I would move this logo bar up right underneath the header/above the fold section. As for the podcast features, could you move them into the about page?

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This passage speaks directly to what we studied under theories of the flesh. When Hartman writes about the “imperative to respect black noise—the shrieks, the moans, the non-sense, and the opacity,” she invokes the idea that the flesh carries forms of knowledge that cannot be translated into the language of the archive or the law (Moraga and Anzaldúa, 2022). Hartman’s insistence on “noise” directly challenges the logic of capitalism as the author states. In class, we discussed Silvia Federici’s work on capital accumulation where, broadly, capitalism emerges through the transformation of people into laboring bodies, units of value, and property (Federici, 2018). However, respecting the “noise” wouldn't allow for that. The shrieks, moans, and non-sense that Hartman describes cannot be translated into value or productivity. Moreover, respecting the pain experienced and humanizing the affected would make it more difficult to justify that exploitation.

Hartman describes her method as an effort to “topple the hierarchy of discourse” a hierarchy built by colonial, patriarchal, and Western epistemologies. This method "...which weaves past, present, and future..." is tied to the Indigenous epistemologies we learned about in class, regarding conceptions of time as non-linear and relational, something lived with, not simply looked back upon. By "...narrating the time of slavery as our present" Hartman engages in a decolonial act, another concept we learned about in class, with this nonlinear and interconnected approach with time.

The archive of slavery exemplifies epistemic and colonial power as we talked about in class in that it is both produced by and exercises control over people and knowledge. As Hartman emphasizes, the archive does not preserve the full humanity of the enslaved. Instead, it records only what the colonial and slave-owning systems deem legible or valuable. The “founding violence” (as Hartman says) of slavery structures knowledge itself, determining which statements are authorized, and which bodies and which lives are treated as dispensable. In this way, the archive produces authority, and reproduces social hierarchies under the guise of neutrality or documentation. The life of Venus cannot be reconstructed through these records alone, because the archive records only the statements that justify her suffering and death. Feminist methods, such as Hartman’s work against this grain, attempting to recover knowledge and to expose the epistemic violence embedded in the archives (Stoler, 2009).

Hartman is again speaking from the margins, trying to restore Venus’s lost voice as they write "...if I could have detailed the small memories banished from the ledger..." Hartman highlights that the ledger (Western epistemology) only preserves names, indictments, and death, not feelings, relationships, or other lived experiences that align with theories of the flesh (Moraga and Anzaldúa, 2022). Through imagining Venus comforting her friend, Hartman values the grief, intimacy, and care denied by the slave ship and official records.

Hartman’s attention to sensory and bodily effects of enslaved life, such as cries, groans, and melodies, is a type of knowledge derived from embodied experience. Theories of the flesh, as described in This Bridge Called My Back argue that lived bodily experience is a vaild form of knowledge (Moraga and Anzaldúa, 2022). Hartman emphasises the physical, emotional, and other bodily sourced information as a way of knowledge and insight that is otherwise absent from the official archives.

In this passage, Hartman highlights the “daily record of abuses” that documents slavery but simultaneously erases the individuality and humanity of these women. Hartman’s struggle to “liberate them from the obscene descriptions” connects directly to the concept of speaking from the margins, as discussed in class with Ann Laura Stoler's book (Stoler, 2009). By focusing on what is missing or suppressed in these records (the margins), Hartman works against the archival grain, and demonstrates how feminist research methods seek to recover histories that exist at the margins.

I interpret this passage as the author trying to respect the humanity and dignity of those who not only could not speak for themselves but also the ones were spoken for (such as the Venus figure she describes and their dehumanization), while also recognizing their own standpoint. Standpoint theory as described by Harding states that everyone's knowledge is a result of their standpoint (the intersection of the many social positions like race or gender someone may have) (Harding, 1987). "What cannot be known" can mean the limits of the author's standpoint.

This passage illustrates exactly what Ann Laura Stoler calls “working against the archival grain” (Stoler, 2009). The historical records are produced by oppressors and reflect the logic, priorities, and worldview of slave owners and colonialists. They erase the individuality and humanity of the enslaved. Feminist methods, as Hartman tries to follow, aim to recover what these archives conceal despite the challenges therefore "working against the archival grain." Hartman raises the critical question of "How do we center the humanity of those whose existence was reduced to subhuman (e.g. property)?"

Here, Hartman reminds me of Sara Ahmed's description of a "feminist killjoy," which is a feminist that is not complicit or silent to injustices even when others may prioritize agreeability (Ahmed, 2017). Like a killjoy, she refuses to allow readers to enjoy a sanitized version of history. By highlighting structural violence, she “kills the joy” of comfortable narratives and exposes what the archive hides.

This passage draws on speculative fiction as a tool for engaging with history, showing how Dana’s time travel in Kindred parallels our engagement with the archive of slavery. Dana's inability to "rescue" her ancestors highlights the interconnection of violence from the past and present as well as limitations when dealing with trauma from the past. Hartman makes a connection between this and Venus, arguing that we too must "bear what cannot be borne" that is, acknowledge the structural constraints and suffering of the past without erasing or simplifying them. This resonates with decolonial approaches from class, which emphasize bearing witness to historical violence and its ongoing effects rather than imposing closure or resolution.

