing. This is necessary, also to reach the popular masses on Twitter and Facebook! Currently, it is a problem, that standardizations within academia driven by STEM standards does not invite for such experiments that would in many cases not even be accepted as examples of academic publication. Also, I want to finish emphasizing that my discussion above is mainly stirred by the qualities of the publication, the important questions and reflections it raises.

References

Andersen, C. U. and S. B. Pold (2014). "Post-digital Books and Disruptive Literary Machines: Digital Literature Beyond the Gutenberg and Google Galaxies." Formules 2014(18): 164-183.<br /> Landow, G. P. (1992). Hypertext the convergence of contemporary critical theory and technology. Baltimore, Md, Johns Hopkins University Press.<br /> Moulthroup, S. (2003 (1991)). You Say You Want a Revolution. The New Media Reader. N. W.-F. N. Montfort. Cambridge, Massachusetts & London, England, The MIT Press: 691-704. <br /> Aarseth, E. J. (1997). Cybertext perspectives on ergodic literature. Baltimore, Md., Johns Hopkins University Press.

Comment by KamilaKuc: 'Altered books tap into our collective heightened interest in books as objects. Physical books, as differentiated from digital versions, tend to trigger memories, both visual and tactile.' (Kuhn, 2013: 11). The question of what will be left behind of the digital is a curious one here, perhaps. While we know what film and a physical book leave behind, the traces of their existence are still present (film strip, video tape, paper), what will be left behind of digital works (books)? What traces do digital forms leave behind?

Comment by KamilaKuc: See the idea of 'altered books', or bookworks, as defined by Doug Beube, as presented in 'Art Made From Books' by Alyson Kuhn, which looks at the conceptual underpinnings of artists books but also art made form books whereby the physical material of the book functions as a material and a platform to exercise ideas.

Comment by KamilaKuc: For the changing guises and forms of a book, see The Book Is Alive blog, which displays book 'as an evolving, open and visual medium' that is curated and alive, thus its shape and content can change.

Comment by KamilaKuc: For the changing guises and forms of a book, see The Book Is Alive blog, which displays book 'as an evolving, open and visual medium' that is curated and alive, thus its shape and content can change.

Comment by friedelitis: This seems like a much more cautious approach compared to the ones before. I would also argue that not all researchers and academics alike are prone (or even exposed to) to the idea of experimentation (see the points on STEM vs HSS made before). Already complex topics could be mystified and therefore not taken seriously in their findings and arguments. This discussion is in danger of becoming a pointless "either or" discussion where it can and should probably be complimentary (and I see how you argue for that).

Comment by friedelitis: I see the point - how would you build the case that the reiteration and reinforcement of established structures is a bad thing, though? Is there an argument to be made that questions around authorship, authority, legitimacy, etc., need to be rethought beyond the idea that (academic) work should be openly accessible to the public? And isn't the idea of linking and rethinking even more powerful once more work is openly available?

Comment by friedelitis: I wonder if this imperative of materiality is not derived from the fact that it is still called a 'book'. A book is in itself regarded to be a (physically) closed, comprehensive representation of a story, a narrative or a question. From a reader's perspective, a book is supposed to 'make sense' on its own, without necessary links to another publication or format. Calling OA books 'books' does therefore not seem helpful if you want to foster a more experimental use of the book.

I would like to further extend this practice-based project both theoretically and practically, by discussing the genealogy and correlations of ‘performative publishing’ with ideas such as ‘technotext’ (Hayles), ‘performative materiality’ (Drucker) and ‘liberature’ (Fajfer), alongside other projects and practices. As part of this I would like to explore the ethical and political challenges towards academic publishing these kind of concepts and practices pose. By using hypothes.is—an open source software/browser extension that enables an annotation layer on top of websites and online files and objects—which for this special disrupted issue of the Journal of Media Practice functions as a way to enable conversations around its processual papers, I would like to draw in these conversations around performative publications by directly setting up a dialogue with various theorists and the works, concepts, practices and values that connect to both this project and to performative publications as I envision them more in general.

What is certain is that poetics in general, and narratology in particular, must not limit itself to accounting for existing forms or themes. It must also explore the field of what is possible or even impossible without pausing too long at that frontier the mapping out of which is not its job. Until now, critics have done no more than interpret literature. Transforming is now the task at hand. That is certainly not the business of theoreticians alone; their role is no doubt negligible. Still, what would theory be worth if it were not also good for inventing practice?

(Genette 1988, 157)

Comment by KamilaKuc: Here the notion of a design as a political tool is also crucial. From the Constructivist practices onwards, the question of how design comments on and engages with contemporary life is definitely manifested in this project as well as in corresponding practices such as Photomediations: An Open Book.. How does the content one wants to present/communicate to the audiences fit the format in which this information is presented/accessed seem to be the key questions.

Comment by KamilaKuc: See for example an experiment concerning gestures of reading and writing, 'unruly gestures.' 'unruly gestures: seven cine-paragraphs on reading/writing practices in our post-digital condition' is a performative essay for 'Shifting Layers. New Perspectives in Media Archaeology Across Digital Media and Audiovisual Arts' edited by Miriam De Rosa and Ludovica Fales (Mimesis International, 2016). In it we aspire to break down preconceptions about gestures of reading/writing that relate to their agency, media-specificity, (linear) historicity and humanism. Informed by Tristan Tzara’s cut-up techniques, where through the gesture of cutting the Dadaists tried to subvert established traditions of authorship, intentionality and linearity, this visual essay has been cut-up into seven semi-autonomous cine-paragraphs, accompanied by text.

Comment by KamilaKuc: It is this idea of critically thinking through making, working through the ideas by employing both critical thinking and making practices and all the processes that are involved in it.

If performative publications are the material expressions or incarnations of specific research projects and processes, entangled with them are various other agencies of production and constraint (i.e. technological, authorial, cultural and discursive agencies, to name just a few). What I want to argue is that performative publications as a specific subset of publications actively interrogate how to align more closely the material form of a publication with its content (in other words, where all publications are performative—i.e. they are knowledge shaping, active agents involved in knowledge production—not all publications are 'performative publications', in the sense that they actively interrogate or experiment with this relation between content and materiality —similar to artist books). Yet in addition to this there is also an openness towards the ongoing interaction between materiality and content which includes entanglements with other agencies, and material forms of constraint and possibility.

This concern for the materiality and form of our publications (and directly related to that the material production and political economy that surrounds a publication) is not a response to what elsewhere as part of a critique of certain tendencies within the field of new materialism is seen as a reaction to ‘the linguistic turn’ (Bruining 2013). On the contrary, I see this as a more direct reaction against perspectives on the digital which perceive digital text as disembodied and as a freeing of data from its material constraints as part of a conversion to a digital environment. However, content cannot be separated that easily from its material manifestations, as many theorist within the digital humanities have already argued (i.e. Hayles, Drucker). Alan Liu classifies this 'database' rhetoric of dematerialization as a religion that is characterised by 'an ideology of strict division between content and presentation' where content is separated from material instantiation or formal presentation as part of an aesthetics of network production and consumption (Liu 2004, 62).

Scholarly poethics is what connects the 'doing' of scholarship with the ethical components of research. Here, ethics and poetics are entangled and an ethical engagement is already from the start involved in the production of scholarship, it informs our scholarship. Whilst formulating a narrative around the idea of a scholarly poetics—what it would look like, what it could mean, imply and do and, perhaps most importantly, what it could potentially achieve—in relation to our publishing practices, I want to argue that we should pay more attention to how we craft our own poetics as scholars.

Just as we have internal discussions about the contents of our scholarship, about the methodologies, theories and politics we use to give meaning and structure to our research, we should similarly have these kinds of discussions about the way we do research. Thus we should also be focusing on the medial forms, the formats and the graphic space in and through which we communicate and perform scholarship (and the discourses that surround these), as well as the structures and institutions that shape and determine our scholarly practices. This ‘contextual’ discussion, focusing on the materiality of our (textual) scholarship and its material modes of production, is and should not in any way be separate from a discussion on the contents of our work. The way we do scholarship informs its ‘outcomes’, what scholarship looks like. It informs the kinds of methodologies, theories and politics we can choose from, and of course, vice versa, these again shape the way we perform our scholarship. A focus on scholarly poethics might therefore be useful in bridging the context/content divide.

So what then is the altered status of a (digital) scholarly poethics today? Which theoretical streams, disciplinary fields, and schools of thought (inside and outside of academia, connecting the arts and the humanities) have specifically incorporated attention to the practices and performances of scholarship and this internal/external divide? Here it would be useful to look to fields such as design, poetry, science and technology studies (STS), feminist theory, the (radical) open access movement, and—in some instances the digital humanities and in cultural and literary studies—where the way we conduct scholarship can be seen to have been at the forefront of academic inquiries. What can we learn from these discussions and how can we add to and expand them to enrich our understanding of what a scholarly poethics could be(come)? As I envision it a scholarly poethics is not one thing, not a specific prescriptive methodology or way of doing scholarship, it is a plural and evolving process in which content and context co-develop. Scholarly poethics thus focuses on the abundant, and continuously changing material-discursive attitudes towards scholarly practices, research, communication media (text/film/audio) and institutions.

Comment by KamilaKuc: A number of recently curated sources explore this idea in a similar manner. See for example Photomediations: An Open Book.

As a term, performative publications have a lot in common with Katherine Hayles’s concept ‘technotexts’. In her book Writing Machines (itself a technotext, beautifully designed by Anne Burdick in a hybrid print and ‘webtake’ version) Hayles introduces the term technotext as an relative and alternative to concepts such as hypertext and cybertext. She defines a technotext as something that comes about ‘when a literary work interrogates the inscription technology that produces it’ (Hayles 2002, 25) and elsewhere as ‘a book that embodies its own critical concepts (Hayles 2002, 140)’. In Writing Machines Hayles then goes on to analyse 3 technotexts, Talan Memmott’s work of electronic literature Lexia to Perplexia (2000), Tom Phillips artist’s book A Humument (1970), and Mark Z. Danielewski’s novel House of Leaves (2000).

For Long performative publications are directly connected to the idea of practice, where following the concept of performativity, he argues that ideas should be put to practice, where practice can further inform and enrich ones ideas again. Long applies these values directly to several of his own performative projects. In his book The Socratic and Platonic Politics: Practicing a Politics of Reading, he shows how Socratic philosophy and Platonic writing was designed to cultivate dialogue and community. By digitally enhancing his publication, Long explores how writing and reading can promote community in a digital context, in specific a community of collaborative readers. As Long argues:

If, however, the book is not to be a mere abstract academic exercise, it will need to be published in a way that performs and enables the politics of collaborative reading for which it argues. (Long 2012)

https://youtu.be/-f9N1n-4cI8

A further extension of this project is a podcast series titled Digital Dialogue which aims to cultivate dialogue in a digital age by engaging other scholars in open conversation online. Long is also involved in the Public Philosophy Journal project, which is specifically set up to crawl the web to find diverse positions on various philosophical subjects and to bring these together in a collaborative writing setting. As Long explains:

The PPJ is designed to crawl the web, listening for conversations in which philosophical ideas and approaches are brought to bear on a wide variety of issues of public concern. Once these conversations are curated and a select number chosen for further development, we will invite participants into a space of collaborative writing so they can work their ideas up into a more fully formulated scholarly article or digital artifact. (Chris Long 2013)

Fajfer and Bazarnik make some interesting observations on how in liberature the book does not contain the work, it is the work. In this sense they don’t see the material book as a representation of the work but as something that actively shapes and determines the work.

Their focus on liberatic works is both a reaction to a previous literary context and a plea to authors to take responsibility for the future becoming of literature. First of all, as a specific response in a Polish context (but more wider too), it rallies against literary traditions that see the materiality of the book as non-significant, that classify literature as ‘disembodied’. As Bazarnik and Fajfer state:

If I emphasise this bodily, material aspect so much, it is because Polish literary studies seem still dominated by scholars indebted to Roman Ingarden, a Polish philosopher who ventured into literary studies to produce a highly influential theory of the literary work of art in which he denied its “material foundation” (as he called it) any significance. It was to be passed over and not interfere with reading (Fajfer and Bazarnik 2010).

Secondly, they present liberature as a way out of the ‘crisis of contemporary literature’, which they say has its roots in the continued focus on the text and its meaning, while neglecting the physical shape and structure of the book. This is delimiting the creative possibilities for the author, they claim. As Fajfer writes:

I believe that it is his responsibility to consider the physical shape of the book and all the matters entailed, just as he considers the text (if not to the same extent, he should at least bear them in mind). The shape of the book should not be determined by generally accepted conventions but result from the author’s autonomous decision just as actions of his characters and the choice of words originate from him (Fajfer 2010, 25).

Liberature is a term, concept and genre coined in 1999 by the Polish avant-garde poet Zenon Fajfer, and further developed by his collaborator: literary scholar and theorist Katarzyna Bazarnik. Liberature is literature in the form of the book. Bazarnik and Fajfer define liberature as ‘a literary genre that integrates text and its material foundation into a meaningful whole' (Bazarnik and Fajfer 2010, 1). In the introduction to Fajfer’s collected essays, Bazarnik describes liberature as literary works in which the artistic message is transmitted not only through the verbal medium, but also through the author ‘speaking’ via the book as a whole (Bazarnik 2010, 7). Liberature is therefore a total approach that reaches beyond the linguistic medium, where the material form of the work is essential to its understanding and forms an organic element of the (inseparable) whole. Both Fajfer and Bazarnik emphasise that in liberature, the material book is no longer a neutral container for a text, but becomes an integral component of the literary work.

Katarzyna Bazarnik, Zenon Fajfer, Oka-leczenie [Eyes-ore] (2000), Liberatura vol. 8, Kraków: Korporacja Ha!art, 2009.

I would like to further extend this practice-based project, both theoretically and practically, by discussing the genealogy and correlations of ‘performative publishing’ with ideas such as ‘technotext’ (Hayles), ‘performative materiality’ (Drucker) and ‘liberature’ (Fajfer), and the ethical and political challenges towards academic publishing these kind of concepts and practices pose.

This article for The disrupted Journal of Media Practice focuses on performative publications and is itself at the same time a performative publication. Written in Hypothes.is this article will hinge upon specific aspects, fragments, and concepts of the original performative project that it engages, entangling the community’s engagements along the way.

eLife assessment

This valuable work investigates the role of boundary elements in the formation of 3D genome architecture. The authors established a specific model system that allowed them to manipulate boundary elements and examine the resulting genome topology. The work yielded the first demonstration of the existence of stem and circle loops in a genome and confirms a model which had been posited based on extensive prior genetic work, providing insights into how 3D genome topologies affect enhancer-promoter communication. The evidence is solid, although the degree of generalization remains uncertain.

Reviewer #1 (Public Review):

In this study, the authors engineer the endogenous left boundary of the Drosophila eve TAD, replacing the endogenous Nhomie boundary by either a neutral DNA, a wildtype Nhomie boundary, an inverted Nhomie boundary, or a second copy of the Homie boundary. They perform Micro-C on young embryos and conclude that endogenous Nhomie and Homie boundaries flanking eve pair with head-to-tail directionality to form a chromosomal stem loop. Abrogating the Nhomie boundary leads to ectopic activation of genes in the former neighboring TAD by eve embryonic stripe enhancers. Replacing Nhomie by an inverted version or by Homie (which pairs with itself head-to-head) transformed the stem loop into a circle loop. An important finding was that stem and circle loops differentially impact endogenous gene regulation both within the eve TAD and in the TADs bracketing eve. Intriguingly, an eve TAD with a circle loop configuration leads to ectopic activation of flanking genes by eve enhancers - indicating compromised regulatory boundary activity despite the presence of an eve TAD with intact left and right boundaries.

The results obtained are of high-quality and are meticulously discussed. This work advances our fundamental understanding of how 3D genome topologies affect enhancer-promoter communication.

This study raises interesting questions to be addressed in future studies.

First, given the unique specificity with which Nhomie and Homie pair (and exhibit "homing" activity), the generalizability of TAD formation by directional boundary pairing remains unclear. Testing whether boundary pairing is a phenomenon restricted to exceptional loci picked for study, rather than a broader rule of TAD formation, would best be done through the development of untargeted approaches to study boundary pairing.

Second, boundary pairing is one of several mechanisms that may form chromosomal contact domains such as TADs. Other mechanisms include cohesin-mediated chromosomal loop extrusion and the inherent tendency of transcriptionally active and inactive chromatin to segregate (or compartmentalize). The functional interplay between these possible TAD-forming mechanisms remains to be further investigated.

Reviewer #2 (Public Review):

This study reports a set of experiments and subsequent analyses focusing on the role of Drosophila boundary elements in shaping 3D genome structure and regulating gene expression. The authors primarily focus on the region of the fly genome containing the even skipped (eve) gene; eve is expressed in a canonical spatial pattern in fly embryos and its locus is flanked by the well-characterized neighbor of homie (nhomie) and homie boundary elements. The main focus of the investigation is the orientation dependence of these boundary elements, which had been observed previously using reporter assays. In this study, the authors use Crispr/Cas9 editing followed by recombination-mediated cassette exchange to create a series of recombinant fly lines in which the nhomie boundary element is either replaced with exongenous sequence from phage 𝝀, an inversion of nhomie, or a copy of homie that has the same orientation as the endogenous homie sequence. The nhomie sequence is also regenerated in its native orientation to control for effects introduced by the transgenesis process.

The authors then perform high-resolution Micro-C to analyze 3D structure and couple this with fluorescent and colorimetric RNA in situ hybridization experiments to measure the expression of eve and nearby genes during different stages of fly development. The major findings of these experiments are that total loss of boundary sequence (replacement with 𝝀 DNA) results in major 3D structure changes and the most prominent observed gene changes, while inversion of the nhomie boundary or replacement with homie resulted in more modest effects in terms of 3D structure and gene expression changes and a distinct pattern of gene expression change from the 𝝀 DNA replacement. As the samples in which the nhomie boundary is inverted or replaced with homie have similar Micro-C profiles at the eve locus and show similar patterns of a spurious gene activation relative to the control, the observed effects appear to be driven by the relative orientation of the nhomie and homie boundary elements to one another.

Collectively, the findings reported in the manuscript are of broad interest to the 3D genome field. Although extensive work has gone into characterizing the patterns of 3D genome organization in a whole host of species, the underlying mechanisms that structure genomes and their functional consequences are still poorly understood. The perhaps best understood system, mechanistically, is the coordinated action of CTCF with the cohesin complex, which in vertebrates appears to shape 3D contact maps through a loop extrusion-pausing mechanism that relies on orientation-dependent sequence elements found at the boundaries of interacting chromatin loops. Despite having a CTCF paralog and cohesin, the Drosophila genome does not appear to be structured by loop extrusion-pausing. The identification of orientation-dependent elements with pronounced structural effects on genome folding thus may shed light on alternative mechanisms used to regulated genome structure, which in turn may yield insights into the significance of particular folding patterns.

On the whole, this study is comprehensive and represents a useful contribution to the 3D genome field. The transgenic lines and Micro-C datasets generated in the course of the work will be valuable resources for the research community. Moreover, the manuscript, while dense in places, is generally clearly written and comprehensive in its description of the work. However, I have a number of comments and critiques of the manuscript, mainly centering on the framing of the experiments and presentation of the Micro-C results and on the manner in which the data are analyzed and reported.

As this document now reflects my review of a revised version of the initial preprint, I will begin to add the new content at this point. As discussed in detail in the following paragraphs, my initial impression of the manuscript has not changed, so I have accordingly left the above text unaltered.

In my initial review, I provided a number of suggestions to improve the quality of the manuscript. These suggestions, which took the form of six major and three minor points, largely focused on 1) altering the writing in certain places to make the story more broadly accessible to the readership and 2) the inclusion of key, missing methodological detail to increase the rigor and reproducibility of the study. No new experiments were requested, and all of the points could be readily addressed with rather straightforward textual changes.

In their revised manuscript, the authors elected to directly address one of the major points and two of the minor points (major point 4, minor points 1 and 3). The remainder of my suggestions remain entirely unaddressed. A similar level of responsiveness was afforded to the very reasonable critiques of the other Reviewer and the Reviewing Editor. The authors have instead largely chosen to respond to the points raised exclusively in the rebuttal document. This document sprawls across >22 pages, includes numerous in-line figures, and cites dozens of references. The tone of this document, in many places, is at best forceful. In a less generous interpretation, many sections are combative, dismissive, and borderline unprofessional.

It is my opinion that the authors are doing the scientific community a disservice with their response. While it is my understanding that readers will be able see the rebuttal letter, I find that end result far from satisfying. How many readers will take the trouble to access that file, versus the manuscript itself? Skirting the review critiques places an unfair burden on readers, who are expecting peer-reviewed science, to dig into the accessory files to follow the critique and response, rather than seeing in reflected in the final product as they accustomed. Intentionally or not, the tactics the authors have chosen detract from what is otherwise a novel and well-intentioned new publishing model. It is also worth pointing out that peer review is done as an act of service to the scientific community, as the senior authors are doubtless aware. The other reviewer, the Reviewing Editor, and I have all taken time away from advancing our own careers and those of our trainees to offer the thoughtful critiques that were so pointedly dismissed.

In summary, as the vast majority of my critiques remain unaddressed, I have simply reproduced them below.

Major Points:

(1) The authors motivate much of the introduction and results with hypothetical "stem loop" and "circle loop" models of chromosome confirmation, which they argue are reflected in the Micro-C data and help to explain the observed ISH patterns. While such structures may possibly form, the support for these specific models vs. the many alternatives is not in any way justified. For instance, no consideration is given to important biophysical properties such as persistence length, packing/scaling, and conformational entropy. As the biophysical properties of chromatin are a very trafficked topic both in terms of experimentation and computational modeling and generally considered in the analysis of chromosome conformation data, the study would be strengthened by acknowledgement of this body of work and more direct integration of its findings.

(2) Similar to Point 1, while there is a fair amount of discussion of how the observed results are or are not consistent with loop extrusion, there is no discussion of the biophysical forces that are thought to underly compartmentalization such as block-polymer co-segregation and their potential influence. I found this absence surprising, as it is generally accepted that A/B compartmentalization essentially can explain the contact maps observed in Drosophila and other non-vertebrate eukaryotes (Rowley, ..., Corces 2017; PMID 28826674). The manuscript would be strengthened by consideration of this phenomenon.

(3) The contact maps presented in the study represent many cells and distinct cell types. It is clear from single-cell Hi-C and multiplexed FISH experiments that chromosome conformation is highly variable even within populations of the same cell, let alone between cell types, with structures such as TADs being entirely absent at the single cell level and only appearing upon pseudobulking. It is difficult to square these observations with the models of relatively static structures depicted here. The authors should provide commentary on this point.

(4) Related to Point 4, the lack of quantitative details about the Micro-C data make it difficult to evaluate if the changes observed are due to biological or technical factors. It is essential that the authors provide quantitative means of controlling for factors like sampling depth, normalization, and data quality between the samples.

(5) The ISH effects reported are modest, especially in the case of the HCR. The details provided for how the imaging data were acquired and analyzed are minimal, which makes evaluating them challenging. It would strengthen the study to provide much more detail about the acquisition and analysis and to include depiction of intermediates in the analysis process, e.g. the showing segmentation of stripes.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, the authors engineer the endogenous left boundary of the Drosophila eve TAD, replacing the endogenous Nhomie boundary by either a neutral DNA, a wildtype Nhomie boundary, an inverted Nhomie boundary, or a second copy of the Homie boundary. They perform Micro-C on young embryos and conclude that endogenous Nhomie and Homie boundaries flanking eve pair with head-to-tail directionality to form a chromosomal stem loop. Abrogating the Nhomie boundary leads to ectopic activation of genes in the former neighboring TAD by eve embryonic stripe enhancers. Replacing Nhomie by an inverted version or by Homie (which pairs with itself head-to-head) transformed the stem loop into a circle loop. An important finding was that stem and circle loops differentially impact endogenous gene regulation both within the eve TAD and in the TADs bracketing eve. Intriguingly, an eve TAD with a circle loop configuration leads to ectopic activation of flanking genes by eve enhancers - indicating compromised regulatory boundary activity despite the presence of an eve TAD with intact left and right boundaries.

Strengths:

Overall, the results obtained are of high-quality and are meticulously discussed. This work advances our fundamental understanding of how 3D genome topologies affect enhancer-promoter communication.

Weaknesses:

Though convincingly demonstrated at eve, the generalizability of TAD formation by directional boundary pairing remains unclear, though the authors propose this mechanism could underly the formation of all TADs in Drosophila and possibly even in mammals. Strong and ample evidence has been obtained to date that cohesin-mediated chromosomal loop extrusion explains the formation of a large fraction of TADs in mammals. 

(1.1) The difficultly with most all of the studies on mammal TADs, cohesin and CTCF roadblocks is that the sequencing depth is not sufficient, and large bin sizes (>1 kb) are needed to visualize chromosome architecture.  The resulting contact profiles show TAD neighborhoods, not actual TADs.

The problem with these studies is illustrated by comparing the contact profiles of mammalian MicroC data sets at different bin sizes in Author response image 1.  In this figure, the darkness of the “pixels” in panels E, F, G and H was enhanced by reducing brightness in photoshop.

Author response image 1.

Mammalian MicroC profiles different bun sizes

Panels A and C show “TADs” using bin sizes typical of most mammalian studies (see Krietenstein et al. (2023) (Krietenstein et al. 2020)).  At this level of resolution, TADs, the “trees” that are the building blocks of chromosomes, are not visible.  Instead, what is seen are TAD neighborhoods or “forests”.  Each neighborhood consists of several dozen individual TADs.  The large bins in these panels also artificially accentuated TAD:TAD interactions, generating a series of “stripes” and “dots” that correspond to TADs bumping into each other and sequences getting crosslinked.  For example, in panel A there is prominent stripe on the edge of a “TAD” (blue arrow).  In panel C, this stripe resolves into a series of dots arranged as parallel, but interrupted “stripes” (green and blue arrows).  At the next level of resolution, it can be seen that the stripe marked by the blue arrow and magenta asterisk is generated by contacts between the left boundary of the TAD indicated by the magenta bar with sequences in a TAD (blue bar) ~180 kb way.  While dots and stripes are prominent features in contact profiles visualized with larger bin sizes (A and C), the actual TADs that are observed with a bin size of 200 bp (examples are underlined by black bars in panel G) are not bordered by stripes, nor are they topped by obvious dots.  The one possible exception is the dot that appears at the top of the volcano triangle underlined with magenta.

The chromosome 1 DNA segment from the MicroC data of Hseih et al. (2023) (Hsieh et al. 2020) shows a putative volcano triangle with a plume (indicated by a V in Author response image 1 panels D, F and H).  Sequences in the V TAD don’t crosslink with their immediate neighbors, and this gives a “plume” above the volcano triangle, as indicate by the light blue asterisk in panels D, F and H.  Interestingly the V TAD does contact two distant TADs, U on the left and W on the right. The U TAD is ~550 kb from V, and the region of contact is indicated by the black arrow.  The W TAD is ~585 kb from V, and the region of contact is indicated by the magenta arrow.  While the plume still seems to be visible with a bin size of 400 bp (light blue asterisk), it is hard to discern when the bin size is 200 bp, as there are not enough reads.

The evidence demonstrating that cohesin is required for TAD formation/maintenance is based on low resolution Hi-C data, and the effects that are observed are on TAD neighborhoods (forests) and not TADs (trees).  In fact, there is published evidence that cohesin is not required in mammals for TAD formation/maintenance.  In an experiment from Goel et al. 2023 the authors depleted the cohesin component Rad21 and then visualized the effects on TAD organization using the high resolution region capture MicroC (RCMC) protocol.  The MicroC contact map in this figure visualizes a ~250 kb DNA segment around the Ppm1pg locus at 250 bp resolution.  On the right side of the diagonal is the untreated control, while the left side shows the MicroC profile of the same region after Rad21 depletion.  The authors indicated that there was a 97% depletion of Rad21 in their experiment.  However, as is evident from a comparison of the experimental and control, loss of Rad21 has no apparent effect on the TAD organization of this mammalian DNA segment.

Several other features are worth noting.  First, unlike the MicroC experiments shown in Author response image 1, there are dots at the apex of the TADs in this chromosomal segment.  In the MicroC protocol, fixed chromatin is digested to mononucleosomes by extensive MNase digestion.  The resulting DNA fragments are then ligated, and dinucleosome-length fragments are isolated and sequenced. 

DNA sequences that are nucleosome free in chromatin (which would be promoters, enhancers, silencers and boundary elements) are typically digested to oligonucleotides in this procedure and won’t be recovered. This means that the dots shown here must correspond to mononucleosome-length elements that are MNase resistant.  This is also true for the dots in the MicroC contact profiles of the Drosophila Abd-B regulatory domain (see Fig. 2B in the paper).  Second, the TADs are connected to each other by 45o stripes (see blue and green arrowheads).  While it is not clear from this experiment whether the stipes are generated by an active mechanism (enzyme) or by some “passive” mechanism (e.g., sliding), the stripes in this chromosomal segment are not generated by cohesin, as they are unperturbed by Rad21 depletion.  Third, there are no volcano triangles with plumes in this chromosomal DNA segment.  Instead, the contact patterns (purple and green asterisks) between neighboring TADs closely resemble those seen for the Abd-B regulatory domains (compare Goel et al. 2023 with Fig. 2B in the paper).  This similarity suggests that the TADs in and around Ppm1g may be circle-loops, not stem-loops.  As volcano triangles with plumes also seem to be rare in the MicroC data sets of Krietenstein et al. (Krietenstein et al. 2020) and Hesih et al. (Hsieh et al. 2020) (with the caveat that these data sets are low resolution: see Author response image 1), it is possible that much of the mammalian genome is assembled into circle-loop TADs, a topology that can’t be generated by the cohesin loop extrusion (bolo tie clip) /CTCF roadblock model.

While Rad21 depletion has no apparent effect on TADs, it does appear to impact TAD neighborhoods.  This is in a supplemental figure in Goel et al. (Goel et al. 2023).  In this figure, TADs in the Ppm1g region of chromosome 5 are visualized with bin sizes of 5 kb and 1 kb.  A 1.2 Mb DNA segment is shown for the 5 kb bin size, while an 800 kb DNA segment is shown for the 1 kb bin size.  As can be seen from comparing the MicroC profiles in Author response image 2 with that in Goel et al. 2023, individual TADs are not visible.  Instead, the individual TADs are binned into large TAD “neighborhoods” that consist of several dozen or more TADs.

Unlike the individual TADs shown in Goel et al. 2023, the TAD neighborhoods in Author response image 2 are sensitive to Rad21 depletion.  The effects of Rad21 depletion can be seen by comparing the relative pixel density inside the blue lines before (above the diagonal) and after (below the diagonal) auxin-induced Rad21 degradation.  The reduction in pixel density is greatest for more distant TAD:TAD contacts (farthest from the diagonal).  By contrast, the TADs themselves are unaffected (Goel et al. 2023), as are contacts between individual TADs and their immediate neighbors.  In addition, contacts between partially overlapping TAD neighborhoods are also lost.  At this point it isn’t clear why contacts between distant TADs in the same neighborhood are lost when Rad21 is depleted; however, a plausible speculation is that it is related to the functioning of cohesin in holding newly replicated DNAs together until mitosis and whatever other role it might have in chromosome condensation.

Author response image 2.

Ppm1g full locus chr5

Moreover, given the unique specificity with which Nhomie and Homie are known to pair (and exhibit "homing" activity), it is conceivable that formation of the eve TAD by boundary pairing represents a phenomenon observed at exceptional loci rather than a universal rule of TAD formation. Indeed, characteristic Micro-C features of the eve TAD are only observed at a restricted number of loci in the fly genome…..

(1.2) The available evidence does not support the claim that nhomie and homie are “exceptional.”  To begin with, nhomie and homie rely on precisely the same set of factors that have been implicated in the functioning of other boundaries in the fly genome.  For example, homie requires (among other factors) the generic boundary protein Su(Hw) for insulation and long-distance interactions (Fujioka et al. 2024).  (This is also true of nhomie: unpublished data.)  The Su(Hw) protein (like other fly polydactyl zinc finger proteins) can engage in distant interactions.  This was first shown by Sigrist and Pirrotta (Sigrist and Pirrotta 1997), who found that the su(Hw) element from the gypsy transposon can mediate long-distance regulatory interactions (PRE dependent silencing) between transgenes inserted at different sites on homologous chromosomes (trans interactions) and at sites on different chromosomes.

The ability to mediate long-distance interactions is not unique to the su(Hw) element, or homie and nhomie.  Muller et al. (Muller et al. 1999) found that the Mcp boundary from the Drosophila BX-C is also able to engage in long-distance regulatory interactions—both PRE-dependent silencing of mini-white and enhancer activation of mini-white and yellow.  The functioning of the Mcp boundary depends upon two other generic insulator proteins, Pita and the fly CTCF homolog (Kyrchanova et al. 2017).  Like Su(Hw) both are polydactyl zinc finger proteins, and they resemble the mammalian CTCF protein in that their N-terminal domain mediates multimerization (Bonchuk et al. 2020; Zolotarev et al. 2016).  Figure 6 from Muller et el. 1999 shows PRE-dependent “pairing sensitive silencing” interactions between transgenes carrying a mini-white reporter, the Mcp and scs’ (Beaf dependent)(Hart et al. 1997) boundary elements, and a PRE closely linked to Mcp.  In this experiment flies homozygous for different transgene inserts were mated and the eye color was examined in their transheterozygous progeny.  As indicated in the figure, the strongest trans-silencing interactions were observed for inserts on the same chromosomal arm; however, transgenes inserted on the left arm of chromosome 3 can interact across the centromere with transgenes inserted on the right arm of chromosome 3. 

Figure 5C (left) from Muller et el. 1999 shows a trans-silencing interaction between w#11.102 at 84D and w#11.16 approximately 5.8 Mb away, at 87D.  Figure 5C (right) shows a trans-silencing interaction across the centromere between w#14.29 on the left arm of chromosome 3 at 78F and w#11.102 on the right arm of chromosome 3 at 84D. The eye color phenotype of mini-white-containing transgenes is usually additive: homozygyous inserts have twice as dark eye color as the corresponding hemizygous inserts.  Likewise, in flies trans-_heterozygous for _mini-white transgenes inserted at different sites, the eye color is equivalent to the sum of the two transgenes.  This is not true when mini-white transgenes are silenced by PREs.  In the combination shown in panel A, the t_rans-_heterozygous fly has a lighter eye color than either of the parents.  In the combination in panel B, the _trans-_heterozygous fly is slightly lighter than either parent.

As evident from the diagram in Figure 6 from Muller et el. 1999, all of the transgenes inserted on the 3rd chromosome that were tested were able to participate in long distance (>Mbs) regulatory interactions.  On the other hand, not all possible pairwise interactions are observed.  This would suggest that potential interactions depend upon the large scale (Mb) 3D folding of the 3rd chromosome.

When the scs boundary (Zw5 dependent) (Gaszner et al. 1999) was added to the transgene to give sMws’, it further enhanced the ability of distant transgenes to find each other and pair.  All eight of the sMws’ inserts that were tested were able to interact with at least one other sMws’ insert on a different chromosome and silence mini-white.  Vazquez et al. () subsequently tagged the sMws’ transgene with LacO sequences (ps0Mws’) and visualized pairing interactions in imaginal discs.  Trans-heterozygous combinations on the same chromosome were found paired in 94-99% of the disc nuclei, while a trans-heterozygous combination on different chromosomes was found paired in 96% of the nuclei (Table 3 from Vazquez et al. 2006).  Vazquez et al. also examined a combination of four transgenes inserted on the same chromosome (two at the same insertion site, and two at different insertion sites).  In this case, all four transgenes were clustered together in 94% of the nuclei (Table 3 from Vazquez et al. 2006).  Their studies also suggest that the distant transgenes remain paired for at least several hours.  A similar experiment was done by Li et al. (Li et al. 2011), except that the transgene contained only a single boundary, Mcp or Fab-7.  While pairing was still observed in trans-heterozygotes, the frequency was reduced without scs and scs’.

It is worth pointing out that there is no plausible mechanism in which cohesin could extrude a loop through hundreds of intervening TADs, across the centromere (ff#13.101_ßà_w#11.102: Figure 6 from Muller et el. 1999; w#14.29_ßà_w#11.02: Figure 6 from Muller et el. 1999 and 5) and come to a halt when it “encounters” Mcp containing transgenes on different homologs.  The same is true for Mcp-dependent pairing interactions in cis (Fig. 7 in Muller et al. (Muller et al. 1999)) or Mcp-dependent pairing interactions between transgenes inserted on different chromosomes (Fig. 8 in Muller et al. (Muller et al. 1999); Line 8 in Table 3 from Vazquez et al. 2006). 

These are not the only boundaries that can engage in long-distance pairing.  Mohana et al. (Mohana et al. 2023) identified nearly 60 meta-loops, many of which appear to be formed by the pairing of TAD boundary elements.  Two examples (at 200 bp resolution from 12-16 hr embryos) are shown in Author response image 3.

Author response image 3.

Metaloops on the 2nd and 3rd chromosomes: circle-loops and multiple stem-loops

One of these meta-loops (panel A) is generated by the pairing of two TAD boundaries on the 2nd chromosome.  The first boundary, blue, (indicated by blue arrow) is located at ~2,006, 500 bp between a small TAD containing the Nplp4 and CG15353 genes and a larger TAD containing 3 genes, CG33543, Obp22a and Npc2a.  Nplp4 encodes a neuropeptide.  The functions of CG15354 and CG33543 are unknown.  Obp22a encodes an odorant binding protein, while Npc2a encodes the Niemann-Pick type C-2a protein which is involved sterol homeostasis.  The other boundary (purple: indicated by purple arrow) is located between two TADs 2.8 Mb away at 4,794,250 bp.  The upstream TAD contains the fipi gene (CG15630) which has neuronal functions in male courtship, while the downstream TAD contains CG3294, which is thought to be a spliceosome component, and schlaff (slf) which encodes a chitin binding protein.  As illustrated in the accompanying diagram, the blue boundary pairs with the purple boundary in a head-to-head orientation, generating a ~2.8 Mb loop with a circle-loop topology.  As a result of this pairing, the multi-gene (CG33543, Obp22a and Npc2a) TAD upstream of the blue boundary interacts with the CG15630 TAD upstream of the purple boundary.  Conversely the small Nplp4:CG15353 TAD downstream of the blue boundary interacts with the CG3294:slf TAD downstream of the purple boundary.  Even if one imagined that the cohesin bolo tie clip was somehow able to extrude 2.8 Mb of chromatin and then know to stop when it encountered the blue and purple boundaries, it would’ve generated a stemloop, not a circle-loop.