This passage emphasizes the continuing effects of slavery and dispossession in the present, what Hartman calls the “afterlife of property.” They frame history not as something behind us, but as ongoing: the violence that produced Venus’s life and death continues to shape Black life today. However, I wonder what Hartman means by "a past that has yet to be done." I assume she is talking about time as cyclical, but how can we then "do" the past?

I found this passage very striking as it refers to the feeling of wanting closure or to find comfort in something tragic, even though the situation is very bleak. Throughout learning about colonial and world history, there is plenty of upsetting, disturbing, and violent subject matter. Wanting to make yourself feel better by changing the narrative or minimizing the event is something I and many others have experienced, just like how Hartman wanted the two girls to be friends despite their horrible circumstances and demise.

Hartman questions the ethics and methods of historical writing. To me, it sounds like they are critiquing Western epistemologies and advocating for something different when they wrote "...how might it be possible to generate a different set of descriptions from this archive?" Traditional Western epistemology as we talked about in class prioritizes “objective” records like legal documents, ship logs, and other sources as legitimate knowledge. These often record only the perspectives of the colonialists and reproduce exploitative depictions of enslaved women. Hartman challenges this by asking whether it is possible to write about Venus ethically, centering her humanity rather than the archive’s focus on violence. Hartman, suggesting that ethical historical writing must go beyond documents to center the humanity, experiences, and agency of those rendered voiceless in official accounts, may benefit from Indigenous epistemologies which validate those lived experiences.

Given what we know about "Venus," as Hartman describes as girls who were deprived of their voice and presence in historical records and could experience abuse/exploitation, how can we trust this description of "consent"? Could women on slave ships, with an obvious power dynamic of master/slave consent?

Hartman raises a central ethical problem in feminist and Black studies: how to represent historical violence without reproducing it. This reminds me of discussions that are made in regards to horror movies or other media that depict sensitive subject matter, which is "what draws the line between accurate portrayal and the glamourization/aestheticization of abuse and suffering?" If the victims, like Emmett Till who was mentioned in the passage, cannot decide that how can historians decide what's appropriate?

I found it be refreshingly pedantic and accurate to refer to it as just a "recommendation" rather than a "requirement" or something... though I'm not actually sure why we call such standard things in the web APIs merely a "recommendation". Can't we just call it a standard at this point?

We are getting feedback that some GAP students struggle to identify organ location as they move on to future courses. Identifying major organs/structures in each abdominopelvic region may be a wise investment of our time here.

Stating that dorsal means toward the spine side may help to differentiate "dorsal" from "posterior."

"Away from the midline" might be clearer here?

I like to point out that palms forward allows radius and ulna to be seen distinctly instead of being partially superimposed as a way to help them remember which direction the palms should be facing.

This is very cool! Is this experiment with wild-type or fmp52∆ cells?

It looks like there's at least one putative UPRE upstream of FMP52 (425 bases upstream of the ATG, 5'-TACGTGT-3')! I'm curious if you tried looking at endogenous Fmp52-GFP with t-2-hex or other ER stress-inducing treatment, as it may get upregulated and be more visible than in unstressed conditions? This would be consistent with your pFMP52-luciferase results.

• Highly Processed Foods & Inflammation: Up to one-third of all cancers are linked to poor nutrition, particularly consuming highly processed foods [00:00:00]. These products (full of artificial additives, hardened fats, processed meats, and sweet snacks) act as "fuel" that drives chronic inflammation, creating an environment conducive to cancer development and growth [00:00:24].

• Sugary Drinks (Napoje Słodzone):

  • Regular consumption of sugary drinks (not natural juices) is linked to a higher risk of several cancers [00:00:57].
  • Studies show an increased risk of breast cancer (14%) in women and prostate cancer (18%) in men [00:01:16].
  • Daily consumption of just one serving of soda (like cola) was linked to an up to 55% higher risk of pancreatic cancer [00:01:35].
  • They are also associated with an average 15% greater risk of colon cancer [00:02:15]. Lab studies suggest the sugar in these drinks can increase the mobility and metastatic ability of cancer cells [00:02:42].

• Drinks with Artificial Sweeteners (Słodziki):

  • While many studies suggest no link to cancer, some research indicates a higher risk of leukemia (15% increase per serving), particularly from drinks containing aspartame [00:04:12].
  • The concern is that aspartame can break down into methanol and then formaldehyde (a recognized carcinogen) [00:04:32], which can damage the genetic material in bone marrow stem cells [00:04:49].

• Fried Foods (Żywność Smażona):

  • Regular consumption of fried foods (especially deep-fried meats and plant products) is linked to a 52% higher risk of stomach cancer [00:05:55] and may increase the risk of prostate cancer by 35% [00:06:04]. This is due to the formation of toxic compounds during frying [00:06:13].

• High-Salt Diet (Dieta Bogata w Sól):

  • A long-term diet high in salt, especially from low-quality processed foods, is strongly associated with an increased risk of stomach cancer (up to 68% higher) [00:07:17].
  • Excess salt directly damages the stomach lining, makes it susceptible to toxins [00:07:42], and promotes the growth of the Helicobacter pylori bacteria, which causes chronic inflammation and ulcers that can lead to cancer [00:08:15].

• BPA and BPS (Canned Foods):

  • Bisfenol A (BPA), used in metal can linings [00:09:40], can migrate into food and is a potential pro-carcinogenic agent because it disrupts hormonal balance [00:09:56]. High exposure may promote hormone-dependent cancers like breast and prostate cancer [00:10:06].
  • Bisfenol S (BPS) is a similar chemical still permitted in food-contact materials and is suggested to be more toxic and equally linked to breast cancer [00:11:24]. The speaker recommends avoiding most canned foods unless marked "BPA Free" [00:11:47].

• Phthalates (Ftalany):

  • These chemicals, which increase plastic flexibility [00:12:19], can migrate from plastic packaging into highly processed foods, especially fast food (e.g., in plastic trays) [00:12:37].
  • Phthalates are suggested to promote cancers of the thyroid, breast, and prostate [00:13:19].

• Alcohol:

  • Alcohol consumption is a confirmed risk factor for cancers of the oral cavity, throat, esophagus, and larynx [00:13:44].
  • Regular consumption also increases the risk of colon cancer by an average of 56% [00:13:53]. Even small, frequent amounts are considered harmful [00:14:02].

While I understand that it's a logical extension to represent the tree as a vector/matrix format as you've done here to obtain summary statistics, it should on principle be feasilble to do something similar using graph neural networks, which can learn tree/graph representations directly from the original phylogeny/graph. Have you considered taking such an approach?

In Decolonizing Feminism: Challenging Connections between Settler Colonialism and Hetero-patriarchy, the authors argue that colonialism, and specifically settler-colonialism where the settlers come to stay, happen through both systemic public acts of violence (e.g., control, labour exploitation), but also more intimate ways (assimilation, cultural erasure, sexual identity policing, linguistic oppression). I think this idea connects to the "everyday-ness of decolonization" mentioned earlier, and how resistance must be grounded in local activities and connections with queer Indigenous people, as much on a larger scale. Otherwise, we risk Indigenous erasure and the enforcement of colonial ideas of "progress."

This is an interesting point because it reminds me of the romanticization of social movements. This romanticization often views the process of liberatory struggles through an idealized and oversimplified lens, and overlooks internal conflicts and flaws. This means that progress is only measured through public-facing spectacles/displays of justice, rather than something that happens simultaneously behind closed doors and isn't easily televisual. In regards to this text, a romanticization of these struggles can often dismiss intellectual labour and progress that takes place in unconventional spaces such as within families, homes, and community spaces, or even within ourselves. The authors are trying to make the point that struggling for liberation and decolonialization isn't a one-size-fits-all-approach.

This is a central theme: the reframing of "queer" beyond its initial identity label. Most of my peers think that queer describes a particular sexual orientation or identity. But to be queer is defined through practices and actions that are inherently anti-oppressive.

This reminds me of a quote from bell hooks, where hook's describe queer as "the self that is at odds with everything around it and has to invent and create and find a place to speak and to thrive and to live." In this way, queerness is not a sexual identity but a method of moving through the world, a method that involves self-creation in opposition to hetero-normativity and patriarchy.

This is the central theme of this piece of work; of how decolonization can take place within intimate spaces such as our communities, families, within friendships, and our everyday work. As emphasized by the authors, decolonial practices must first take place within our private, personal experiences, before they can take shape through our laws, policies, and systems. The latter can be a slow process, but the former can start today.

It is interesting to me that even at a specialized school for top performing students, that the teachers were not more educated that these might be common traits for the students they teach.

I can think of many examples of students like these in my own schooling experience and it makes me wonder if they were really an unruly, disrespectful kid or if something else was going on. It is hard as a teacher when you have so many students to dive in deep like this but it SO important for the student if you do.

I am curious to know what the science is behind people being able to communicate through music before speech. I think there must be something very primal about it.

I think this is the case for a lot of people. It is a long process to get any intervention or type of assistance when kids are this young. I wonder why this is.

I found this interesting because how can you have it both ways by separating the gifted kids but including average to below-average students? Who do you pick?

I think this is great point that teachers should recognize more. One of my biggest questions when it comes to teaching students like this who need this differentiation is how do we deal with the social issue that might arise? If other students see that Johnny is getting different work and make a fuss about it, how do we redirect it without making it a big deal?

This disparity shocked me at first but then when I reflected on my own experiences, I couldn't recall a time where I saw a gifted or talented student receive services.

This unfortunately does not surprise me. In all of my studies thus far, it is evident that socioeconomic class plays a HUGE role in what services people receive.

I don't know how to feel about this. I guess there is not one standard so each child can get the services they need. But what happens to a child who moves and who no longer qualifies for services because they don't meet the new score requirement?

I think it is interesting that it took this long to coin the term gifted. Along with other differences, it still fascinates me all of this research and academic stride forward is so recent.

I remember hearing people use this phrase when I was in elementary school.

Is there a way that the conductor could’ve had a little more grace with David?