The second meta-loop (panel B) is more complicated as it is generated by pairing interactions between four boundary elements.  The blue boundary (blue arrow) located ~4,801,800 bp (3L) separates a large TAD containing the RhoGEF64C gene from a small TAD containing CG7509, which encodes a predicted subunit of an extracellular carboxypeptidase.  As can be seen in the MicroC contact profile and the accompanying diagram, the blue boundary pairs with the purple boundary (purple arrow) which is located at ~7,013, 500 (3L) just upstream of the 2nd internal promoter (indicated by black arrowhead) of the Mp (Multiplexin) gene.  This pairing interaction is head-to-tail and generates a large stem-loop that spans ~2.2 Mb.  The stem-loop brings sequences upstream of the blue boundary and downstream of the purple boundary into contact (the strings below a bolo tie clip), just as was observed in the boundary bypass experiments of Muravyova et al. (Muravyova et al. 2001) and Kyrchanova et al. (Kyrchanova et al. 2008).  The physical interactions result in a box of contacts (right top) between sequences in the large RhoGEF64C TAD and sequences in a large TAD that contains an internal Mp promoter.  The second pairing interaction is between the brown boundary (brown arrow) and the green boundary (green arrow).  The brown boundary is located at ~4 805,600 bp (3L) and separates the TAD containing CG7590 from a large TAD containing CG1808 (predicted to encode an oxidoreductase) and the Dhc64C (Dynein heavy chain 64C) gene.  The green boundary is located at ~6,995,500 bp (3L), and it separates a TAD containing CG32388 and the biniou (bin) transcription factor from a TAD that contains the most distal promoter of the Mp (Multiplexin) gene (blue arrowhead).  As indicated in the diagram, the brown and green boundaries pair with each other head-to-tail, and this generates a small internal loop (and the final configuration would resemble a bolo tie with two tie clips).  This small internal loop brings the CG7590 TAD into contact with the TAD that extends from the distal Mp promoter to the 2nd internal Mp promoter.  The resulting contact profile is a rectangular box with diagonal endpoints corresponding to the paired blue:purple and brown:green boundaries.  The pairing of the brown:green boundaries also brings the TADs immediately downstream of the brown boundary and upstream of the green boundary into contact with each other, and this gives a rectangular box of interactions between the Dhc64C TAD, and sequences in the bin/CG3238 TAD.  This box is located on the lower left side of the contact map.

Since the bin and Mp meta-loops in Author response image 3B are stem-loops, they could have been generated by “sequential” cohesin loop extrusion events.  Besides the fact that cohesin extrusion of 2 Mb of chromatin and breaking through multiple intervening TAD boundaries challenges the imagination, there is no mechanism in the cohesion loop extrusion/CTCF roadblock model to explain why cohesion complex 1 would come to a halt at the purple boundary on one side and the blue boundary on the other, while cohesin complex 2 would instead stop when it hits the brown and green boundaries.  This highlights another problem with the cohesin loop extrusion/CTCF roadblock model, namely that the roadblocks are functionally autonomous: they have an intrinsic ability to block cohesin that is entirely independent of the intrinsic ability of other roadblocks in the neighborhood.  As a result, there is no mechanism for generating specificity in loop formation.  By contrast, boundary pairing interactions are by definition non-autonomous and depend on the ability of individual boundaries to pair with other boundaries: specificity is built into the model. The mechanism for pairing, and accordingly the basis for partner preferences/specificity, are reasonably well understood.  Probably the most common mechanism in flies is based on shared binding sites for architectural proteins that can form dimers or multimers (Bonchuk et al. 2021; Fedotova et al. 2017).  Flies have a large family of polydactyl zinc finger DNA binding proteins, and as noted above, many of these form dimers or multimers and also function as TAD boundary proteins.  This pairing principle was first discovered by Kyrchanova et al. (Kyrchanova et al. 2008).  This paper also showed that orientation-dependent pairing interactions is a common feature of endogenous fly boundaries.  Another mechanism for pairing is specific protein:protein interactions between different DNA binding factors (Blanton et al. 2003).  Yet a third mechanism would be proteins that bridge different DNA binding proteins together.  The boundaries that use these different mechanisms (BX-C boundaries, scs, scs’) depend upon the same sorts of proteins that are used by homie and nhomie.  Likewise, these same set of factors reappear in one combination or another in most other TAD boundaries.  As for the orientation of pairing interactions, this is most likely determined by the order of binding sites for chromosome architectural proteins in the partner boundaries.

…and many TADs lack focal 3D interactions between their boundaries.

(1.3) The idea that flies differ from mammals in that they “lack” focal 3D interactions is simply mistaken.  One of the problems with drawing this distinction is that most all of the “focal 3D interactions” seen mammalian Hi-C experiments are a consequence of binning large DNA segments in low resolution restriction enzyme-dependent experiments.  This is even true in the two “high” resolution MicroC experiments that have been published (Hsieh et al. 2020; Krietenstein et al. 2020).  As illustrated above in Author response image 1, most of the “focal 3D interactions” (the dots at the apex of TAD triangles) seen with large bin sizes (1 kb and greater) disappear when the bin size is 200 bp and TADs rather than TAD neighborhoods are being visualized.

As described in point #1.1, in the MicroC protocol, fixed chromatin is first digested to mononucloesomes by extensive MNase digestion, processed/biotinylated, and ligated to give dinucleosome-length fragments, which are then sequenced.  Regions of chromatin that are nucleosome free (promoters, enhancers, silencers, boundary elements) will typically be reduced to oligonucleotides in this procedure and will not be recovered when dinucleosome-length fragments are sequenced.  The loss of sequences from typical paired boundary elements is illustrated by the lar meta-loop shown in Author response image 4 (at 200 bp resolution).  Panels A and B show the contact profiles generated when the blue boundary (which separates two TADs that span  the Lar (Leukocyteantigen-related-like) transcription unit interacts with the purple boundary (which separates two TADs in a gene poor region ~620 kb away).  The blue and purple boundaries pair with each other head-to-head, and this pairing orientation generates yet another circle-loop.  In the circle-loop topology, sequences in the TADs upstream of both boundaries come into contact with each other, and this gives the small dark rectangular box to the upper left of the paired boundaries (Author response image 4A).  (Note that this small box corresponds to the two small TADs upstream of the blue and purple boundaries, respectively. See panel B.)  Sequences in the TADs downstream of the two boundaries also come into contact with each other, and this gives the large box to the lower right of the paired boundaries.  While this meta-loop is clearly generated by pairing interactions between the blue and purple boundaries, the interacting sequences are degraded in the MicroC protocol, and sequences corresponding to the blue and purple boundaries aren’t recovered.  This can be seen in panel B (red arrow and red arrowheads).  When a different Hi-C procedure is used (dHS-C) that captures nucleosome-free regions of chromatin that are physically linked to each other (Author response image 4C & D), the sequences in the interacting blue and purple boundaries are recovered and generate a prominent “dot” at their physical intersection (blue arrow in panel D).

Author response image 4.

Lar metaloop. Panels A & bB: MicroC. Panels C & D: dHS-C

While sequences corresponding to the blue and purple boundaries are lost in the MicroC procedure, there is at least one class of elements that engage in physical pairing interactions whose sequences are (comparatively) resistant to MNase digestion.  This class of elements includes many PREs ((Kyrchanova et al. 2018); unpublished data), the boundary bypass elements in the Abd-B region of BX-C (Kyrchanova et al. 2023; Kyrchanova et al. 2019a; Kyrchanova et al. 2019b; Postika et al. 2018), and “tethering” elements (Batut et al. 2022; Li et al. 2023).  In all of the cases tested, these elements are bound in nuclear extracts by a large (>1000 kD) GAGA factor-containing multiprotein complex called LBC.  LBC also binds to the hsp70 and eve promoters (unpublished data).  Indirect end-labeling experiments (Galloni et al. 1993; Samal et al. 1981; Udvardy and Schedl 1984) indicate that the LBC protects a ~120-180 bp DNA segment from MNase digestion.  It is likely that this is the reason why LBC-bound sequences can be recovered in MicroC experiments as dots when they are physically linked to each other.  One such example (based on the ChIP signatures of the paired elements) is indicated by the green arrow in panel B and D of Author response image 4.  Note that there are no dots corresponding to these two LBC elements within either of the TADs immediately downstream of the blue and purple boundaries.  Instead the sequences corresponding to the two LBC elements are only recovered when the two elements pair with each other over a distance of ~620 kb.  The fact that these two elements pair with each other is consistent with other findings which indicate that, like classical boundaries, LBC elements exhibit partner preferences.  In fact, LBC elements can sometimes function as TAD boundaries.  For example, the Fab-7 boundary has two LBC elements, and full Fab-7 boundary function can be reconstituted with just these two elements (Kyrchanova et al. 2018).

Reviewer #2 (Public Review):

"Chromatin Structure II: Stem-loops and circle-loops" by Ke*, Fujioka*, Schedl, and Jaynes reports a set of experiments and subsequent analyses focusing on the role of Drosophila boundary elements in shaping 3D genome structure and regulating gene expression. The authors primarily focus on the region of the fly genome containing the even skipped (eve) gene; eve is expressed in a canonical spatial pattern in fly embryos and its locus is flanked by the well-characterized neighbor of homie (nhomie) and homie boundary elements. The main focus of investigation is the orientation dependence of these boundary elements, which had been observed previously using reporter assays. In this study, the authors use Crispr/Cas9 editing followed by recombination-mediated cassette exchange to create a series of recombinant fly lines in which the nhomie boundary element is either replaced with exongenous sequence from phage 𝝀, an inversion of nhomie, or a copy of homie that has the same orientation as the endogenous homie sequence. The nhomie sequence is also regenerated in its native orientation to control for effects introduced by the transgenesis process.

The authors then perform high-resolution Micro-C to analyze 3D structure and couple this with fluorescent and colorimetric RNA in situ hybridization experiments to measure the expression of eve and nearby genes during different stages of fly development. The major findings of these experiments are that total loss of boundary sequence (replacement with 𝝀 DNA) results in major 3D structure changes and the most prominent observed gene changes, while inversion of the nhomie boundary or replacement with homie resulted in more modest effects in terms of 3D structure and gene expression changes and a distinct pattern of gene expression change from the 𝝀 DNA replacement. As the samples in which the nhomie boundary is inverted or replaced with homie have similar Micro-C profiles at the eve locus and show similar patterns of a spurious gene activation relative to the control, the observed effects appear to be driven by the relative orientation of the nhomie and homie boundary elements to one another.

Collectively, the findings reported in the manuscript are of broad interest to the 3D genome field. Although extensive work has gone into characterizing the patterns of 3D genome organization in a whole host of species, the underlying mechanisms that structure genomes and their functional consequences are still poorly understood. The perhaps best understood system, mechanistically, is the coordinated action of CTCF with the cohesin complex, which in vertebrates appears to shape 3D contact maps through a loop extrusion-pausing mechanism that relies on orientation-dependent sequence elements found at the boundaries of interacting chromatin loops.

(2.1) The notion that mammalian genome is shaped in 3D by the coordinate action of cohesin and CTCF has achieved the status of dogma in the field of chromosome structure in vertebrates.  However, as we have pointed out in #1.1, the evidence supporting this dogma is far from convincing.  To begin with, it is based on low resolution Hi-C experiments that rely on large bin sizes to visualize so-called “TADs.”  In fact, the notion that cohesin/CTCF are responsible on their own for shaping the mammalian 3D genome appears to be a result of mistaking a series of forests for the actual trees that populate each of the forests.

As illustrated in Author response image 1 above, the “TADs” that are visualized in these low resolution data sets are not TADs at all, but rather TAD neighborhoods consisting of several dozen or more individual TADs.  Moreover, the “interesting” features that are evident at low resolution (>1 kb)—the dots and stripes—largely disappear at resolutions appropriate for visualizing individual TADs (~200 bp).

In Goel et al. 2023, we presented data from one of the key experiments in Goel et al. (Goel et al. 2023).  In this experiment,  the authors used RCMC to generate high resolution (~250 bp) MicroC contact maps before and after Rad21 depletion.  Contrary to dogma, Rad21 depletion has absolutely no effect on TADs in a ~250 kb DNA segment—and these TADs look very much like the TADs we observe in the Drosophila genome, in particular in the Abd-B region of BX-C that is thought to be assembled into a series of circle-loops (see Fig. 2B).

While Goel et al. (Goel et al. 2023) observed no effect of Rad21 depletion on TADs, they found that loss of Rad21 disturbs long-distance (but not short-distance) contacts in large TAD neighborhoods when their RCMC data set is visualized using bin sizes of 5 kb and I kb.  This is shown in Author response image 2.  The significance of this finding is, however, uncertain.  It could mean that the 3D organization of large TAD neighborhoods have a special requirement for cohesin activity.  On the other hand, since cohesin functions to hold sister chromosomes together after replication until they separate during mitosis (and might also participate in mitotic condensation), it is also possible that the loss of long-range contacts in large TAD neighborhoods when Rad21 is depleted is simply a reflection of this particular activity.  Further studies will be required to address these possibilities.

As for CTCF: a careful inspection of the ChIP data in Goel et al. 2023 indicates that CTCF is not found at each and every TAD boundary.  In fact, the notion that CTCF is the be-all and end-all of TAD boundaries in mammals is truly hard to fathom.  For one, the demands for specificity in TAD formation (and in regulatory interactions) are likely much greater than those in flies, and specificity can’t be generated by a single DNA binding protein.  For another, several dozen chromosomal architectural proteins have already been identified in flies.  This means that (unlike what is thought to be true in mammals) it is possible to use a combinatorial mechanism to generate specificity in, for example, the long distance interactions in RFig 6 and 7.  As noted in #2.1 above, many of the known chromosomal architectural proteins in flies are polydactyl zinc finger proteins (just like CTCF).  There are some 200 different polydactyl zinc finger proteins in flies, and the function of only a hand full of these is known at present.  However, it seems likely that a reasonable fraction of this class of DNA binding proteins will ultimately turn out to have an architectural function of some type (Bonchuk et al. 2021; Fedotova et al. 2017).  The number of different polydactyl zinc finger protein genes in mammals is nearly 3 times that of flies.  It is really possible that of these, only CTCF is involved in shaping the 3D structure of the mammalian genome?

Despite having a CTCF paralog and cohesin, the Drosophila genome does not appear to be structure by loop extrusion-pausing. The identification of orientation-dependent elements with pronounced structural effects on genome folding thus may shed light on alternative mechanisms used to regulated genome structure, which in turn may yield insights into the significance of particular folding patterns.

(2.2) Here we would like to draw the reviewer’s and reader’s attention to Author response image 3, which shows that orientation-dependent pairing interactions have a significant impact on physical interactions between different sequences.  We would also refer the reader to two other publications.  One of these is Kyrchanova et al. (Kyrchanova et al. 2008), which was the first to demonstrate that orientation of pairing interactions matters.  The second is Fujioka et al. (Fujioka et al. 2016), which describes experiments indicating that nhomie and homie pair with each other head-to-tail and with themselves head-to-head.

On the whole, this study is comprehensive and represents a useful contribution to the 3D genome field. The transgenic lines and Micro-C datasets generated in the course of the work will be valuable resources for the research community. Moreover, the manuscript, while dense in places, is generally clearly written and comprehensive in its description of the work. However, I have a number of comments and critiques of the manuscript, mainly centering on the framing of the experiments and presentation of the Micro-C results and on manner in which the data are analyzed and reported. They are as follows:

Major Points:

(1) The authors motivate much of the introduction and results with hypothetical "stem loop" and "circle loop" models of chromosome confirmation, which they argue are reflected in the Micro-C data and help to explain the observed ISH patterns. While such structures may possibly form, the support for these specific models vs. the many alternatives is not in any way justified. For instance, no consideration is given to important biophysical properties such as persistence length, packing/scaling, and conformational entropy. As the biophysical properties of chromatin are a very trafficked topic both in terms of experimentation and computational modeling and generally considered in the analysis of chromosome conformation data, the study would be strengthened by acknowledgement of this body of work and more direct integration of its findings.

(2.3) The reviewer is not correct in claiming that “stem-loops” and “circle-loops” are “hypothetical.”  There is ample evidence that both types of loops are present in eukaryotic genomes, and that loop conformation has significant readouts in terms of not only the physical properties of TADs but also their functional properties.  Here we would draw the reviewer’s attention to Author response image 3 and Author response image 4 for examples of loops formed by the orientation-dependent pairing of yet other TAD boundary elements.  As evident from the MicroC data in these figures, circle-loops and stem-loops have readily distinguishable contact patterns.  The experiments in Fujioka et al. (Fujioka et al. 2016) demonstrate that homie and nhomie pair with each other head-to-tail, while they pair with themselves head-to-head.  The accompany paper (Bing et al. 2024) also provides evidence that loop topology is reflected both in the pattern of activation of reporters and in the MicroC contact profiles.  We would also mention again Kyrchanova et al. (Kyrchanova et al. 2008), who were the first to report orientation-dependent pairing of endogenous fly boundaries.

At this juncture it would premature to try to incorporate computational modeling of chromosome conformation in our studies.  The reason is that the experimental foundations that would be essential for building accurate models are lacking.  As should be evident from RFigs. 1-3 above, studies on mammalian chromosomes are simply not of high enough resolution to draw firm conclusions about chromosome conformation: in most studies only the forests are visible.  While the situation is better in flies, there are still too many unknown.  As just one example, it would be important to know the orientation of the boundary pairing interactions that generate each TAD.  While it is possible to infer loop topology from how TADs interact with their neighbors (a plume versus clouds), a conclusive identification of stem- and circle-loops will require a method to unambiguously determine whether a TAD boundary pairs with its neighbor head-to-head or headto-tail.

(2) Similar to Point 1, while there is a fair amount of discussion of how the observed results are or are not consistent with loop extrusion, there is no discussion of the biophysical forces that are thought to underly compartmentalization such as block-polymer co-segregation and their potential influence. I found this absence surprising, as it is generally accepted that A/B compartmentalization essentially can explain the contact maps observed in Drosophila and other non-vertebrate eukaryotes (Rowley, ..., Corces 2017; PMID 28826674). The manuscript would be strengthened by consideration of this phenomenon.

(2.4) Compartments in mammals have typically been identified and characterized using lowresolution data sets, and these studies have relied on visualizing compartments using quite large bin sizes (>>1 kb).  Our experiments have nothing to do with the large-scale compartments seen in these Hi-C experiments.  Instead, we are studying the properties of individual TADs: how TADs are formed, the relationship between TAD topology and boundary:boundary pairing, and the impact of TAD topology on interactions between TADs in the immediate neighborhood.  There is no evidence to date that these large compartments or “block polymer co-segregation” have a) any impact on the properties of individual boundary elements, b) have a role in determining which boundary elements actually come together to form a given TAD, c) impact the orientation of the interactions between boundaries that generate the TAD or d) determine how TADs tend to interact with their immediate neighbors.  