I’ve seen some choirs do this but maybe having a gifted student conduct a piece and rehearse the piece with their peers. This can really challenge their creativity by controlling how the ensemble plays. This will also keep them engaged longer with them thinking about conducting.

Maybe planning more for the gifted students might help. For example, if their task is to learn the Eb concert scale maybe have the gifted students play it in more rhythms or starting on different scale degrees. To try and get them to be occupied for longer.

I think this is a good idea not just for gifted students. Especially when teaching general music and the students sometimes say the most random things.

This surprised me. The fact that so much time is wasted for gifted students.

What is the best way to challenge students while not leaving other students behind?

I haven’t actually thought about the range of being gifted and how some students will show it differently.

I wonder if people easily got on board with what he was saying.

This addresses a broader issue than just Hannah. I feel like this happens a lot in a concert band setting of course not as extreme as Hannah. But when you have one concert band in the entire high school how do you program pieces that will be playable for the freshman but challenge the seniors.

homonyms

A blank English pipeline would be nlp_blank = spacy.blank("en")

in

Here and throughout confusion with "homonyms".

prevent

considerations

extracts

• The author describes a comprehensive setup to block virtually all online advertising across devices and services.
• They focus on network-level filtering instead of per-device ad blockers, so that phones, TVs, and other clients benefit automatically.
• The core of the solution is running a self-hosted DNS-based blocker (like Pi-hole or AdGuard Home) to sinkhole common ad and tracker domains.
• Additional blocklists are layered on top to handle more aggressive tracking and region-specific ad domains, trading a bit of breakage for increased privacy.
• For services that hardcode ad endpoints or use techniques that bypass DNS blocking, the author uses more advanced tools such as proxying or firewall rules.
• Some apps and sites break when ads are blocked; in those cases, the author selectively whitelists domains or uses per-device exceptions rather than relaxing global rules.
• On mobile, encrypted DNS and VPN-like tunneling are configured so that all traffic still flows through the home-level blocking setup even on the go.
• The author argues that this configuration significantly improves page load times, reduces bandwidth usage, and makes devices feel faster and less cluttered.
• They acknowledge an ethical gray area with ad blocking but conclude that user safety, privacy, and mental comfort outweigh the downsides of depriving low-quality ad networks of revenue.
• The piece emphasizes that the goal is not absolute perfection but a sustainable setup that requires minimal maintenance once deployed.

Hacker News Discussion

• Commenters discuss additional tools like SponsorBlock for skipping in-video sponsorships on platforms such as YouTube, highlighting that traditional ad blockers do not remove creator-embedded promos.
• Several users point out that DNS-level blocking does not stop ads injected directly by streaming services, noting that such platforms often use certificate pinning or app-level tricks that make proxying and MITM approaches difficult or impossible.
• A highly upvoted comment recommends using a user-agent switcher to bypass sites that block non-Chrome browsers, with examples where services claim to be incompatible with Firefox but run better once the browser “pretends” to be Chrome.
• Participants criticize websites that enforce brittle user-agent checks instead of feature detection, arguing that this needlessly breaks otherwise compatible browsers and punishes privacy-conscious users.
• Some users express skepticism about privacy-focused browsers that are built on or dependent on codebases controlled by ad-driven companies, calling out an inherent tension between privacy promises and ad-based business models.

second

unclear where on the website this list can be found

necessarily

This breaks my heart

test

Je propose d'ajouter une virgule avant :

"…aux individus, mais"

Though you already report high accuracy with your method, do you think the result would improve or change if you also included the CH stretch region (~2700-2900 cm-1 or so)?

Thanks for sharing this work! Did you notice any alteration in Raman signal of cells due to laser exposure? How did you select the acquisition parameters?

Because these mutants were generated via random mutagenesis, it's difficult to interpret phenotypic variability of each mutant's lipid metabolism in the context of their varied genetic backgrounds. It may be worth showing lipid-productivity data in mutants that have similar levels of starch content to wild-type to show a clear baseline for interpretation.

It would help to show more of the screening process that led to the five chosen lines. For example, reporting how many colonies were screened, the growth conditions used during screening (e.g., BBM vs BBM−N, duration, light), how starch phenotypes were called from iodine staining, and how many candidates fell into each class (low starch / high starch). A semi-quantitative summary of iodine phenotypes would let readers estimate the probability of generating starch phenotypes via this mutagenesis protocol (an informative result on its own).

Here biomass productivity is calculated as u (growth rate) x B_end, but the more common way to calculate BP is B_end - B_start / time. u x B could overstate productivity and/or make comparisons phase-sensitive. Sometimes those early timepoints may be harder to collect/quantify, but is it known that the cultures are in exponential phase for the duration of the experiment?

How many mutants did you have to screen to find five starch high/low phenotypes? Did you also consider mutants that had spatially abnormal starch distribution?

eLife Assessment

This important study presents novel data on temporal variation in sperm whale communication, contributing to a richer understanding of the social transmission of vocal styles across neighbouring clans. The evidence is solid, although some terminology limits comparisons to other taxa. This research will be of interest to bioacoustics and cetacean communication specialists, particularly those working on social learning and culture.