In more recent publications (c.f., Harris et al. 2023) compartments have shrunk in size and instead of being units of several hundred kb, the median length of the “compartmental” unit in mammalian cells is about12 kb. This is not too much different from the size of fly TADs.  However, the available evidence does not support the idea that block polymer co-segregation/co-repulsion drive the TAD:TAD interactions seen in MicroC experiments.  For example, according to this “micro-compartment” model, the specific patterns of interaction between TADs in the CG3294 meta-loop in Author response image 3 would be driven by block polymer co-segregation and co-repulsion. In this model, the TAD upstream of the blue boundary (which contains CG33543, the odorant binding protein gene Obp22a and the Npc2a gene which encodes a protein involved in sterol homeostasis) would share the same chromatin state/biophysical properties as the TAD upstream of the purple boundary, which has the fipi gene. While it is true that CG33543, Obp22a and also the fipi gene are not expressed in embryos, Npc2a is expressed at high levels during embryogenesis, yet it is part of the TAD that interacts with the fipi TAD.  The TAD downstream of the blue boundary contains CG15353 and Nplp4 and it interacts with the TAD downstream of the purple boundary which contains CG3294 and slf.  CG15353 and Nplp4 are not expressed in the embryo and as such should share a compartment with a TAD that is also silent. However, slf is expressed at a high level in 1216 hr embryos, while CG3294 is expressed at a low level.  In neither case would one conclude that the TADs upstream and downstream of the blue and purple boundaries, respectively, interact because of shared chromatin/biophysical states that drive block polymer co-segregation corepulsion. 

One might also consider several gedanken experiments involving the long-range interactions that generate the CG3294 meta-loop in Author response image 3.    According to the micro-compartment model the patchwork pattern of crosslinking evident in the CG3294 meta-loop arises because the interacting  TADs share the same biochemical/biophysical properties, and this drives block polymer cosegregation and co-repulsion.  If this model is correct, then this patchwork pattern of TAD:TAD interactions would remain unchanged if we were to delete the blue or the purple boundary.  However, given what we know about how boundaries can find and pair with distant boundaries (c.f., Figure 6 from Muller et el. 1999 and the discussion in #1.2), the result of these gedanken experiments seem clear: the patchwork pattern shown in Author response image 3A will disappear.  What would happen if we inverted the blue or the purple boundary? Would the TAD containing CG33543, Obp22a and Npc2a still interact with fipi as would be expected from the compartment model?  Or would the pattern of interactions flip so that the CG33543, Obp22a and Npc2a TAD interacts with the TAD containing CG3294 and slf?  Again we can anticipate the results based on previous studies: the interacting TADs will switch when the CG3294 meta-loop is converted into a stem-loop.  If this happened, the only explanation possible in the compartment model is that the chromatin states change when the boundary is inverted so that TAD upstream of blue boundary now shares the same chromatin state as the TAD downstream of the purple boundary, while the TAD downstream of the blue boundary shares same state as the TAD upstream of the purple boundary.  However, there is no evidence that boundary orientation per se can induce a complete switch in “chromatin states” as would be required in the compartment model. 

While we have not done these experimental manipulations with the CG3294 meta-loop, an equivalent experiment was done in Bing et al. (Bing et al. 2024).  However, instead of deleting a boundary element, we inserted a homie boundary element together with two reporters (gfp and LacZ) 142 kb away from the eve TAD.  The result of this gedanken “reverse boundary deletion” experiment is shown in Author response image 5.  Panel A shows the MicroC contact profile in the region spanning the transgene insertion site and the eve TAD in wild type (read “deletion”) NC14 embryos.  Panel B shows the MicroC contact profile from 12-16 hr embryos carrying the homie dual reporter transgene inserted at -142 kb.  Prior to the “deletion”, the homie element in the transgene pairs with nhomie and homie in the eve TAD and this generates a “mini-metaloop.”  In this particular insert, the homie boundary in the transgene (red arrow) is “pointing” in the opposite orientation from the homie boundary in the eve TAD (red arrow).  In this orientation, the pairing of the transgene homie with eve nhomie/homie brings the LacZ reporter into contact with sequences in the eve TAD.  Since a mini-metaloop is formed by homie_à _nhomie/homie pairing, sequences in TADs upstream and downstream of the transgene insert interact with sequences in TADs close to the eve TAD (Author response image 5B).  Taken together these interactions correspond to the interaction patchwork that is typically seen in “compartments” (see boxed region and inset).  If this patchwork is driven as per the model, by block polymer co-segregation and co-repulsion, then it should still be present when the transgene is deleted.  However, panel A shows that the interactions linking the transgene and the sequences in TADs next to the transgene to eve and TADs next to eve disappear when the homie boundary (plus transgene) is “deleted” in wild type flies.

Author response image 5.

Boundary deletion and compartments

A second experiment would be to invert the homie boundary so that instead of pointing away from eve it points towards eve.  Again, if the compartmental patchwork is driven by block polymer co-segregation and co-repulsion, inverting the homie boundary in the transgene should have no effect on the compartmental contact profile.  Inspection of Fig. 7 in Bing et al. (Bing et al. 2024) will show that this prediction doesn’t hold either.  When homie is inverted, sequences in the eve TAD interact with the gfp reporter not the LacZ reporter.  In addition, there are corresponding changes in how sequences in TADs to either side of eve interact with sequences to either side of the transgene insert.  

Yet another “test” of compartments generated by block polymer co-segregation/co-repulsion is provided by the plume above the eve volcano triangle.  According to the compartment model, sequences in TADs flanking the eve locus form the plume above the eve volcano triangle because their chromatin shares properties that drive block polymer co-segregation.  These same properties result in repulsive interactions with chromatin in the eve TAD, and this would explain why the eve TAD doesn’t crosslink with its neighbors.  If the distinctive chromatin properties of eve and the neighboring TADs drive block polymer co-segregation and co-repulsion, then inverting the nhomie boundary or introducing homie in the forward orientation should have absolutely no effect on the physical interactions between chromatin in the eve TAD and chromatin in the neighboring TADs.  However, Figures 4 and 6 in this paper indicate that boundary pairing orientation, not block polymer co-segregation/co-repulsion, is responsible for forming the plume above the eve TAD. Other findings also appear to be inconsistent with the compartment model. (A) The plume topping the eve volcano triangle is present in NC14 embryos when eve is broadly expressed (and potentially active throughout the embryo).  It is also present in 12-16 hr embryos when eve is only expressed in a very small subset of cells and is subject to PcG silencing everywhere else in the embryo.  B) According to the compartment model the precise patchwork pattern of physical interactions should depend upon the transcriptional program/chromatin state that is characteristic of a particular developmental stage or cell type.  As cell fate decisions are just being made during NC14 one might expect that most nuclei will share similar chromatin states throughout much of the genome.  This would not be true for 12-16 hr embryos.  At this stage the compartmental patchwork would be generated by a complex mixture of interactions in cells that have quite different transcriptional programs and chromatin states.  In this case, the patchwork pattern would be expected to become fuzzy as a given chromosomal segment would be in compartment A in one group of cells and in compartment B in another.   Unlike 12-16 hr embryos,  larval wing discs would be much more homogeneous and likely give a distinct and relatively well resolved compartmental pattern. We’ve examined the compartment patchwork of the same chromosomal segments in NC14 embryos, 12-16 hr embryos and larval wing disc cells.  While there are some differences (e.g., changes in some of the BX-C TADs in the wing disc sample) the compartmental patchwork patterns are surprisingly similar in all three cases. Nor is there any “fuzziness” in the compartmental patterns evident in 12-16 hr embryos, despite the fact that there are many different cell types at this stage of development.  C) TAD interactions with their neighbors and compartmental patchworks are substantially suppressed in salivary gland polytene chromosomes.  This would suggest that features of chromosome structure might be the driving force behind many of the “compartmental” interactions as opposed to distinct biochemical/biophysical of properties of small chromosomal segments that drive polymer co- segregation/co-repulsion.  

(3) The contact maps presented in the study represent many cells and distinct cell types. It is clear from single-cell Hi-C and multiplexed FISH experiments that chromosome conformation is highly variable even within populations of the same cell, let alone between cell types, with structures such as TADs being entirely absent at the single cell level and only appearing upon pseudobulking. It is difficult to square these observations with the models of relatively static structures depicted here. The authors should provide commentary on this point.

(2.5) As should be evident from Author response image 1, single-cell Hi-C experiments would not provide useful information about the physical organization of individual TADs, TAD boundaries or how individual TADs interact with their immediate neighbors.  In addition, since they capture only a very small fraction of the possible contacts within and between TADs, we suspect that these single-cell studies aren’t likely to be useful for making solid conclusions about TAD neighborhoods like those shown in Author response image 1 panels A, B, C and D, or Author response image 2.  While it might be possible to discern relatively stable contacts between pairs of insulators in single cells with the right experimental protocol, the stabilities/dynamics of these interactions may be better judged by the length of time that physical interactions are seen to persist in live imaging studies such as Chen et al. (2018), Vazquez et al. (2006) and Li et al. (2011).

The in situ FISH data we’ve seen also seems problematic in that probe hybridization results in a significant decondensation of chromatin.  For two probe sets complementary to adjacent ~1.2 kb DNA sequences, the measured center-to-center distance that we’ve seen was ~110 nM.  This is about 1/3rd the length that is expected for a 1.2 kb naked DNA fragment, and about 1.7 times larger than that expected for a beads-on-a-string nucleosome array (~60 nM).  However, chromatin is thought to be compacted into a 30 nM fiber, which is estimated to reduce the length of DNA by at least another ~6 fold.  If this estimate is correct, FISH hybridization would appear to result in a ~10 fold decompaction of chromatin.  A decompaction of this magnitude would necessarily be followed by a significant distortion in the actual conformation of chromatin loops.

(4) The analysis of the Micro-C data appears to be largely qualitative. Key information about the number of reads sequenced, reaps mapped, and data quality are not presented. No quantitative framework for identifying features such as the "plumes" is described. The study and its findings would be strengthened by a more rigorous analysis of these rich datasets, including the use of systematic thresholds for calling patterns of organization in the data.

Additional information on the number of reads and data quality have been included in the methods section. 

(5) Related to Point 4, the lack of quantitative details about the Micro-C data make it difficult to evaluate if the changes observed are due to biological or technical factors. It is essential that the authors provide quantitative means of controlling for factors like sampling depth, normalization, and data quality between the samples.

In our view the changes in the MicroC contact patterns for the eve locus and its neighbors when the nhomie boundary is manipulated are not only clear cut and unambiguous but are also readily evident in the Figs that are presented in the manuscript.  If the reviewer believes that there aren’t significant differences between the MicroC contact patterns for the four different nhomie replacements, it seems certain that they would also remain unconvinced by a quantitative analysis.

The reviewer also suggests that biological and/or technical differences between the four samples could account for the observed changes in the MicroC patterns for the eve TAD and its neighbors.  If this were the case, then similar changes in MicroC patterns should be observed elsewhere in the genome.  Since much of the genome is analyzed in these MicroC experiments there is an abundance of internal controls for each experimental manipulation of the nhomie boundary.  For two of the nhomie replacements, nhomie reverse and homie forward, the plume above the eve volcano triangle is replaced by clouds surrounding the eve volcano triangle.  If these changes in the eve MicroC contact patterns are due to significant technical (or biological) factors, we should observe precisely the same sorts of changes in TADs elsewhere in the genome that are volcano triangles with plumes.   Author response image 6 shows the MicroC contact pattern for several genes in the Antennapedia complex.  The deformed gene is included in a TAD which, like eve, is a volcano triangle topped by a plume.  A comparison of the deformed MicroC contact patterns for nhomie forward (panel B) with the MicroC patterns for nhomie reverse (panel C) and homie forward (panel D) indicates that while there are clearly technical differences between the samples, these differences do not result in the conversion of the deformed plume into clouds as is observed for the eve TAD.  The MicroC patterns elsewhere in Antennapedia complex are also very similar in all four samples.  Likewise, comparisons of regions elsewhere in the fly genome indicate that the basic contact patterns are similar in all four samples.   So while there are technical differences which are reflected in the relative pixel density in the TAD triangles and the LDC domains, these differences do not result in converting plumes into clouds nor do the alter the basic patterns of TAD triangles and LDC domains.  As for biological differences— the embryos in each sample are at roughly the same developmental stage and were collected and processed using the same procedures. Thus, the biological factors that could reasonably be expected to impact the organization of specific TADs (e.g., cell type specific differences) are not going to impact the patterns we see in our experiments. 

Author response image 6.

(6) The ISH effects reported are modest, especially in the case of the HCR. The details provided for how the imaging data were acquired and analyzed are minimal, which makes evaluating them

challenging. It would strengthen the study to provide much more detail about the acquisition and analysis and to include depiction of intermediates in the analysis process, e.g. the showing segmentation of stripes.

The imaging analysis is presented in Fig. 5 is just standard confocal microscopy.  Individual embryos were visualized and scored.  An embryo in which stripes could be readily detected was scored as ‘positive’ while an embryo in which stripes couldn’t be detected was scored as ‘negative.’   

Recommendations for the authors:

Editor comments:

It was noted that the Jaynes lab previously published extensive genetic evidence to support the stem loop and circle loop models of Homie-Nhomie interactions (Fujioka 2016 Plos Genetics) that were more convincing than the Micro-C data presented here in proof of their prior model. Maybe the authors could more clearly summarize their prior genetic results to further try to convince the reader about the validity of their model.

Reviewer #1 (Recommendations For The Authors):

Below, I list specific comments to further improve the manuscript for publication. Most importantly, I recommend the authors tone down their proposal that boundary pairing is a universal TAD forming mechanism.

(1) The title is cryptic.

(2) The second sentence in the abstract is an overstatement: "In flies, TADs are formed by physical interactions between neighboring boundaries". Hi-C and Micro-C studies have not provided evidence that most TADs in Drosophila show focal interactions between their bracketing boundaries. The authors rely too strongly on prior studies that used artificial reporter transgenes to show that multimerized insulator protein binding sites or some endogenous fly boundaries can mediate boundary bypass, as evidence that endogenous boundaries pair.

Please see responses #1.1 and #1.3 and figures Author response image 1 and Author response image 3.  Note that using dHS-C, most TADs that we’ve looked at so far are topped by a “dot” at their apex.

(3) Line 64: the references do not cite the stated "studies dating back to the '90's'".

The papers cited for that sentence are reviews which discussed the earlier findings.  The relevant publications are cited at the appropriate places in the same paragraph.  

(4) Line 93: "On the other hand, while boundaries have partner preferences, they are also promiscuous in their ability to establish functional interactions with other boundaries." It was unclear what is meant here.

Boundaries that a) share binding sites for proteins that multimerized, b) have binding sites for proteins that interact with each other, or c) have binding sites for proteins that can be bridged by a third protein can potentially pair with each other.  However, while these mechanisms enable promiscuous pairing interactions, they will also generate partner preferences (through a greater number of a, b and/or c).

(5) It could be interesting to discuss the fact that it remains unclear whether Nhomie and Homie pair in cis or in trans, given that homologous chromosomes are paired in Drosophila.

The studies in Fujioka et al. (Fujioka et al. 2016) show that nhomie and homie can pair both in cis and in trans.  Given the results described in #1.2, we imagine that they are paired in both cis and trans in our experiments.

(6) Line 321: Could the authors further explain why they think that "the nhomie reverse circle-loop also differs from the nhomie deletion (λ DNA) in that there is not such an obvious preference for which eve enhancers activate expression"?

The likely explanation is that the topology/folding of the altered TADs impacts the probability of interactions between the various eve enhancers and the promoters of the flanking genes.  

(7) The manuscript would benefit from shortening the long Discussion by avoiding repeating points described previously in the Results.

(8) Line 495: "If, as seems likely, a significant fraction of the TADs genome-wide are circle loops, this would effectively exclude cohesin-based loop extrusion as a general mechanism for TAD formation in flies". The evidence provided in this manuscript appears insufficient to discard ample evidence from multiple laboratories that TADs form by compartmentalization or loop extrusion. Multiple laboratories have, for example, demonstrated that cohesin depletion disrupts a large fraction of mammalian TADs. 

Points made here and in #9 have been responded to in #1.1, #2.1 and #2.4 above.  We would suggest that the evidence for loop extrusion falls short of compelling (as it is based on the analysis of TAD neighborhoods, not TADs—that is forests, not trees) and given the results reported in Goel et al. (in particular Fig. 4 and Sup Fig. 8) is clearly suspect. This is not to mention the fact that cohesin loop-extrusion can’t generate circle-loops TADs, yet circle-loops clearly exist.  Likewise, as discussed in #2.4, it is not clear to us that the shared chromatin states, polymer co-segregation and co-repulsion account for the compartmental patchwork patterns of TAD;TAD interactions. The results from the  experimental manipulations in this paper and the accompanying paper, together with studies by others (e.g., Kyrchanova et al. (Kyrchanova et al. 2008), Mohana et al. (Mohana et al. 2023) would also seem to be at odds with the model for compartments as currently formulated.  

The unique properties of Nhomie and Homie, namely the remarkable specificity with which they physically pair over large distances (Fujioka et al. 2016) may rather suggest that boundary pairing is a phenomenon restricted to special loci. Moreover, it has not yet been demonstrated that Nhomie or Homie are also able to pair with the TAD boundaries on their left or right, respectively.

Points made here were discussed in detail in #1.2.  As described in detail in #1.2, It is not the case that nhomie and homie are in “unique” or “special.”  Other fly boundaries can do the same things.  As for whether nhomie and homie pair with their neighbors:  We haven’t done transgene experiments (e.g., testing by transvection or boundary bypass).  Likewise, in MicroC experiments there are no obvious dots at the apex of the neighboring TADs that would correspond to nhomie pairing with the neighboring boundary to the left and homie pairing with the neighboring boundary to the right. However, this is to be expected. As we discussed in in #1.3 above, only MNase resistant elements will generate dots in standard MicroC experiments.  On the other hand, when boundary:boundary interactions are analyzed by dHS-C (c.f., Author response image 4), there are dots at the apex of both neighboring TADs.  This would be direct evidence that nhomie pairs with the neighboring boundary to the left and homie pairs with the neighboring boundary to the right.

(9) The comment in point 8 also applies to the concluding 2 sentences (lines 519-524) of the Discussion.

See response to 8 above. Otherwise, the concluding sentences are completely accurate. Validation of the cohesin loop extrusion/CTCF roadblock model will required demonstrating a) that all TADs are either stem-loops or unanchored loops and b) that TAD endpoints are always marked by CTCF. 

The likely presence of circle-loops and evidence that TAD boundaries that don’t have CTCF (c.f.,Goel et al. 2023) already suggests that this model can’t (either fully or not all) account for TAD formation in mammals. 

(10) Figs. 3 and 6: It would be helpful to add the WT screenshot in the same figure, for direct comparison.

It is easy enough to scroll between Figs-especially since nhomie forward looks just like WT.

(11) Fig. 6: It would be helpful to show a cartoon view of a circle loop to the right of the Micro-C screenshot, as was done in Fig. 3.

Good idea.   Added to the Fig.

(12) Fig. 5: It would be helpful to standardize the labelling of the different genotypes throughout the figures and panels ("inverted" versus "reverse" versus an arrow indicating the direction).

Fixed.

Reviewer #2 (Recommendations For The Authors):

Minor Points:

(1) The Micro-C data does not appear to be deposited in an appropriate repository. It would be beneficial to the community to make these data available in this way.

This has been done.

(2) Readers not familiar with Drosophila development would benefit from a gentle introduction to the stages analyzed and some brief discussion on how the phenomenon of somatic homolog pairing might influence the study, if at all.