Reviewer #2 (Public review):

Summary:

The current article adapts standard rhythmic measures to describe the temporal organisation of whale song units.

Strengths:

The detailed description of the internal temporal structure of whale songs is something that has thus far been lacking.

Weaknesses:

Conceptual and terminological bases of the paper are problematical and hamper comparison with other taxa, including humans. According to signal theory, codas are indexical rather than symbolic. They signal an individual's group identity. Borrowing from humans and linguistics, coda inter-group variation represents a case of accents -- phonologically different varieties of the same call -- not dialects, confirming they are an index. Moreover, symbolism is not a feature detectable or confirmed through rhythmic analyses or temporal characterisation. This raises serious doubt whether alleged "dialects," "symbolism" and similarity between whales and humans is factual. The comparative scope and relevance of this paper for the broader field is limited and evolutionary claims are potentially misleading and perilous.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #2 (Public review):

Summary:

The current article presents a new type of analytical approach to the sequential organisation of whale song units.

Strengths:

The detailed description of the internal temporal structure of whale songs is something that has been thus far lacking.

Weaknesses:

The conceptual and terminological bases of the paper are problematical and hamper comparison with other taxa, including humans. According to signal theory, codas are indexical rather than symbolic. They signal an individual's group identity. Borrowing from humans and linguistics, coda inter-group variation represents a case of accents - phonologically different varieties of the same call - not dialects, confirming they are an index. This raises serious doubt about whether alleged "symbolism" and similarity between whale and human vocal behaviour is factual.

We respect that the reviewer does not agree with describing codas as symbolic markers of cultural identity in sperm whales, but ultimately we find the quantitative evidence presented in Hersh et al. (2022) compelling, and stand by the framing of our manuscript, which builds on this foundation.

The same applies to the difference between ICIs (inter-click interval) and IOIs (inter-onset interval). If the two are equivalent, variation in click duration needs to be shown so small that can be considered negligible. This raises serious doubt about whether the alleged variation in whale codas is indeed rhythmic in nature and prevents future efforts for comparison with the vocal capacities of other species. The scope and relevance of this paper for the broader field is limited.

We believe there has been a miscommunication. Coda inter-click intervals are calculated as the time between the onsets of sequential clicks within a coda. This is identical to definitions of inter-onset intervals in many publications, including:

• Burchardt and Knörnschild (2020): “the duration between the beginning of one element and the next”

• Friberg and Battel (2002): “the time interval between the onset of the tone and the onset of the immediately following tone”

• De Gregorio et al. (2021): “the time between the onset of a note and the next one”

In response to a comment from this reviewer in the first round of revisions, we made the point that we do not believe rhythm analyses need be restricted to inter-onset intervals alone. Regardless of that stance, we did analyze inter-onset intervals in this manuscript and accordingly are capturing aspects of rhythm in our analyses. We have removed a poorly worded sentence in our introduction and apologize for any confusion it caused. We have also made this explicit in lines 30–35: “This classification is based on the total number of clicks and their rhythm and tempo extrapolated from the time interval between the onsets of consecutive clicks: the inter-click interval (ICI) [15, 16] (Fig. 1A). This measure is equivalent to the inter-onset intervals (IOIs) often used in rhythm analyses [17, 18, 19] but for the sake of compatibility with studies on sperm whale acoustics, we use ICI terminology throughout this paper.”

In our analyses, inter-click intervals and inter-onset intervals are equivalent measures.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

My concerns regarding interdisciplinary terminology and methods remain unaddressed. The study's inaccurate terminology hinders reliable comparison with other taxa, including humans. Being "symbolic" bears no weight on the new method that the authors present, thus, the unwillingness for compatibility is limiting and perplexing. The authors state that codas have been previously described as being symbolic, but just because poor terminology has been used before doesn't justify perpetuating it, especially when it confounds and conflicts with broader comparative efforts.

We agree that being symbolic bears no weight on the new method we present, but we believe it does bear weight on our interpretation of what our method reveals about patterns in sperm whale communication. For that reason, we have opted to maintain the current framing of our manuscript.

The same applies to the difference between ICIs and IOIs. The authors resist amending terminology, even though they state the two represent the same measure. If so, want prevents the correct use of IOIs?

We have opted to use ICI throughout the paper because it is standard terminology in sperm whale acoustics, but we have now made the ICI/IOI equivalence explicitly clear in the introduction.

References:

Burchardt LS, Knörnschild M. 2020. Comparison of methods for rhythm analysis of complex animals’ acoustic signals. PLoS Computational Biology 16. doi:10.1371/journal.pcbi.1007755

De Gregorio C, Valente D, Raimondi T, Torti V, Miaretsoa L, Friard O, Giacoma C, Ravignani A, Gamba M. 2021. Categorical rhythms in a singing primate. Current Biology 31:R1379–R1380. doi:10.1016/j.cub.2021.09.032

Friberg A, Battel GU. 2002. Structural communication In: Parncutt R, McPherson G, editors. The Science & Psychology of Music Performance: Creative Strategies for Teaching and Learning. Oxford University Press. doi:10.1093/acprof:oso/9780195138108.001.0001

Hersh TA, Gero S, Rendell L, Cantor M, Weilgart L, Amano M, Dawson SM, Slooten E, Johnson CM, Kerr I, Payne R, Rogan A, Andrews O, Ferguson EL, Hom-Weaver CA, Norris TF, Barkley YM, Merkens KP, Oleson EM, Doniol-Valcroze T, Pilkington J, Gordon J, Fernandes M, Guerra M, Hickmott L, Whitehead H. 2022. Evidence from sperm whale clans of symbolic marking in non-human cultures. Proceedings of the National Academy of Sciences 119:e2201692119. doi:10.1073/pnas.2201692119

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

1. First, the authors have not convincingly shown that skin cells, or more specifically skin ECs, are a major source of circulating G-CSF in the psoriasis model as stated in the title and abstract. The data in Figure 4 show selective upregulation of Csf3 gene in skin ECs and their ability to secrete G-CSF upon IMQ treatment in vitro. However, the provided data do not address to what degree the skin EC-derived G-CSF contributes to the elevated level of circulating G-CSF. Additional experiments to selectively deplete G-CSF in skin ECs, or at least in skin cells of the affected site, are warranted to support the authors' claim. Does intradermal injection of G-CSF neutralizing antibody into the psoriatic skin reduce circulating levels of G-CSF?

Author's response:

Thank you for reviewer's comment. We agree with the Reviewer#1 that it is important to directly block G-CSF to the skin via intradermal injection and measure the G-CSF level in the serum afterwards. Therefore, we will perform intradermal injection of IgG-isotype or anti-G-CSF antibody into the IMQ-induced psoriatic mice.

Another concern is insufficient demonstration of G-CSF-mediated emergency granulopoiesis in the psoriasis model. All data in Figure 5 were obtained from experiments with only n=3, and adding more replicates, in particular to those in Figure 5B, which show quite some variation in MPP numbers, is recommended. The relatively small reduction of BM granulocyte numbers (Figure 5C) compared to greater depletion of circulating granulocytes (Figure S5A) raises the possibility that it is the mobilization effect rather than granulopoiesis-stimulating effect that skin-derived G-CSF exerts to promote supply of circulating neutrophils that eventually infiltrate into the affected skin. This could also explain the negligible effect of IL-1blockade (Figure S4), which selectively shut off myelopoiesis-stimulating effect of IL-1 (Pietras et al. Nat Cell Biol 2016, PMID: 27111842). Are the HSPCs in the psoriasis model more cycling? Do they show myeloid-skewed differentiation when cultured ex vivo or upon transplantation?

Author's response: Thank you for these critical comments. We agree to do the following experiments to address them:

1) HSPCs quantification in Figure 5 especially the MPPs will be added with more replicates.

2) We will assess cycling status of HSPCs by flow cytometric analysis of Ki67and Propidium Iodide to characterize G0, G1 and G2/M cell cycle phase.

3) To test myeloid-skewed differentiation, Lin- c-Kit+ Sca-1+ cells containing HSPCs will be isolated from bone marrow of Vas/IMQ-treated mice and transplanted into lethally irradiated syngeneic mice.

The authors' claim that skin-derived G-CSF "induces" neutrophil infiltration warrants further clarification. Alternative explanation is that the upregulated neutrophil-attracting chemokines (Figure S1D) could induce infiltration, whereas G-CSF increase the number of neutrophils to circulate in the vessels near the psoriatic skin. This notion seems supported elsewhere (Moos et al. J Invest Dermatol. 2019, PMID: 30684554). Can the infiltration be inhibited by systemically injecting neutralizing antibody of their receptor, CXCR2?

Author's response: The manuscript focuses on the skin-derived G-CSF function as a long-distance signal for emergency granulopoiesis in the bone marrow upon psoriasis, not the chemoattractant property of it. The sentence of interest is "We found that upon psoriasis induction, skin-resident endothelial cells are activated to produce G-CSF which activates emergency granulopoiesis in bone marrow and induces cutaneous infiltration and accumulation of neutrophil that are functionally inflammatory." in line 28-30. In agreement with point #2 from Reviewer#2, the fact that neutrophil recruitment factors (CXCL1, CXCL2, and CXCL5) were upregulated in psoriatic skin (Figure S1D), suggesting a CXCL-mediated neutrophil recruitment. The sentence of concern need to be changed to "We found that upon psoriasis induction, skin-resident endothelial cells are activated to produce G-CSF which activates emergency granulopoiesis in bone marrow, leading to cutaneous accumulation of neutrophil that are functionally inflammatory.". This revised sentence has omitted the proposal that G-CSF directly dictates neutrophils mobilization to the skin, which is not the key message of the study. Therefore, we found that the CXCR2 (CXCLs receptor) blockade experiment may be of the benefit of future studies.

It remains unclear how skin-derived G-CSF accumulates pathogenic neutrophils. The authors state "pathogenic granulopoiesis," but are the circulating neutrophils in the psoriatic mice already "pathogenic" or do they acquire pathogenic phenotype after cutaneous infiltration? Additional RNA-seq to compare circulating and infiltrated neutrophils would answer this question.