We included a rough description the stages that were analyzed for both the in situs and MicroC. We thought that an actual description of what is going on at each of the stages wasn’t necessary as the process of development is not a focus of this manuscript.  In other studies, we’ve found that there are only minor differences in MicroC patterns between the blastoderm stage and stage 12-16 embryos.  While these minor differences are clearly interesting, we didn’t discuss them in the text.   In all of experiments chromosomes are likely to be paired.  In NC14 embryos (the stage for visualizing eve stripes and the MicroC contact profiles in Fig. 2) replication of euchromatic sequences is thought to be quite rapid.  While homolog pairing is incomplete at this stage, sister chromosomes are paired.  In stage 12-16 embryos, homologs will be paired and if the cells are arrested in G2, then sister chromosome will also be paired.  So in all of experiments, chromosomes (sisters and/or homologs) are paired. However, since we don’t have examples of unpaired chromosomes, our experiments don’t provide any info on how chromosome pairing might impact MicroC/expression patterns.

(3) "P > 0.01" appears several times. I believe the authors mean to report "P < 0.01".

Fixed.  

References for Response

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Reply to the reviewers

We would like to thank all reviewers for their detailed and constructive feedback, which substantially helped improve the manuscript. We apologise for the time taken for the revisions, which was partially due to the first author (successfully) writing and defending her PhD thesis in the same time frame. We would like to point out already here that, based on reviewers' feedback, main figure 6 is completely redone and the conclusions of this figure have changed substantially. We no longer suggest RNA chaperoning activity (it was identified as being due to the high concentration of TEV protease, in a control suggested by the reviewers). Instead, our refined assay conditions with lower TEV protease concentration identified ribonuclease activity of membrane-bound full-length 2C, which is consistent with a publication from 2022 (PMID: 35947700).

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Evidence, reproducibility, and clarity

Summary:

In this study by Shankar and colleagues, the authors aim to understand the structure and function of the enterovirus 2C protein, a putative viral helicase with AAA+ ATPase activity. Using poliovirus (as a model enterovirus) 2C, the author's propose the protein contains two amphipathic helices (AH1 and AH2) at the N-terminus that are divided by a conserved glycine. Using purified MBP-tagged 2C and N-terminal 2C truncations, their data suggests AH1 is primarily responsible for clustering at membranes, whilst AH2 is the main mediator of 2C oligmerisation and membrane binding. Furthermore, 2C was suggested to be able to recruit RNA to membranes, with a preference for dsRNA, and the author's data implies that the helicase activity of 2C is ATP-independent. Instead, the ATP activity appears to be required for 2C hexamer formation or chaperone activity. The manuscript is generally well written /presented and the author's present very interesting data which raises several questions, some of which require additional experimentation to help support the author's conclusions. Specific comments are as follows.

We thanks the reviewer for the overall positive assessment, as well as the specific comments below.

Major Comments:

1. The authors use four main constructs throughout the paper: full-length 2C, 2C with deletion of AH1 (ΔAH1), 2C with both AH1 and AH2 deleted (ΔMBP) and 2C with an extended N-terminal deletion. From this, the author's draw conclusions on the function of both AH1 and AH2. One of the author's main conclusions is that AH2 is the main mediator of 2C membrane association (e.g., in line 169). However, is it possible to conclude the relative importance of AH1 vs AH2 without testing a construct containing the deletion of AH2 only (ΔAH2)? This should be generated and used alongside this data to fully define the relative importance of AH1 and AH2 in these assay and remove the possibility that the deletion of AH1 changes the structure and/or function of AH2, which could also result in the observed differences.

This was a very good suggestion. We expressed and purified the ΔAH2 protein requested by the reviewer and characterized its oligomeric state as well as its membrane binding. It turns out, as suspected, that the ΔAH2 protein behaves very similarly to the ΔMBD protein (i.e. it does not form higher order oligomers and does not bind membranes). The changes in the manuscript due to this addition are many but can primarily be found in main figures 2-3 and their associated supplementary figures.

Previous structural predictions of 2C do not appear to have two separate AHs at the N-terminus. Are the AH1 and AH2 structures predicted to be formed in the context of the entire 2C protein, 2BC precursors and polyprotein? Are there structural approaches that could provide experimental evidence for two separate AH at the N-terminus?

This is a good point. Previous predictions were not that detailed, partially since they were done in the pre-alphafold era. Unfortunately, we cannot think of a tractable experimental method that could verify the split nature of the amphipathic helix in the only context that would matter: the protein bound to a membrane. A long-term goal would be in situ structures of full-length 2C on membranes using cryo-electron tomography, but our current sample and data sets are not sufficient for this. We added a mention of the long-term need for experimental structures of full-length 2C on lines 315-318 in the discussion.

Why are the 2C dimers (lines 137-138) not apparent on the mass photometry data presented (figure 2)?

Different constructs were measured by mas photometry and SEC-MALS. Also, the required concentration is 100-1000x lower for mass photometry which will affect a dynamic equilibrium in case the same construct were measured by the two methods.

It appeared that binding of ΔMBD-2C was better when POPS is in the membrane (line 174). What is the explanation for this and was this finding significant?

Well spotted. It may mean that 2C has a second, lower affinity membrane-binding site which is charge-dependent somewhere outside the MBD. We now added a mention of this in the discussion, lines 321-323.

From the author's data on lipid drop clustering they conclude ΔAH1 is more effective for clustering, however, the ΔAH1 construct produces pentamers not hexamers (from Figure 2). Is formation of hexamers related to or required for membrane clustering?

ΔAH1 is LESS effective at clustering, not more. As for the mention of pentamers in the original submission: we now think this was an unfortunate choice of words. The mass photometry data for 2C(ΔAH1) could more parsimoniously be interpreted as a mix of hexamers and other (unknown to us) smaller oligomers such as trimers. We have removed all mentions of pentamers.

The replicon data presented in Figure 7 should include a replication-defective control (e.g., polymerase mutant), in order to compare how defective in replication ΔAH1 and ΔMBP deletions are compared to a fully-defective construct. Likewise, deletion of ΔAH1 in this construct is likely to affect processing of the viral polyprotein where several previous studies with picornaviruses have demonstrated that the residues in the P2'-P4' positions can change cleavage efficiency (e.g., PMID: 2542331), or the structure of 2C, leading to the reduction of replication.

Thanks for these good comments. We made the polymerase-dead (GDD-to-GAA) replicon and remeasured it side by side with the 2C replicons. It has a similar luciferase activity indicating that no replication takes place in the 2C deletion replicons. This is shown in the new figure 7. As for the possibility or processing defects, we mentioned this in the original discussion and have now cited the reference suggested by the reviewer in this context (line 324).

How does the author's model of ATPase-independent helicase activity and an APT-dependent required RNA chaperone activity fit with 2 step model for RNA binding and ATPase activity suggested by Yeager et al (PMID: 36399514)?

Acting upon comments from other reviewers, we completely redid the "helicase assay" in the revised manuscript. It turns out that the ATP-independent unwinding activity in the original submission was an artefact of the assay conditions (specifically, of the TEV protease at the higher concentration we used in the old assay). In our improved assay we neither see helicase activity nor ATP-independent RNA chaperoning activity.

Optional major comments that would increase the significance of the work:

All of the optional comments below are exceptionally interesting. But given the long time needed for the several major changes to this manuscript (e.g. the ΔAH2 protein characterization and reoptimisation of the helicase assay) we believe it is more sensible to address them in future studies, for which the 2C reconstitution system can be used.

The preference for dsRNA over ssRNA appears to be quite small (Figure 5d). In the context of a viral infection where ssRNA is likely to outnumber dsRNA at different times during infection is this preference physiologically relevant? In relation to this, what size stretch of dsRNA is required for preference, and could this correspond to cis-acting RNA structural elements, dsRNA as it escapes 3D polymerase or as part of the RF and RI forms (PMID: 9343205)? What is the proposed mechanism of how dsRNA outcompetes membrane tethering of 2C? OPTIONAL The author's study has been conducted in the absence of other viral non-structural proteins. What is the physiological importance of the observations, such as membrane interaction/clustering or RNA binding when presented in the context of the other replication machinery. OPTIONAL Do 2C monomers, dimers and hexamers have different functions in viral replication perhaps at different stages of replication and which of these forms are relevant during viral infection or can they all be detected during infection? Can any suggested separate functional arrangements be separated by genetic complementation experiments? OPTIONAL

Minor comments:

1. The author's appear to interchange between naming/nomenclature of the constructs which makes it confusing to follow (for example, ΔMBD is the same as 2C(41-329) likewise, 2C(Δ115) is sometimes called 2C(116-329)). It would be much easier to follow if the naming of constructs was consistent throughout (unless I am misunderstanding some subtlety in the difference between such constructs).

Thanks very much for spotting this. We have fixed it.

The author's suggest a pentamer arrangement for the ΔAH1 construct, however in the mass photometry data (figure 2D), a hexamer is indicated with the arrow. It would be helpful to change the label to indicate the size of the pentamer where this is being generated, not the hexamer.

As mentioned above, we think the "pentamer" designation of the original manuscript was unfortunate. It is more parsimonious to interpret this as a mix of states, hexamer and undefined snaller.

In most figures, data for full-length 2C, ΔAH1 and ΔMBP is shown. However data for ΔMBP is missing in Figure 4. Using ΔMBP may demonstrate even lower clustering, hinting that AH2 is also involved in this process.

Thanks for this comment. In our view, it can be derived from figure 3 (which shows lack of binding to PC/PE membranes) that the ΔMBD construct would not cluster membranes under the conditions of the assay (clustering requires concomitant binding to two membranes). We now describe our rationale for this on lines 220-222. However, we did include the ΔMBD protein in the new negative staining TEM supplementary figure where it and ΔAH2 show no signs of clustering (figure S10).

I think it would be better for normalise the data in the flotation experiments such that the percentage of 2C in the upper faction is presented as relative to the amount of lipid in the upper fraction (presented in Figure S4).

The change suggested by the reviewer would make it impossible to show the important no-liposome control (leftmost bar in Fig. 3C) in the same plot as the other measurements. We believe that would unnecessarily complicate the figure. Thus, we opted to keep the measurement that are normalised by lipid fluorescence in the supplementary figure. Instead, we now added another mention of this supplementary figure in the legend to main figure 3.

At several places (e.g., lines 232 and 272) the author's refer to "realistic systems". I think the term "physiologically relevant" might be more appropriate.

Agreed and changed throughout.

Line 237: I think "y" is a typo and should read "by".

Thanks. This text was reworked due to the major changes to figure 6.

Reviewer #1 (Significance (Required)):

Significance

I have limited expertise with structural biology but specialise my research on positive-sense RNA virus replication, structure and function. This research is of interest to a broad audience of researchers investigating many positive-sense RNA viruses, which extends beyond the viral family studied here. The work utilises novel techniques to begin to understand the specific roles of 2C in poliovirus replication. The author's data add important incremental new insight into recent studies on viral helicase proteins as referenced in the study, however, a key limitation is understanding the importance/relevance of their observations during a viral infection.

We thanks the reviewer for this positive and nuanced appraisal of our work.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors present an alternative assay system to investigate picornavirus 2C, a protein that is tricky to analyze biochemically in its full length form because of an amphipathic helix at the N-terminus. Poliovirus 2C is expressed with an N-terminal MBP tag, a 50kD protein that helps with solubility as is commonly used for 2C investigations. A difference here is that liposomes are included to mimic membranes for 2C attachment. The key findings are that 2C induces clustering of of liposomes, that double stranded RNA binding by 2C impacts this clustering effect and that a free N-terminus (after cleavage of MBP by TEV protease) is needed for RNA binding and an ATP independent (ie non helicase) RNA duplex separation activity.

Major:

In the floatation assays in figure 3 the authors use a system where MBP-2C is fluorophore-labeled with ATTO488 on exposed cysteines. Poliovirus and other enterovirus 2C has a very well characterized zinc finger domain that has cysteines coordinating a zinc ion. Mutation experiments previously showed that these cysteines are necessary for viral replication and 2C stability. Have the authors controlled for disruption of the zinc finger domain by the labelling of cysteines with ATT0488 and checked if the protein remains folded?

We completely agree with the reviewer and apologise for the omission in the original submission. We have now included a Zn content measurement, which shows unchanged levels between labelled and unlabelled 2C protein (Figure S7). Also, we now in the revised manuscript explicitly describe our original reasoning for labelling on native cysteines: the presence of two cysteines which are not necessary for viral replication and which are more solvent exposed-exposed (and thus more likely to be labelled) in the crystal structure of the soluble fragment of 2C (lines 176-181).

In the analysis of the amphipathic helix, did the authors include membranes in their structural predictions o just the free helix? How does inclusion of membranes impact the predictions? In the predictions in Figure D, only 2 of 4 show a kink and there doesn't seem to be a correlation between those that predict a kink or not and whether the hydrophobic side is aligned in Figure S1.

Unfortunately, predicting a protein structure with the interacting membrane is beyond what is currently doable with protein prediction methods (one would have to combine protein structure predictions with molecular dynamics simulations including a membrane). Based on general principles of protein structure, it is likely that there is some flexibility around G17. Thus there may not be a single "kink angle" for any given virus, but we believe that the presence of the kink (and offset hydrophobic surfaces) for a number of viruses lends credibility and robustness to the observation. We added some descriptions of this thinking on lines 126-127.

Based on previous structures of 2C from different viruses the N-terminal amphipathic helix containing region is predicted to localize on one face of the predicted hexametric structure tethering 2C to the membrane. How does the authors hypothesized model explain 2C dependent clustering? is there evidence that 2C hexamers can oligomerize further into dodecamers for example, maintaining separate faces to enable N-terminal interaction with different membranes? What is the distance between the liposomes in figure 4 at the points of density attributed to 2C? How does this compare to the size of 2C determined in previous structural studies? Is it consistent with one hexamer/2 hexamers sitting on top of one another?

These are very interesting questions but we believe it is prudent to limit our speculation at this point. Eventually, we hope that larger data sets of cryo-electron tomography, coupled to subtomogram averaging, may provide a more definitive answer. What we managed to do with our current cryo-electron tomography data set is to estimate the volume of individual protein densities, and from the volume calculate an estimated molecular mass of the individual complexes seen in the tomograms. This correlates very well with 2C hexamers (new figure 4D).

In the Discussion lines 278-285 the authors suggest that having MBP attached may reflect the polyprotein condition. Can they make a construct with MBP-2B2C to examine interaction with liposomes and assess 2C function?

This is a highly relevant question, but the biochemistry of 2BC is even more challenging than 2C, and we are unfortunately nowhere near being able to work with purified 2BC at the moment.

Discussion lines 293-296, the possibility of two different populations of 2C, binding RNA or membranes cannot be excluded, there is much more 2C around late in infection that present in early infection- the model in figure 8 doesn't acknowledge/capture this.

We have changed the model figure such that more 2C is seen later, and the clustering function is also seen late in infection. The original discussion text referred to (which is unchanged) talks about a "preferential role in RNA replication and particle assembly at later time points" specifically for this reason. We hope the new figure 8 is better at conveying this message.

Discussion lines 313-317, the authors don't reference a study where a mutant of foot-and-mouth disease virus 2C lacking the n-terminal amphipathic helix that could bind but not hydrolyze ATP, hexamerized in the presence of RNA that seems pertinent here (PMID: 20507978).

Thanks for the suggestion. However, after the extensive changes we made to the revised to figure 6 based on excellent reviewer comments (essentially: the RNA chaperoning activity turned out to be an artefact, the improved assay shows no sign of RNA unwinding but instead of 2C-mediated ribonuclease activity), these sentence of the original discussion lost most of their context and we opted to remove them.

Some evidence of MBP-2C cleavage by TEV in the different assays used should be presented as this is a major focus of discussion and currently no gels show TEV cleavage is happening.

Thanks for the suggestion - we agree. We now show these in the new supplementary figures S5 and S12.

Reviewer #2 (Significance (Required)):

The work presents an additional methodology to investigate a a protein that has previously been difficult to study. The authors acknowledge that there is still a lot of 2C biology that remains to be discovered.

Thanks, we agree.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The manuscript provides insights into the role of the N-terminus in membrane binding and its importance in the various functions of 2C.

Major issues

Line 103-119. Is this novel? I thought people had done a lot of bioinformatic analysis of PV 2C (especially Wimmer) who also did mutational work to analyse the importance of various amino acids in the N-terminal helix. I feel like the paper in general, and this section in particular, underplays the large body of work that has been done on the amphipathic helix by various groups.

We apologise if our original manuscript didn't sufficiently acknowledge previous work in the field. In the first sentence of the mentioned paragraph (now lines 112-113) , we did however cite several papers that have previously addressed the amphipathic nature of the N-terminus of 2C. We have now added two more references along the same line, and changed the wording in a way that we hope better bring across that the amphipathic nature per se has been studies before. We would be happy to add more specific references if the reviewer has any suggestions. However, the rest of our analysis IS indeed novel for the following reasons: (i) we show that the amphipathic region is not a simple, single amphipathic helix, but instead has a conserved glycine (helix breaker/destabiliser residue) and two distinct amphipathic stretches before and after this region, (ii) we use alphafold2 (not available at the time of the earlier work) to provide the first reliable structural models of the membrane-binding domain. These models consistently, across several enterovirus 2C proteins, reveal that the hydrophobic surfaces of the first and second amphipathic regions, on either side of the conserved glycine 17, are offset from one another. This lends additional credibility to the distinct nature of these regions which have not previously been identified as such and which we also show in the biochemical assays to be functionally distinct. We have now also added a clarification to the Discussion that the N-terminus of 2C had previously been identified as its membrane-binding domain and we cite references for this. We hope that these changes will sufficiently acknowledge earlier work in the field while clearly pointing out the advance that our paper makes.

Line 132. Did you validate your column with known MW standards? The peak for full length and deltaAH1 look fairly standard for 2C, in that you have a mixture of species. Not sure you can say it is a hexamer when it is such a broad peak. C doesn't really help you too much since the counts at 400 (pentamer) and 480 (hexamer) are almost the same with quite large error bars. Like most people that have worked with 2C I think the best you can say is that you are making some kind of oligomerized 2C that includes hexamer, pentamer, etc. Why no dimer for MBP-2C and MBP-2C(delta AH1) when compared to the other constructs?

We did not calibrate the gel filtration column since the outcome would anyway be a more crude estimate of molecular mass than the mass photometry and SEC-MALS measurements. But we do agree with the reviewer on the broad mass photometry peaks. To address this experimentally, we compared the existing MBP-2C spectra to new recordings on apoferritin, a highly stable homomultimeric protein complex of a similar mass to aa MBP-2C hexamer. The apoferritin mass estimate is overlayed with the full-length MBP-2C in the new figure 2D and the corresponding supplementary figure S3. This indeed shows that the MBP-2C peak is broader, i.e. consistent with a mix of species which are predominantly but not only hexamers. We describe and discuss this on lines 145-149. As for the mention of pentamers in the original submission: we now think this was an unfortunate choice of words. The mass photometry data for 2C(ΔAH1) could more parsimoniously be interpreted as a mix of hexamers and other (unknown to us) smaller oligomers such as trimers. We have removed all mentions of pentamers.

Line 143. Does your data show that there are two amphipathic helices? Bioinformatics suggests it but your experiments just show the importance of the two areas in oligomerization, not that it is forming two helices.

We agree that the choice of words was not idea and have now changed it to "structure predictions indicate" (lines 162).

Figure S2. Your preps are still relatively dirty, which isn't ideal for biochemical assays. Especially lane 3, where you are looking at 50-60% purity. I don't want you to re-run experiments but I think you need to comment on the purity of the protein you are working with. Also I don't like that you removed the top and bottom of the SDS-PAGE. How much protein never entered the gel. Is there a big fat band at 20 kDa? You need to have the full gel here. Did you measure 260 nm of the preps as well to see if you had bound RNA to the 2C?

Thanks for the comment, we agree that our original submission lacked detail in the description of the protein purification. This is now addressed with the new figure S2 which shows size exclusion chromatograms of the fluorophore-labelled proteins (same chromatograms as in figure 2) and the corresponding uncropped gels imaged both in the stain-free channel (showing all proteins) and in the fluorescence channel. The A260/A280 ratio measured for all proteins shows that they are free of nucleic acids at the point of imaging. The protein preps are not 100% homogeneous but we do believe that they are more than 50-60% pure.

Lines 170. Wasn't this done in the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles"? I don't see it referenced. What is novel about the current work when compared to that paper? Any differences?

Thanks for pointing this out. The referenced study worked with a synthesized, isolated peptide corresponding to AH2 (i.e. not with full protein). An amphipathic peptide outside the context of its protein cannot be expected to recapitulate the properties of the entire protein, e.g. since it is not spatially constrained in how it interactis with membranes. As one example (relating to the title of that paper) we don't see full-length 2C protein tubulating membranes the way the isolated peptide does. As for the reviewer's question about novelty, the paper mentioned does not identify the split nature of the amphipathic region, does not consider the role of AH1, does not characterise the membrane-binding properties of full-length 2C with respect to liposome membrane composition and size, does not identify and characterise the membrane clustering properties of 2C, nor its interactions with nucleic acid when bound to a membrane. However, we do agree that we should have cited the paper in our manuscript. We now cite it in the discussion, lines 320-321.

I'm surprised by the lack of electron microscopy (negative stain mostly) of both the oligomerized 2C and the various liposomes. I know the Carlson group is a microscopy group so why the lack of validation using electron microscopy of the various DLS experiments? I know you did cryo-ET for one of the constructs but I think negative stain electron microscopy of other constructs would be useful.

Thanks for the suggestion. As suggested, we have now expanded the analysis with negative staining EM of several more constructs studied by DLS. It can be found in the new supplementary figure S10.

Figure 4C. What evidence is there that this is 2C apart from you added it to the liposomes? It also comes back to the relative impurity of your protein prep. Could this be E.coli contamination?

Thanks for this comment. We have now added a new supplementary figure (S5) showing SDS-PAGE gels of the reactions used for flotation and DLS assays - which are identical to the cryo-ET samples. In addition, we estimated the molecular mass of the individual, putative 2C desities in the cryo-electron tomograms by measuring their volume. This analysis, which can be found in the new figure 4D, shows that the estimated mass of individual protein densities is consistent with a hexamer of full-length 2C. In addition, we mention in the discussion the long-term need to determine high-resolution structures of membrane-bound 2C using cryo-ET and subtomogram averaging (lines 315-318).

Figure 8. Is this model supported by the data in this paper? Your cryo-ET says that 2C is there but that isn't supported by any other data. How is the dsRNA protected from the innate immune system in this model? is it just sat out in the cytosol? How is the nascent ssRNA packeged into the capsid? Is there competition between the dsRNA and capsid for 2C binding (which your model suggests)? I know it sounds like I am being overly critical of the model but in my opinion there are still too many unanswered questions in the field to come up with a half decent model.

Thanks for this comment. We are the first to agree that our understanding of the roles of 2C is far from complete! We should have been more clear that the model figure represents some of the roles of 2C identified to date, and does not claim to be complete. However we do feel that a model figure serves a purpose of putting our findings into a context, and also providing testable hypotheses for future research . As for the question, some of the roles of 2C shown in the model figure (in particular, particle assembly) are rather supported but earlier work of ourselves and others. We have now produced a new model figure and changed the figure legend to better reflect the incompleteness of the current understanding, and the origin of the different parts of the model figure. In addition, we extended the final paragraph of the discussion (which lists still-unknown aspects of 2C) with the reviewer's mention of dsRNA shielding from innate immunity (lines 374-375). The other aspects mentioned by the reviewer as not yet fully understood are already mentioned in that paragraph.

Minor issues

Lines 43-45: I feel like you underplay the success of the poliovirus vaccination program. Approximately 30 of WPV1 in 2022 and the full eradication of WPV2 and 3. Vaccine derived polio is still an issue but even that is relatively low compared to where the world was in the 1950s.

We agree that the previous wording was not ideal. We replaced it and added another recent reference - related to the type 2 vaccine switch (lines 47-49).

Line 66. I agree there are 11 individual proteins but I feel like this leaves out the fact that some of the uncleaved precursors appear to have some functions, for example 2BC.

Good point. We have now added a mention of 2BC and the fact that it has distinct functions to the introduction (lines 70-71). 2BC is also mentioned in the legend of the model figure (figure 8).

Line 56: LD needs to be defined.

Well spotted thanks. Since the abbreviation was not used anywhere else we opted to spell it out instead (line 59).

Line 75. I think you have misrepresented Xia et al here. They clearly say that in their study that they show helicase and chaperone activity. I never managed to repeat that work but you should still report what they claim. One major thing is that they used insect expressed protein, whereas most people (including myself and in the paper under review) use E.coli expressed protein. Do post translational modifications play an important role in function?

You are right that the reference to their paper for this statement was incorrect. We have now made this part of the introduction more explicit (lines 82-83) and we also in the new discussion mention the possibility of e.g. post-translational modifications affecting 2C helicase activity, under reference to Xia et al (lines 359-361)

Line 103. Need to make it clear here it is poliovirus 2C.

Thanks, we added it (line 112).

Line 135. I assume you mean kDa instead of uM?

It should actually be μM. It is the solution concentration at which the assay was performed. We added some words to clarify this (line 154).

Figure 3. What do you mean by "Only 2C"? Is that MBP-2C? Maybe I am reading the data wrong but adding TEV does nothing? How do you know TEV is removing the MBP? It looks like MBP-2C binds to the liposomes just the same as cleaved MBP-2C. I see in line 165 you acknowledge this. Could an alternative conclusion for line 168 be that MBP isn't being cleaved off but that AH2 is too small to be exposed in that construct? Did you do that construct without MBP being cleaved? I think you need to confirm that MBP is being cleaved off.

Thanks for spotting this mistake. It should indeed be MBP-2C (in the absence of liposomes). We corrected figure 3. Also, in response to this comment and similar ones, we have now added a new supplementary figure showing SDS-PAGE gels of the reaction loaded onto flotation assays and DLS (figure S5). It shows that MBP-2C is cleaved.

Line 184. Is there a reason you use the 2019 paper as a reference instead of the far earlier Bienz et al papers? I'd suggest they are the seminal papers on 2C membrane association. Once again how is this work different from the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles" paper?

See our response above of the paper mentioned here (which we have now cited). As for why we cite the 2019 paper here: our statement pertains specifically to the contact sites between lipid droplets and replication organelles, not to the membrane binding of 2C per se. We have now added a more general mention of membrane remodelling by non-structural proteins in the introduction, where we cite on of the Bienz papers (lines 75-77).

Figure 5D. So only 1-3% of RNA is found in the upper fraction? Is that significant enough to say that dsRNA was recruited significantly more than ssRNA? How confident are you in your quantification of the starting amounts of RNA?

We agree that the fraction is low, however, the fluorescence signal is very clearly above background. We are thus confident in the measurement. The low percentage at the end of the experiment likely has a simple physico-chemical explanation: in a dynamic equilibrium in a density gradient, whatever RNA dissociates during the run will migrate away from the 2C-vesicle fraction and not be able to rebind. We still tried to address this concern by a complementary experiment where we used fluorescence anisotropy to measure binding of RNA to 2C on vesicles. While the measurements showed the same tendency, they curves were not clean enough to be published, which we think is due to the complex system with 2C bound to vesicles and clusters of vesicles. Still, in view of the relatively low percentage of measured recruitment we opted to adjust the paper title and the title of figure 5 (including the subheading related to figure 5) to put less emphasis on the dsRNA recruitment.

Line 223. Any idea why the MBP needs to be cleaved off? Clearly the MDB is accessible or it would not bind to the liposomes.

Since we have no data directly supporting this we prefer not to speculate in the paper. But one guess would be that the NTD of 2C, as implicated by previous publications, has a dual role in membrane binding and RNA binding. It may be that it can bind membrane while conjugated to MBP, but needs MBP to be removed in order to simultaneously bind membrane and RNA.

Line 237: missing "b" in "by"

Thanks. This paragraph was rewritten in the light of the changes to figure 6.

Figure 6. I don't fully understand the results here. Earlier you showed that the delta MBD didn't really bind SUV. So presumably it isn't really membrane bound. Why does it have similar activity to full-length MBP in your helicase assay if membrane is important? Did you do SUV and TEV protease only control?

We are very grateful to this reviewer (and others) for pointing out the need for a TEV control. When performing the control, we found that the TEV protease, at the high concentrations initially used, surprisingly had an artefactual RNA chaperone-like effect on its own. We then proceeded to titrate down the TEV protease concentration to the point where it no longer interfered. At this TEV protease concentration, although 2C was substantially cleaved (see the new supplementary figure S12), we could no longer detect an RNA chaperone activity. Thus, the contents of the new figure 6, and its conclusions, have been substantially changed. We now focused our attention on the remaining effect that 2C has on RNA: single-strand ribonuclease activity. These experiments were all conducted in the presence of RNase inhibitors, and the presence of Mg2+-dependent ribonuclease activity parallels a recent publication that found this for truncated 2C from hepatitis A and several enteroviruses.

Line 257: "staring"?

Thanks, corrected. A staring glycine would indeed be something strange.

Line 336. Need to change the u to mu.

Thanks, corrected.

Any discussion on your observation in Figure 1D that EV71 and CVB3 don't appear to have AH1 and AH2 or do you think that the domains are conserved across the different viruses?

Thanks for bringing this up. Based on this and a comment from another reviewer, we have now clarified our thinking around this. Since the glycine will introduce some flexibility between AH1 and AH2, we cannot say from the single alphafold predictions that this is THE kink angle. The presence of the kink in the predictions of several MBDs lends more credibility to the robustness of the observation, but most importantly the hydrophobic surfaces in AH1 and AH2 are non-aligned for ALL sequences we looked at. This is now described on lines 126-128.

Table 1 (and possibly elsewhere): an apostrophe is not the prime symbol. 5' compared to 5′.

Thanks, we corrected this throughout.

Line 702 "and" should be "an".

Thanks, corrected.

I couldn't open one of the movies (140844_0_supp_2820374_a2g272.avi).

Sorry to hear this, we will check the movie again.

Reviewer #3 (Significance (Required)):

Overall I liked the paper and is worth publishing. One of the issues in the 2C field is the difficulty in making pure 2C and carrying out in vitro assays that correlate with what is observed in the natural infection. I think this paper suffers from similar struggles with a 2C preparation that doesn't appear that pure. I think it also suffers from not having 2C from a wild-type infection. I don't think that it is feasible to get that kind of 2C but by once again using a recombinant protein from E.coli we are left with another manuscript that provides conflicting evidence of the functions of 2C without a definitive answer. The experiments are well done, although are missing some controls and the manuscript is laid out in a logical manner and is relatively easy to follow.

We thanks the reviewer for these comments. We believe that we have now provided better information regarding the purification of the recombinant 2C protein, and we do think that the controls present in the original manuscript and the revised manuscript alleviate the concerns about lack of specificity. Of course, isolating 2C vesicles from wildtype infection would be another interesting way of approaching its function, but such an approach would come with its own set of challenges related e.g. to the presence of confounding host factors.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

This is an interesting manuscript that reports the development of an in vitro membrane assay for probing the biochemical functions of the enterovirus 2C protein. The technique is interesting because it can be applied to 2C proteins from other members of the picornavirus family, an important group of mammalian pathogens. It has the capacity to probe different functions (e.g. membrane clustering, ATPase activity, RNA-binding and manipulation activities).

Overall, the manuscript is well written and gives a clear account of the work undertaken. It adds insight to previous studies of enteroviral (and picornaviral) 2C proteins, providing confirmation of some earlier work in a more physiological context and some new insights, particularly into the membrane and RNA binding aspects of 2C.

That said, there are a number of places where some amendment of the claims made is required to provide a more precise statement of the findings of this work. These are listed below.

We thank the reviewer for this positive feedback on our work, as well as for the specific comments below.

Line 21 (Abstract) - The authors claim to have shown that a conserved glycine divides the N-terminal membrane-binding domain into 2 helices. I would suggest instead what they have produced are computational predictions that this is the case - some way short of an experimental demonstration. Sequence analysis predicts helical secondary structure in the N-terminus and indeed Alphafold2 also predicts a helical structure, but these predictions require experimental verification. The authors should therefore rewrite sections that claim to have shown the presence of 2 helices. In doing so, they should perhaps also comment on the fact that Alphafold2 does not predict 2 helices in this region for all enteroviruses (see Fig 1D). Moreover, the sequence analysis in Fig. S1 shows the presence of two Lys residues in the segment 17-38; it would be interesting for the reader to have these indicated in the figures showing the Alphafold2 prediction - do they in any way interrupt the hydrophobic face of the predicted helix?

Thanks very much for this comment, which is in line with what other reviewers also wrote. We agree, and changed the abstract sentence. We have also rewritten the manuscripts in several places to address the limits of structure predictions and the eventual need for an experimental structure of full-length membrane-bound 2C (lines 126-128 and 315-318).

Line 82 (Introduction) - The authors write that the membrane binding domain (MBD) of poliovirus has been shown to mediate hexamerisation, citing Adams et al (2009) - reference 43. However, that is not what this paper shows. Rather it provides evidence of aggregation of an MBP-2C fusion protein into forms that ranged from tetramer to octamer, but no evidence that these aggregates assume functional forms (e.g. the presumed hexameric ring structure characteristic of the AAA+ ATPase family to which 2C belongs). As far as I am aware the first demonstration of hexameric ring formation by a picornaviral 2C protein was for the 2C of foot-and-mouth disease virus (see Sweeney et al, JBC, 2010). Although this is not an enterovirus, this finding was later confirmed for Echovirus 30 (ref 51). I should declare an interest here: the Sweeney paper is from my lab. I will leave it to the editor and the authors to determine how to write a more precise account of the early observations of hexamerisation in picornaviral and enteroviral 2C proteins.

Thanks very much for this insightful comment. As a response to this and other similar comments, we are much more cautious about our wording in the revised manuscript (see also response to comment below. In the part of the introduction discussed here (now lines 89-91) we now use the original wording of the Adams paper ("oligomerization"). In the context of that new text we didn't feel that Sweeney et al paper was a suitable reference, but we now cite it in the later mention of 2C's oligomeric/hexameric state in the first part of the Results (lines 137-138 ).

Line 132 - the authors used mass photometry to investigate oligomeric forms of their MBP-2C constructs and state that for the full length 2C protein "the high-mass peak closely corresponds to a hexamer". While it is true that the peak shown in Fig 2C aligns with the expected MW for an MBP-2C hexamer, the peak is very broad, indicative of the presence of other oligomeric states with lower and higher numbers of monomers. This should be commented on. Indeed, the finding seems to echo the early findings of Adams et al (ref 43) with poliovirus MBP-2C.

Thanks for this comment, which was also made by another reviewer. We cite here what we replied to that reviewer

...we do agree with the reviewer on the broad mass photometry peaks. To address this experimentally, we compared the existing MBP-2C spectra to new recordings on apoferritin, a highly stable homomultimeric protein complex of a similar mass to aa MBP-2C hexamer. The apoferritin mass estimate is overlayed with the full-length MBP-2C in the new figure 2D and the corresponding supplementary figure S3. This indeed shows that the MBP-2C peak is broader, i.e. consistent with a mix of species which are predominantly but not only hexamers. We describe and discuss this on lines 145-149.

Line 143 - for the reasons given above, this summary paragraph represents too strong a statement of what has been observed.

We agree, and changed the paragraph. It now only refers to "oligomerization" (lines 162-164).

Line 197 - I note that the authors did not test the membrane clustering capabilities of the 2C(41-329) construct. Although the 2C(deltaAH1) construct had already shown a significant loss of activity, the shorter construct could still have been a useful control. I don't think it is necessary for this experiment to be done, but if the authors have a rationale for not performing the experiment, perhaps they could include it in a revised manuscript.

Thanks for the suggestion. The rationale is that a protein that doesn't bind a membrane in the first place will also not cluster them (an action that requires binding TWO membranes). We now describe our reasoning on lines 220-222. Nevertheless, we did test these constructs in the new supplementary figure showing negative staining TEM (figure S10).

Line 223 - typo. I think you mean MBD.

Thanks! Corrected (now line 257).

Line 215 - the authors observed that the presence of ssDNA reduced membrane clustering and conclude that "nucleic acid binding partially outcompetes membrane tethering activity". Two things: (1) although I agree is it likely that this effect is due to binding of DNA to 2C, binding has not been demonstrated experimentally so the authors should be more careful in how they describe their result; (2) there is no data presented to show that RNA binding reduces membrane tethering so at best I think the conclusion has to be that the data are consistent with the notion that DNA binding reduces membrane tethering. It would of course be interesting to see the effects of RNA and I'm curious to know why the assay was not performed.

Thanks for the comment. The honest answer is that previous publications (primarily Yeager et al, NAR 2022) convinced us that the outcome should be near-identical with DNA, so we chose DNA oligos because they are cheaper and easier to work with. But we agree with the reviewer that RNA is of course more relevant. We now present a comparison at 5 μM of ssDNA and ssRNA, which in fact shows a slightly stronger effect on membrane clustering by RNA (figure 5C). In the light of this additional experiment, we feel that some of the text changes suggested by the reviewer may no longer be necessary.

Line 237 - typo: by, not y

Thanks. In the light of the extensive changes to figure 6 this text was removed.

Line 284 - the authors claim that 2C may only bind RNA after the N-terminus is liberated from 2B in infected cells, since cleavage of the MBP tag from their construct was needed for 2C to bind RNA in their in vitro assay. However, this does not automatically follow given the large structural differences between MBP and 2B and the fact that the authors have not tested the RNA binding capacity of a 2BC fusion protein. Their claim here is too strong and should be re-written.

We agree, and have added a discussion along the lines suggested by the reviewer (line 330-332).

Line 293 - The authors speculate that RNA binding might cause a shift between the membrane clustering activities and the role of the protein in RNA replication. However, since they have not shown that RNA binding reduces membrane clustering, this is too speculative.

In our revised manuscript we have studied the effect of RNA on membrane binding, thus we feel that this text is relevant in the context of the extended experiments.

Line 299-317 - within this discussion is the assumption that in their assay system enterovirus 2C adopts the ring-like hexameric structure typical of AAA+ ATPases. While I agree this may well be the case, it has not been demonstrated in this study so the authors should make clear they are making this assumption. The same applies to the legend of Fig 8.