Author's response: We appreciate this valuable comment. We will perform RNA-seq with the peripheral blood-circulating neutrophils (CD45+ CD11b+ Ly6G+ Ly6Cmid) versus skin-infiltrating neutrophils from both Vas/IMQ mice.

In addition, how the accumulated pathogenic neutrophils exacerbate the psoriatic changes remains obscure. Although the authors have attempted to correlate Il17a gene expression in infiltrated neutrophils with psoriatic skin changes, the data do not address to what degree it contributes to cutaneous IL-17A protein levels. The data that cutaneous neutrophil depletion leads to subtle decrease in skin IL-17A expression (Figure 2H) rather supports alternative possibilities. For instance, as indicated elsewhere, IL-17A cutaneous tone could be enhanced by neutrophil-mediated augmentation of Th17 or gamma/delta T cell function (Lambert et al. J Invest Dermatol. 2019, PMID: 30528823). Does neutrophil depletion or G-CSF neutralization alter cell numbers or function of cutaneous Th17 and gamma/delta T cells?

Author's response: Thank you for this insightful comment. To better understand the relative contribution of neutrophils to the cutaneous IL-17A tone in the psoriatic skin, we will perform flowcytometric analysis of Th17 and gamma/delta T cells which are widely known as the major source of IL-17 in psoriatic skin of IMQ-induced mice following injection of isotype-matched or anti-Ly6G antibody.

Finally, as the above conclusions rely solely on the IMQ-induced acute psoriasis model, it would be informative if they could be derived from another psoriasis model. IMQ is known to induce unintended systemic inflammation due to grooming-associated ingestion (Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514), and "pathological crosstalk between skin and BM in psoriatic inflammation" could be strengthened by an intradermal injection model.

Author's response: We appreciate the reviewer for bringing this important point. Regarding the systemic inflammation upon psoriasis, the above-cited study reported increased IFN-B expression in the intestines of IMQ-ingested animal (Grine L et al. Sci Rep. 2016, PMID: 26818707 in Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514). We examined several pro-inflammatory cytokines including IFN-b, IFN-g, and IL-6 and in contrast, found no systemic increase in all these cytokines, except for IFN-g downregulation (Explanation Figure 1), which suggests no evidence of grooming-associated ingestion.

We also examined the Csf3 expression across several distinctively located tissues which showed a selective upregulation in the skin (Figure 4C), suggesting a skin-restricted perturbation. In addition, one study showed that IMQ-ingestion didn't alter number of gut injury-associated CXCR3+ macrophages nor did it aggravate skin inflammation (Pinget et al. Cell Reports. 2022, PMID: 35977500). Together, these findings support that IMQ-induced psoriasis by topical cutaneous application used in our study elicit a local inflammation but not systemic inflammation.

The authors, however, realize that testing alternative psoriasis model such as intradermal injection of IL-23 (Chan et al. J Exp Med. 2006, PMID: 17074928) will strengthen the skin-local insults within the psoriasis model employed, and should be tested in the future.

Minor comments

Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.

Author's response: We appreciate the Reviewer#1 pointing this issue. As mentioned by the Reviewer#1, the elongated structures detected in the intravital microscopy are not neutrophils, but autofluorescence from the skin bulge regions (Wun et al. J Invest Dermatol. 2005, PMID: 15816847). We have eliminated these unspecific signals from the transformation and quantification (Figure 1F, S1G, and S1H). We will also add an explanatory sentence in Materials and Methods section "Of note, the fluorescent signal with elongated structures resembling hair bulge were autofluorescence and thus removed from further analysis." to be more precise about our methods.

In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.

Author's response: Thank you for the comment, we will correct this misspelling.

Author's response: We appreciate that Reviewer#1 bring up this point. We examined the kinetics of the bone marrow cellularity and GMPs across 4 days of psoriasis induction in mice. The bone marrow cell number was lowered along that span with lowermost count at 2 days. Consistent to the BM-cellularity, the GMP number was also lowered about one-third in the first 2 days of psoriasis. This kinetic is consistent with the previous report showing a rapid reduction of GMPs in the bone marrow within 2 days following systemic G-CSF administration driven emergency granulopoiesis (Hirai et al. Nat. Immunol. 2006, PMID: 16751774). From 2 days to 4 days, the GMP number rapidly increased to slightly above basal number (Explanation Figure 2). This timely coordinated expansion suggests a significant supply of GMPs from the differentiating upstream myeloid progenitors (Figure 3B).

When the psoriatic mice with elevated G-CSF is injected with anti-G-CSF or IgG-isotype antibody, the bone marrow cellularity and GMP numbers at 4 days were (Explanation Figure 3). Firstly, as psoriasis reduced bone marrow cellularity (Explanation Figure 2), the unchanged number after anti-G-CSF injection indicates that administration of 10µg/day for 4 days does not significantly affect mobilization of psoriatic bone marrow cells. Secondly, the similar GMP numbers at 4 days psoriasis is plausibly due to snapshot analysis when it has already in the numerical recovery period (Explanation Figure 2). Importantly, the notion that anti-G-CSF injection to psoriatic mice reduced granulocytes in the bone marrow, peripheral blood, and skin suggesting G-CSF as a key mediator in psoriatic driven emergency granulopoiesis on top of unlikely case of ineffective anti-G-CSF treatment.