This part of the discussion was extensively rewritten after our changes to figure 6. We now only refer to "hexamer" once in the corresponding part of the discussion, where we talk about structural models of hexamers produced by other groups who have crystallised fragments of 2C. There we believe we should refer to hexamers to accurately cite their work.

We are not sure what the reviewer is referring to when it comes to the legend for figure 8: the original legend had no reference to the oligomeric state of 2C. We have substantially changed figure 8 and its legend and the new figure and legend make no references to hexamers/oligomers.

Line 302 - the authors claim to have shown that 2C is 'selective' for dsRNA. I think at best they have shown a preference for binding dsRNA over ssRNA.

We changed the wording (line 349). We have also changed the title of the paper where we removed "double-stranded".

Line 313 - The sentence starting "A recent study..." needs a reference.

The revised discussion no longer contains this sentence.

Line 332 - the full sequence of the synthetic gene used in this study should be made available (e.g. as supplementary information or a deposited sequence with an accession number). This is a critical point before the paper can be published.

We will of course submit the sequences as supplementary data. Thanks for the reminder.

Line 362 - the authors should describe the likely points of attachment of fluorophores and comment on how this labelling might affect 2C function.

Thanks for the comment. In response to this and a similar comment from another reviewer, we discuss the likely conjugation site of the fluorophore (lines 175-181), and also (due to the proximity to the Zn finger) provide a new measurement showing that equal amounts of Zn can be detected in the labelled and unlabelled protein (figure S7).

Line 372 - Is a single protein standard (BSA) sufficient to calibrate the SEC-MALS system?

Yes, it is the recommended procedure (note that SEC-MALS is only dependent on scattering, not elution volumes etc).

Reviewer #4 (Significance (Required)):

As stated above this is an interesting study that presents findings from a novel assay. It will be of interest to picornavirologists and the wider community interested in the mechanisms of AAA+ ATPases.

We thanks the reviewer for this positive appraisal of our work.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #4

Evidence, reproducibility and clarity

This is an interesting manuscript that reports the development of an in vitro membrane assay for probing the biochemical functions of the enterovirus 2C protein. The technique is interesting because it can be applied to 2C proteins from other members of the picornavirus family, an important group of mammalian pathogens. It has the capacity to probe different functions (e.g. membrane clustering, ATPase activity, RNA-binding and manipulation activities).

Overall, the manuscript is well written and gives a clear account of the work undertaken. It adds insight to previous studies of enteroviral (and picornaviral) 2C proteins, providing confirmation of some earlier work in a more physiological context and some new insights, particularly into the membrane and RNA binding aspects of 2C.

That said, there are a number of places where some amendment of the claims made is required to provide a more precise statement of the findings of this work. These are listed below.

Line 21 (Abstract) - The authors claim to have shown that a conserved glycine divides the N-terminal membrane-binding domain into 2 helices. I would suggest instead what they have produced are computational predictions that this is the case - some way short of an experimental demonstration. Sequence analysis predicts helical secondary structure in the N-terminus and indeed Alphafold2 also predicts a helical structure, but these predictions require experimental verification. The authors should therefore rewrite sections that claim to have shown the presence of 2 helices. In doing so, they should perhaps also comment on the fact that Alphafold2 does not predict 2 helices in this region for all enteroviruses (see Fig 1D). Moreover, the sequence analysis in Fig. S1 shows the presence of two Lys residues in the segment 17-38; it would be interesting for the reader to have these indicated in the figures showing the Alphafold2 prediction - do they in any way interrupt the hydrophobic face of the predicted helix?

Line 82 (Introduction) - The authors write that the membrane binding domain (MBD) of poliovirus has been shown to mediate hexamerisation, citing Adams et al (2009) - reference 43. However, that is not what this paper shows. Rather it provides evidence of aggregation of an MBP-2C fusion protein into forms that ranged from tetramer to octamer, but no evidence that these aggregates assume functional forms (e.g. the presumed hexameric ring structure characteristic of the AAA+ ATPase family to which 2C belongs). As far as I am aware the first demonstration of hexameric ring formation by a picornaviral 2C protein was for the 2C of foot-and-mouth disease virus (see Sweeney et al, JBC, 2010). Although this is not an enterovirus, this finding was later confirmed for Echovirus 30 (ref 51). I should declare an interest here: the Sweeney paper is from my lab. I will leave it to the editor and the authors to determine how to write a more precise account of the early observations of hexamerisation in picornaviral and enteroviral 2C proteins. Line 132 - the authors used mass photometry to investigate oligomeric forms of their MBP-2C constructs and state that for the full length 2C protein "the high-mass peak closely corresponds to a hexamer". While it is true that the peak shown in Fig 2C aligns with the expected MW for an MBP-2C hexamer, the peak is very broad, indicative of the presence of other oligomeric states with lower and higher numbers of monomers. This should be commented on. Indeed, the finding seems to echo the early findings of Adams et al (ref 43) with poliovirus MBP-2C.

Line 143 - for the reasons given above, this summary paragraph represents too strong a statement of what has been observed.

Line 197 - I note that the authors did not test the membrane clustering capabilities of the 2C(41-329) construct. Although the 2C(deltaAH1) construct had already shown a significant loss of activity, the shorter construct could still have been a useful control. I don't think it is necessary for this experiment to be done, but if the authors have a rationale for not performing the experiment, perhaps they could include it in a revised manuscript.

Line 223 - typo. I think you mean MBD.

Line 215 - the authors observed that the presence of ssDNA reduced membrane clustering and conclude that "nucleic acid binding partially outcompetes membrane tethering activity". Two things: (1) although I agree is it likely that this effect is due to binding of DNA to 2C, binding has not been demonstrated experimentally so the authors should be more careful in how they describe their result; (2) there is no data presented to show that RNA binding reduces membrane tethering so at best I think the conclusion has to be that the data are consistent with the notion that DNA binding reduces membrane tethering. It would of course be interesting to see the effects of RNA and I'm curious to know why the assay was not performed.

Line 237 - typo: by, not y

Line 284 - the authors claim that 2C may only bind RNA after the N-terminus is liberated from 2B in infected cells, since cleavage of the MBP tag from their construct was needed for 2C to bind RNA in their in vitro assay. However, this does not automatically follow given the large structural differences between MBP and 2B and the fact that the authors have not tested the RNA binding capacity of a 2BC fusion protein. Their claim here is too strong and should be re-written.

Line 293 - The authors speculate that RNA binding might cause a shift between the membrane clustering activities and the role of the protein in RNA replication. However, since they have not shown that RNA binding reduces membrane clustering, this is too speculative.

Line 299-317 - within this discussion is the assumption that in their assay system enterovirus 2C adopts the ring-like hexameric structure typical of AAA+ ATPases. While I agree this may well be the case, it has not been demonstrated in this study so the authors should make clear they are making this assumption. The same applies to the legend of Fig 8.

Line 302 - the authors claim to have shown that 2C is 'selective' for dsRNA. I think at best they have shown a preference for binding dsRNA over ssRNA.

Line 313 - The sentence starting "A recent study..." needs a reference.

Line 332 - the full sequence of the synthetic gene used in this study should be made available (e.g. as supplementary information or a deposited sequence with an accession number). This is a critical point before the paper can be published.

Line 362 - the authors should describe the likely points of attachment of fluorophores and comment on how this labelling might affect 2C function.

Line 372 - Is a single protein standard (BSA) sufficient to calibrate the SEC-MALS system?

Significance

As stated above this is an interesting study that presents findings from a novel assay. It will be of interest to picornavirologists and the wider community interested in the mechanisms of AAA+ ATPases.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

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Referee #3

Evidence, reproducibility and clarity

The manuscript provides insights into the role of the N-terminus in membrane binding and its importance in the various functions of 2C.

Major issues

Line 103-119. Is this novel? I thought people had done a lot of bioinformatic analysis of PV 2C (especially Wimmer) who also did mutational work to analyse the importance of various amino acids in the N-terminal helix. I feel like the paper in general, and this section in particular, underplays the large body of work that has been done on the amphipathic helix by various groups.

Line 132. Did you validate your column with known MW standards? The peak for full length and deltaAH1 look fairly standard for 2C, in that you have a mixture of species. Not sure you can say it is a hexamer when it is such a broad peak. C doesn't really help you too much since the counts at 400 (pentamer) and 480 (hexamer) are almost the same with quite large error bars. Like most people that have worked with 2C I think the best you can say is that you are making some kind of oligomerized 2C that includes hexamer, pentamer, etc. Why no dimer for MBP-2C and MBP-2C(delta AH1) when compared to the other constructs?

Line 143. Does your data show that there are two amphipathic helices? Bioinformatics suggests it but your experiments just show the importance of the two areas in oligomerization, not that it is forming two helices.

Figure S2. Your preps are still relatively dirty, which isn't ideal for biochemical assays. Especially lane 3, where you are looking at 50-60% purity. I don't want you to re-run experiments but I think you need to comment on the purity of the protein you are working with. Also I don't like that you removed the top and bottom of the SDS-PAGE. How much protein never entered the gel. Is there a big fat band at 20 kDa? You need to have the full gel here. Did you measure 260 nm of the preps as well to see if you had bound RNA to the 2C?

Lines 170. Wasn't this done in the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles"? I don't see it referenced. What is novel about the current work when compared to that paper? Any differences?

I'm surprised by the lack of electron microscopy (negative stain mostly) of both the oligomerized 2C and the various liposomes. I know the Carlson group is a microscopy group so why the lack of validation using electron microscopy of the various DLS experiments? I know you did cryo-ET for one of the constructs but I think negative stain electron microscopy of other constructs would be useful.

Figure 4C. What evidence is there that this is 2C apart from you added it to the liposomes? It also comes back to the relative impurity of your protein prep. Could this be E.coli contamination?

Figure 8. Is this model supported by the data in this paper? Your cryo-ET says that 2C is there but that isn't supported by any other data. How is the dsRNA protected from the innate immune system in this model? is it just sat out in the cytosol? How is the nascent ssRNA packeged into the capsid? Is there competition between the dsRNA and capsid for 2C binding (which your model suggests)? I know it sounds like I am being overly critical of the model but in my opinion there are still too many unanswered questions in the field to come up with a half decent model.

Minor issues

Lines 43-45: I feel like you underplay the success of the poliovirus vaccination program. Approximately 30 of WPV1 in 2022 and the full eradication of WPV2 and 3. Vaccine derived polio is still an issue but even that is relatively low compared to where the world was in the 1950s.

Line 66. I agree there are 11 individual proteins but I feel like this leaves out the fact that some of the uncleaved precursors appear to have some functions, for example 2BC.

Line 56: LD needs to be defined.

Line 75. I think you have misrepresented Xia et al here. They clearly say that in their study that they show helicase and chaperone activity. I never managed to repeat that work but you should still report what they claim. One major thing is that they used insect expressed protein, whereas most people (including myself and in the paper under review) use E.coli expressed protein. Do post translational modifications play an important role in function?

Line 103. Need to make it clear here it is poliovirus 2C.

Line 135. I assume you mean kDa instead of uM?

Figure 3. What do you mean by "Only 2C"? Is that MBP-2C? Maybe I am reading the data wrong but adding TEV does nothing? How do you know TEV is removing the MBP? It looks like MBP-2C binds to the liposomes just the same as cleaved MBP-2C. I see in line 165 you acknowledge this. Could an alternative conclusion for line 168 be that MBP isn't being cleaved off but that AH2 is too small to be exposed in that construct? Did you do that construct without MBP being cleaved? I think you need to confirm that MBP is being cleaved off.

Line 184. Is there a reason you use the 2019 paper as a reference instead of the far earlier Bienz et al papers? I'd suggest they are the seminal papers on 2C membrane association. Once again how is this work different from the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles" paper?

Figure 5D. So only 1-3% of RNA is found in the upper fraction? Is that significant enough to say that dsRNA was recruited significantly more than ssRNA? How confident are you in your quantification of the starting amounts of RNA?

Line 223. Any idea why the MBP needs to be cleaved off? Clearly the MDB is accessible or it would not bind to the liposomes.

Line 237: missing "b" in "by"

Figure 6. I don't fully understand the results here. Earlier you showed that the delta MBD didn't really bind SUV. So presumably it isn't really membrane bound. Why does it have similar activity to full-length MBP in your helicase assay if membrane is important? Did you do SUV and TEV protease only control?

Line 257: "staring"?

Line 336. Need to change the u to mu.

Any discussion on your observation in Figure 1D that EV71 and CVB3 don't appear to have AH1 and AH2 or do you think that the domains are conserved across the different viruses?

Table 1 (and possibly elsewhere): an apostrophe is not the prime symbol. 5' compared to 5′.

Line 702 "and" should be "an".

I couldn't open one of the movies (140844_0_supp_2820374_a2g272.avi).

Significance

Overall I liked the paper and is worth publishing. One of the issues in the 2C field is the difficulty in making pure 2C and carrying out in vitro assays that correlate with what is observed in the natural infection. I think this paper suffers from similar struggles with a 2C preparation that doesn't appear that pure. I think it also suffers from not having 2C from a wild-type infection. I don't think that it is feasible to get that kind of 2C but by once again using a recombinant protein from E.coli we are left with another manuscript that provides conflicting evidence of the functions of 2C without a definitive answer. The experiments are well done, although are missing some controls and the manuscript is laid out in a logical manner and is relatively easy to follow.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #2

Evidence, reproducibility and clarity

The authors present an alternative assay system to investigate picornavirus 2C, a protein that is tricky to analyze biochemically in its full length form because of an amphipathic helix at the N-terminus. Poliovirus 2C is expressed with an N-terminal MBP tag, a 50kD protein that helps with solubility as is commonly used for 2C investigations. A difference here is that liposomes are included to mimic membranes for 2C attachment. The key findings are that 2C induces clustering of of liposomes, that double stranded RNA binding by 2C impacts this clustering effect and that a free N-terminus (after cleavage of MBP by TEV protease) is needed for RNA binding and an ATP independent (ie non helicase) RNA duplex separation activity.

Major:

In the floatation assays in figure 3 the authors use a system where MBP-2C is fluorophore-labeled with ATTO488 on exposed cysteines. Poliovirus and other enterovirus 2C has a very well characterized zinc finger domain that has cysteines coordinating a zinc ion. Mutation experiments previously showed that these cysteines are necessary for viral replication and 2C stability. Have the authors controlled for disruption of the zinc finger domain by the labelling of cysteines with ATT0488 and checked if the protein remains folded?

In the analysis of the amphipathic helix, did the authors include membranes in their structural predictions o just the free helix? How does inclusion of membranes impact the predictions? In the predictions in Figure D, only 2 of 4 show a kink and there doesn't seem to be a correlation between those that predict a kink or not and whether the hydrophobic side is aligned in Figure S1.

Based on previous structures of 2C from different viruses the N-terminal amphipathic helix containing region is predicted to localize on one face of the predicted hexametric structure tethering 2C to the membrane. How does the authors hypothesized model explain 2C dependent clustering? is there evidence that 2C hexamers can oligomerize further into dodecamers for example, maintaining separate faces to enable N-terminal interaction with different membranes? What is the distance between the liposomes in figure 4 at the points of density attributed to 2C? How does this compare to the size of 2C determined in previous structural studies? Is it consistent with one hexamer/2 hexamers sitting on top of one another?

In the Discussion lines 278-285 the authors suggest that having MBP attached may reflect the polyprotein condition. Can they make a construct with MBP-2B2C to examine interaction with liposomes and assess 2C function?

Discussion lines 293-296, the possibility of two different populations of 2C, binding RNA or membranes cannot be excluded, there is much more 2C around late in infection that present in early infection- the model in figure 8 doesn't acknowledge/capture this.

Discussion lines 313-317, the authors don't reference a study where a mutant of foot-and-mouth disease virus 2C lacking the n-terminal amphipathic helix that could bind but not hydrolyze ATP, hexamerized in the presence of RNA that seems pertinent here (PMID: 20507978).

Some evidence of MBP-2C cleavage by TEV in the different assays used should be presented as this is a major focus of discussion and currently no gels show TEV cleavage is happening.

Significance

The work presents an additional methodology to investigate a a protein that has previously been difficult to study. The authors acknowledge that there is still a lot of 2C biology that remains to be discovered.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In this study by Shankar and colleagues, the authors aim to understand the structure and function of the enterovirus 2C protein, a putative viral helicase with AAA+ ATPase activity. Using poliovirus (as a model enterovirus) 2C, the author's propose the protein contains two amphipathic helices (AH1 and AH2) at the N-terminus that are divided by a conserved glycine. Using purified MBP-tagged 2C and N-terminal 2C truncations, their data suggests AH1 is primarily responsible for clustering at membranes, whilst AH2 is the main mediator of 2C oligmerisation and membrane binding. Furthermore, 2C was suggested to be able to recruit RNA to membranes, with a preference for dsRNA, and the author's data implies that the helicase activity of 2C is ATP-independent. Instead, the ATP activity appears to be required for 2C hexamer formation or chaperone activity. The manuscript is generally well written /presented and the author's present very interesting data which raises several questions, some of which require additional experimentation to help support the author's conclusions. Specific comments are as follows.

Major Comments:

1. The authors use four main constructs throughout the paper: full-length 2C, 2C with deletion of AH1 (ΔAH1), 2C with both AH1 and AH2 deleted (ΔMBP) and 2C with an extended N-terminal deletion. From this, the author's draw conclusions on the function of both AH1 and AH2. One of the author's main conclusions is that AH2 is the main mediator of 2C membrane association (e.g., in line 169). However, is it possible to conclude the relative importance of AH1 vs AH2 without testing a construct containing the deletion of AH2 only (ΔAH2)? This should be generated and used alongside this data to fully define the relative importance of AH1 and AH2 in these assay and remove the possibility that the deletion of AH1 changes the structure and/or function of AH2, which could also result in the observed differences.
2. Previous structural predictions of 2C do not appear to have two separate AHs at the N-terminus. Are the AH1 and AH2 structures predicted to be formed in the context of the entire 2C protein, 2BC precursors and polyprotein? Are there structural approaches that could provide experimental evidence for two separate AH at the N-terminus?
3. Why are the 2C dimers (lines 137-138) not apparent on the mass photometry data presented (figure 2)?
4. It appeared that binding of ΔMBD-2C was better when POPS is in the membrane (line 174). What is the explanation for this and was this finding significant?
5. From the author's data on lipid drop clustering they conclude ΔAH1 is more effective for clustering, however, the ΔAH1 construct produces pentamers not hexamers (from Figure 2). Is formation of hexamers related to or required for membrane clustering?
6. The replicon data presented in Figure 7 should include a replication-defective control (e.g., polymerase mutant), in order to compare how defective in replication ΔAH1 and ΔMBP deletions are compared to a fully-defective construct. Likewise, deletion of ΔAH1 in this construct is likely to affect processing of the viral polyprotein where several previous studies with picornaviruses have demonstrated that the residues in the P2'-P4' positions can change cleavage efficiency (e.g., PMID: 2542331), or the structure of 2C, leading to the reduction of replication.
7. How does the author's model of ATPase-independent helicase activity and an APT-dependent required RNA chaperone activity fit with 2 step model for RNA binding and ATPase activity suggested by Yeager et al (PMID: 36399514)? Optional major comments that would increase the significance of the work:
8. The preference for dsRNA over ssRNA appears to be quite small (Figure 5d). In the context of a viral infection where ssRNA is likely to outnumber dsRNA at different times during infection is this preference physiologically relevant? In relation to this, what size stretch of dsRNA is required for preference, and could this correspond to cis-acting RNA structural elements, dsRNA as it escapes 3D polymerase or as part of the RF and RI forms (PMID: 9343205)? What is the proposed mechanism of how dsRNA outcompetes membrane tethering of 2C? OPTIONAL
9. The author's study has been conducted in the absence of other viral non-structural proteins. What is the physiological importance of the observations, such as membrane interaction/clustering or RNA binding when presented in the context of the other replication machinery. OPTIONAL
10. Do 2C monomers, dimers and hexamers have different functions in viral replication perhaps at different stages of replication and which of these forms are relevant during viral infection or can they all be detected during infection? Can any suggested separate functional arrangements be separated by genetic complementation experiments? OPTIONAL

Minor comments:

1. The author's appear to interchange between naming/nomenclature of the constructs which makes it confusing to follow (for example, ΔMBD is the same as 2C(41-329) likewise, 2C(Δ115) is sometimes called 2C(116-329)). It would be much easier to follow if the naming of constructs was consistent throughout (unless I am misunderstanding some subtlety in the difference between such constructs).
2. The author's suggest a pentamer arrangement for the ΔAH1 construct, however in the mass photometry data (figure 2D), a hexamer is indicated with the arrow. It would be helpful to change the label to indicate the size of the pentamer where this is being generated, not the hexamer.
3. In most figures, data for full-length 2C, ΔAH1 and ΔMBP is shown. However data for ΔMBP is missing in Figure 4. Using ΔMBP may demonstrate even lower clustering, hinting that AH2 is also involved in this process.
4. I think it would be better for normalise the data in the flotation experiments such that the percentage of 2C in the upper faction is presented as relative to the amount of lipid in the upper fraction (presented in Figure S4).
5. At several places (e.g., lines 232 and 272) the author's refer to "realistic systems". I think the term "physiologically relevant" might be more appropriate.
6. Line 237: I think "y" is a typo and should read "by".

Significance

I have limited expertise with structural biology but specialise my research on positive-sense RNA virus replication, structure and function. This research is of interest to a broad audience of researchers investigating many positive-sense RNA viruses, which extends beyond the viral family studied here. The work utilises novel techniques to begin to understand the specific roles of 2C in poliovirus replication. The author's data add important incremental new insight into recent studies on viral helicase proteins as referenced in the study, however, a key limitation is understanding the importance/relevance of their observations during a viral infection.

Résumé de la vidéo [00:00:00][^1^][1] - [00:27:18][^2^][2]:

Cette vidéo présente une conférence sur l'éducation et l'évaluation des performances en mathématiques, en se concentrant sur les Olympiades de mathématiques et l'impact de la participation à ces compétitions sur les carrières scientifiques futures. Les intervenants discutent de la sélection et de la préparation des participants, ainsi que des disparités sociales et de genre dans l'accès à ces compétitions.

Points forts: + [00:00:30][^3^][3] Introduction de la conférence * Présentation des intervenants et du sujet * Importance des réactions suscitées par les interventions précédentes * Annonce du thème des Olympiades de mathématiques + [00:01:10][^4^][4] Parcours académique et recherche * Présentation du parcours académique de Colomb Sayar * Objectifs de sa thèse sur l'éducation en mathématiques * Rôle des concours périscolaires dans la démocratisation de la discipline + [00:03:01][^5^][5] Les Olympiades internationales de mathématiques * Présentation des Olympiades et de leur fonctionnement * Discussion sur la sous-représentation des filles dans les compétitions * Impact de la participation sur la formation et le choix de carrière en mathématiques + [00:09:01][^6^][6] Histoire et évolution des Olympiades * Croissance du nombre de participants et de pays au fil des années * Analyse des trajectoires de réussite de différentes équipes nationales * Stratégies de recrutement et de préparation des participants + [00:15:59][^7^][7] Ancienneté et réutilisation des candidats * Étude de l'ancienneté des participants et de leur réutilisation par les équipes nationales * Analyse des gains individuels liés à la participation multiple * Absence de corrélation entre la réutilisation des candidats et le succès de l'équipe + [00:26:55][^8^][8] Le cas spécifique de la France * Histoire des procédures de sélection et de préparation de l'équipe française * Transition d'un modèle de détection de talent à un modèle de développement de potentiel * Rôle des enseignants dans l'accès à la participation aux Olympiades

Résumé de la vidéo [00:27:21][^1^][1] - [00:43:36][^2^][2] : Cette vidéo présente une analyse détaillée des méthodes de sélection et de préparation des participants français aux Olympiades internationales de mathématiques. Elle examine l'évolution des pratiques depuis les années 90, soulignant l'importance de l'équité et de l'efficacité dans le recrutement et la formation des candidats. L'intervenant discute également du rôle des enseignants et des changements institutionnels qui ont influencé les performances de la France dans ces compétitions.

Points saillants: + [00:27:21][^3^][3] Les débuts de la sélection * Sélection initiale très individualisée et peu formalisée * Recrutement restreint à des élèves spécifiques du lycée Louis Legrand * Préparation rudimentaire pour les Olympiades + [00:29:39][^4^][4] Chute des performances * Diminution significative des scores moyens des participants français dans les années 90 * Changement des critères de sélection et d'envoi des participants + [00:32:00][^5^][5] Création de l'association Animath * Réponse à la baisse des performances et mobilisation de la communauté mathématique * Formalisation et institutionnalisation des procédures de sélection et de préparation + [00:37:01][^6^][6] Impact de la préparation sur la scolarité * Recrutement de plus en plus précoce des participants potentiels * Interaction entre la préparation aux Olympiades et la scolarité des élèves + [00:39:46][^7^][7] Disparités dans le recrutement * Disparités observées dès les premières étapes de la Coupe Animath * Importance de l'information et de la préparation par les enseignants + [00:42:24][^8^][8] Amélioration des procédures * Nécessité de mieux doter les enseignants en ressources pour préparer les élèves * Questionnement sur l'évolution de l'association Animath pour une meilleure efficacité et équité

Résumé de la vidéo [00:00:00][^1^][1] - [00:20:49][^2^][2] : Cette vidéo présente une conférence de Jérôme Dovioot, professeur de sociologie à l'École normale supérieure et chercheur, sur l'éducation et l'évaluation des performances en lecture. Il aborde les méthodes d'enseignement de la lecture en France, les résultats en baisse des élèves en primaire, et l'impact des différentes méthodes sur les inégalités scolaires.

Points forts : + [00:00:23][^3^][3] Introduction de Jérôme Dovioot * Expert en transmission des savoirs et apprentissage * Auteur d'ouvrages sur l'école et la lecture * Membre du conseil scientifique de l'éducation nationale + [00:01:35][^4^][4] Les pratiques enseignantes et les inégalités scolaires * Baisse continue des résultats en lecture * Importance de la vitesse d'automatisation de la lecture * Liens entre méthodes d'enseignement et réussite des élèves + [00:05:16][^5^][5] Débat sur les méthodes d'enseignement de la lecture * Opposition entre approches phonique synthétique et idéovisuelle * Impact historique et sociologique des principes idéovisuels * Invalidation des arguments par la recherche + [00:09:08][^6^][6] Pratiques d'enseignement de la lecture en France * Méthode phonique synthétique strict et méthodes mixtes * Analyse des manuels d'enseignement et pratiques déclarées * Influence des méthodes sur les performances des élèves + [00:13:11][^7^][7] Enquête sur les méthodes d'enseignement et résultats des élèves * Utilisation des évaluations nationales pour l'analyse * Caractéristiques des enseignants et résultats des élèves * Prédiction des performances en lecture basée sur diverses variables + [00:17:39][^8^][8] Efficacité des méthodes d'enseignement * Meilleurs résultats avec la méthode synthétique strict * Écarts de performance plus importants pour les élèves fragiles * Nécessité d'adapter les méthodes aux niveaux des élèves

Résumé de la vidéo [00:20:51][^1^][1] - [00:32:25][^2^][2]:

Cette partie de la vidéo aborde l'éducation et l'évaluation des performances des élèves en lecture, en se concentrant sur l'impact des méthodes d'enseignement et le contexte social des écoles. L'étude présentée montre que les méthodes synthétiques strictes sont plus efficaces, surtout pour les élèves issus de milieux sociaux défavorisés et ceux ayant une maîtrise initiale faible du principe alphabétique.

Points forts: + [00:20:51][^3^][3] Disparités dans l'apprentissage * Les écarts de performance augmentent avec le niveau social de l'école * Les méthodes synthétiques strictes sont plus efficaces pour les élèves fragiles * L'impact des méthodes varie selon le niveau initial des élèves + [00:23:24][^4^][4] Méthodes d'enseignement et efficacité * La méthode synthétique stricte favorise une meilleure compréhension de la lecture * Les élèves ayant une bonne maîtrise du principe alphabétique sont moins affectés par les méthodes mixtes * Les méthodes mixtes peuvent introduire de la confusion chez les élèves avec une maîtrise faible + [00:26:08][^5^][5] Influence du contexte social * Les méthodes efficaces profitent davantage aux élèves de milieux populaires * L'apprentissage familial joue un rôle crucial dans l'acquisition de la lecture * Les interventions parentales compensent les méthodes d'enseignement moins efficaces + [00:29:07][^6^][6] Conséquences et recommandations * La méthode d'enseignement a un poids décisif sur les inégalités sociales d'apprentissage * Une politique de lutte contre les inégalités doit améliorer l'efficacité pédagogique * La formation des enseignants est essentielle pour l'amélioration de l'enseignement de la lecture

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:01][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'éducation et l'évaluation des performances cognitives, avec un accent sur l'inhibition cognitive et son rôle dans l'apprentissage. Olivier Houdé, un expert en psychologie du développement, discute des mécanismes internes du cerveau qui apprend, soulignant l'importance de la métacognition et de l'autoévaluation.

Points saillants: + [00:00:23][^3^][3] Introduction d'Olivier Houdé * Ancien instituteur, devenu professeur de psychologie * Spécialiste de la catégorisation et de l'inhibition cognitive + [00:02:36][^4^][4] L'évaluation et l'auto-évaluation des performances * Importance de comprendre le fonctionnement du cerveau * Nécessité de connaître les mécanismes internes pour une éducation efficace + [00:04:35][^5^][5] Le cerveau, théâtre de l'éducation * Le cerveau est encore l'angle mort de l'éducation nationale * L'éducation se fait souvent sans comprendre les mécanismes internes du cerveau + [00:09:45][^6^][6] Technologies d'imagerie cérébrale * Utilisation de l'IRM et d'autres outils pour observer le cerveau en développement * Importance de la dynamique du cerveau dans l'apprentissage + [00:17:01][^7^][7] Dynamique du cerveau et apprentissage * Apprentissage par reconfiguration antéro-postérieur et postéro-anérieur * Rôle de l'inhibition cognitive dans la correction des biais cognitifs + [00:20:03][^8^][8] L'inhibition cognitive comme outil d'apprentissage * L'inhibition permet de contrôler et de corriger les automatismes et les biais * L'importance de l'inhibition dans l'éducation et le développement cognitif

Résumé de la vidéo [00:24:06][^1^][1] - [00:46:31][^2^][2]:

Cette partie de la vidéo aborde l'éducation et l'évaluation des performances, en se concentrant sur les processus d'apprentissage et d'inhibition dans le cerveau humain. Pierre-Michel Menger discute de la manière dont l'apprentissage implique l'élimination de connexions synaptiques inutiles, ce qui est crucial pour comprendre les erreurs des enfants à l'école. Il souligne l'importance de l'inhibition cognitive et comment elle peut expliquer les biais cognitifs chez les adultes, même ceux qui sont bien éduqués.

Points forts: + [00:24:06][^3^][3] Dynamiques d'apprentissage * L'apprentissage comme élimination synaptique * Importance de l'inhibition dans l'apprentissage * Influence de l'inhibition sur les performances scolaires + [00:28:00][^4^][4] Maturation du cortex préfrontal * Développement lent et impact sur l'inhibition * Relation avec les biais cognitifs chez les adultes * Fragilité persistante de l'inhibition chez l'adulte + [00:32:00][^5^][5] Développement cognitif non linéaire * Comparaison avec le modèle linéaire de développement * Importance des stratégies cognitives variées * Nécessité de l'inhibition pour choisir la bonne stratégie + [00:38:00][^6^][6] Entraînement à l'inhibition * Différence entre l'entraînement logique et inhibiteur * Impact de l'inhibition sur la performance cognitive * Utilisation de l'inhibition dans l'éducation moderne + [00:42:00][^7^][7] Intelligence artificielle et inhibition * Parallèles entre le cerveau humain et l'IA * Rôle de l'inhibition dans le contrôle des systèmes IA * Importance de l'inhibition pour éviter les dérives de l'IA

Résumé de la vidéo [00:46:32][^1^][1] - [00:48:37][^2^][2]:

Cette partie de la vidéo discute de l'éducation et de l'évaluation des performances à travers des outils pédagogiques innovants. Pierre-Michel Menger présente des méthodes pour enseigner la métacognition aux enfants, en utilisant des personnages et des jeux pour développer la flexibilité cognitive.

Points forts: + [00:46:32][^3^][3] Outils pédagogiques pour l'éducation * Création de personnages et posters métacognitifs * Utilisation de jeux pour enseigner la flexibilité cognitive + [00:47:00][^4^][4] La coéducation à la maison * Les jeux sont traduits en plusieurs langues * La maison comme lieu d'éducation complémentaire + [00:47:38][^5^][5] Livres pour enfants et adolescents * "Explore ton cerveau" pour les maternels * "C'est pas moi, c'est mon cerveau" pour les ados

Résumé de la vidéo [00:48:49][^1^][1] - [00:49:35][^2^][2]:

Cette partie de la vidéo aborde l'éducation et l'évaluation de ses performances. Pierre-Michel Menger discute de la valorisation croissante des performances et de la fragilité de cette perception due au scepticisme envers l'objectivité quantitative.

Points forts : + [00:48:49][^3^][3] La valorisation des performances * De plus en plus reconnues * De plus en plus fragiles * Scepticisme croissant + [00:49:01][^4^][4] L'importance de la recherche * Améliorer le système éducatif * Utiliser les connaissances produites * Perspective pragmatique + [00:49:20][^5^][5] Ressources pour approfondir * Références suggérées * Pour ceux qui veulent en savoir plus * Remerciements et fin de la présentation

Résumé de la vidéo [00:00:00][^1^][1] - [00:24:01][^2^][2]:

Cette vidéo présente une conférence de Pierre-Michel Menger sur l'éducation et l'évaluation des performances cognitives, avec un accent sur l'inhibition cognitive et son rôle dans l'apprentissage. Olivier Houdé, un expert en psychologie du développement, discute des mécanismes internes du cerveau qui apprend, soulignant l'importance de la métacognition et de l'autoévaluation.

Points saillants: + [00:00:23][^3^][3] Introduction d'Olivier Houdé * Ancien instituteur, devenu professeur de psychologie * Spécialiste de la catégorisation et de l'inhibition cognitive + [00:02:36][^4^][4] L'évaluation et l'auto-évaluation des performances * Importance de comprendre le fonctionnement du cerveau * Nécessité de connaître les mécanismes internes pour une éducation efficace + [00:04:35][^5^][5] Le cerveau, théâtre de l'éducation * Le cerveau est encore l'angle mort de l'éducation nationale * L'éducation se fait souvent sans comprendre les mécanismes internes du cerveau + [00:09:45][^6^][6] Technologies d'imagerie cérébrale * Utilisation de l'IRM et d'autres outils pour observer le cerveau en développement * Importance de la dynamique du cerveau dans l'apprentissage + [00:17:01][^7^][7] Dynamique du cerveau et apprentissage * Apprentissage par reconfiguration antéro-postérieur et postéro-anérieur * Rôle de l'inhibition cognitive dans la correction des biais cognitifs + [00:20:03][^8^][8] L'inhibition cognitive comme outil d'apprentissage * L'inhibition permet de contrôler et de corriger les automatismes et les biais * L'importance de l'inhibition dans l'éducation et le développement cognitif

Résumé de la vidéo [00:24:06][^1^][1] - [00:46:31][^2^][2]:

Cette partie de la vidéo aborde l'éducation et l'évaluation des performances, en se concentrant sur les processus d'apprentissage et d'inhibition dans le cerveau humain. Pierre-Michel Menger discute de la manière dont l'apprentissage implique l'élimination de connexions synaptiques inutiles, ce qui est crucial pour comprendre les erreurs des enfants à l'école. Il souligne l'importance de l'inhibition cognitive et comment elle peut expliquer les biais cognitifs chez les adultes, même ceux qui sont bien éduqués.

Points forts: + [00:24:06][^3^][3] Dynamiques d'apprentissage * L'apprentissage comme élimination synaptique * Importance de l'inhibition dans l'apprentissage * Influence de l'inhibition sur les performances scolaires + [00:28:00][^4^][4] Maturation du cortex préfrontal * Développement lent et impact sur l'inhibition * Relation avec les biais cognitifs chez les adultes * Fragilité persistante de l'inhibition chez l'adulte + [00:32:00][^5^][5] Développement cognitif non linéaire * Comparaison avec le modèle linéaire de développement * Importance des stratégies cognitives variées * Nécessité de l'inhibition pour choisir la bonne stratégie + [00:38:00][^6^][6] Entraînement à l'inhibition * Différence entre l'entraînement logique et inhibiteur * Impact de l'inhibition sur la performance cognitive * Utilisation de l'inhibition dans l'éducation moderne + [00:42:00][^7^][7] Intelligence artificielle et inhibition * Parallèles entre le cerveau humain et l'IA * Rôle de l'inhibition dans le contrôle des systèmes IA * Importance de l'inhibition pour éviter les dérives de l'IA

Résumé de la vidéo [00:46:32][^1^][1] - [00:48:37][^2^][2]:

Cette partie de la vidéo discute de l'éducation et de l'évaluation des performances à travers des outils pédagogiques innovants. Pierre-Michel Menger présente des méthodes pour enseigner la métacognition aux enfants, en utilisant des personnages et des jeux pour développer la flexibilité cognitive.

Points forts: + [00:46:32][^3^][3] Outils pédagogiques pour l'éducation * Création de personnages et posters métacognitifs * Utilisation de jeux pour enseigner la flexibilité cognitive + [00:47:00][^4^][4] La coéducation à la maison * Les jeux sont traduits en plusieurs langues * La maison comme lieu d'éducation complémentaire + [00:47:38][^5^][5] Livres pour enfants et adolescents * "Explore ton cerveau" pour les maternels * "C'est pas moi, c'est mon cerveau" pour les ados

Résumé de la vidéo [00:48:49][^1^][1] - [00:49:35][^2^][2]:

Cette partie de la vidéo aborde l'éducation et l'évaluation de ses performances. Pierre-Michel Menger discute de la valorisation croissante des performances et de la fragilité de cette perception due au scepticisme envers l'objectivité quantitative.

Points forts : + [00:48:49][^3^][3] La valorisation des performances * De plus en plus reconnues * De plus en plus fragiles * Scepticisme croissant + [00:49:01][^4^][4] L'importance de la recherche * Améliorer le système éducatif * Utiliser les connaissances produites * Perspective pragmatique + [00:49:20][^5^][5] Ressources pour approfondir * Références suggérées * Pour ceux qui veulent en savoir plus * Remerciements et fin de la présentation