Taken together, these data suggest a G-CSF mediated emergency granulopoiesis occurrence in the IMQ-induced psoriasis. We will put these data into a revised Figure.

In Figures 6B, in which cluster of human skin cells IL-17A expression would be enriched?

Author's response: Thank you for this important point. The IL-17A expression is found in the T-cell cluster (Explanation Figure 4). We also expected to see IL-17A contribution from other cell subset(s), in particular neutrophil. However, due to the fragile nature of neutrophils and thereby, technical difficulty to get their sequencing reads, this dataset (GSE173706) doesn't contain neutrophils, but rather monocytes, macrophages, and dendritic cells among the myeloid subset (Explanation Figure 5). With this, it leaves open the question on what potential contribution of IL-17A produced by neutrophils is in human psoriasis (Reich et al. Exp. Dermatol. 2015, PMID: 25828362).

Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.

Author's response: We appreciate the Reviewer#1 pointing this issue. As mentioned by the Reviewer#1, the elongated structures detected in the intravital microscopy are not neutrophils, but autofluorescence from the skin bulge regions (Wun et al. J Invest Dermatol. 2005, PMID: 15816847). We have eliminated these unspecific signals from the transformation and quantification (Figure 1F, S1G, and S1H). We will also add an explanatory sentence in Materials and Methods section "Of note, the fluorescent signal with elongated structures resembling hair bulge were autofluorescence and thus removed from further analysis." to be more precise about our methods.

In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.

Author's response: Thank you for the comment, we will correct this misspelling.

Reviewer#2

1. Interpretation of neutrophil transcriptomic changes (Figure 2)

The RNA-seq analysis reveals substantial downregulation of several canonical pro inflammatory pathways in neutrophils from psoriatic skin, including IL-6, IL-1, and type II interferon signaling. The authors should discuss the functional relevance of this unexpected transcriptional repression. For example, does this indicate a shift toward specialized effector functions rather than classical cytokine responsiveness? More importantly, the most striking transcriptional change is the upregulation of NADPH oxidase-related genes (e.g., Nox1, Nox3, Nox4, Enox2). This suggests an oxidative stress-driven pathogenic mechanism, potentially more relevant than IL-17A production. Yet this aspect is not explored in the manuscript. Assessing ROS levels or oxidative neutrophil effector functions in this model would considerably strengthen the mechanistic link. Conversely, although IL-17A is upregulated in neutrophils, neutrophil depletion reduces total Il17a expression in skin only partially. This indicates that neutrophils are unlikely to be the dominant IL-17A source in the lesion. The authors' focus on neutrophil-derived IL 17A therefore seems overstated. A more rigorous assessment-e.g., conditional deletion of Il17a specifically in neutrophils-would be required to establish its true contribution. Taken together, the data suggest that oxidative programs, rather than IL-17A production, may represent the principal pathogenic axis downstream of neutrophils, and this deserves deeper discussion.

Author's response: Thank you for raising this valuable views. We have agreed to address these critical points by the following approaches:

1) To address the changes in NADPH oxidase-related gene signature, we will measure ROS production in the neutrophils from skin and peripheral blood with DHR123.

2) Responding to the IL17A contribution by neutrophils, we will flow cytometrically assess the Th17 and gamma/delta T cell population in the skin of psoriatic mice treated with anti-Ly6G or isotype-matched antibody as was suggested by Reviewer#1.

3) We will discuss downregulation of the canonical pro inflammatory and IL-17 pathways in the psoriatic neutrophils in the discussion.

Human data reanalysis (Figure 6):

The re-analysis of bulk and single-cell RNA-seq datasets is valuable but incomplete. Several mechanistically relevant questions could be addressed with the available data:

2.1. GM-CSF (CSF2) is also strongly upregulated in psoriatic lesions (bulk RNA-seq). It would be informative to determine whether endothelial cells also express CSF2 in the scRNA-seq dataset, as this would suggest coordinated regulation of myeloid-supporting cytokines.

2.2. Myeloid cell subsets should be examined more closely. A comparison of human myeloid transcriptomes with the mouse neutrophil RNA-seq would clarify whether similar IL-17A-related or NADPH oxidase-related signatures occur in human disease. In particular, which cell types express IL17A in human lesions?

2.3. Chemokine production should be attributed to specific cell types. Bulk RNA-seq confirms strong induction of CXCL1, CXCL2, CXCL5, but the scRNA-seq dataset allows determining whether these chemokines originate from endothelial cells or other stromal/immune populations. This information is important for defining whether endothelial cells coordinate both neutrophil recruitment and granulopoiesis.

Addressing these points would make the human-mouse comparison substantially stronger.

Author's response: Thank you for pointing these important issues. By reanalyzing the dataset, we found several points regarding the comments, as follows:

2.1) CSF2 is expressed by T-cell cluster in the human skin dataset (Explanation Figure 4), in agreement with previous murine study (Hartwig et al. Cell Reports. 2018, PMID: 30590032). We will add this data in the revised manuscript.

2.2) In line with point#10 from Reviewer#1, the dataset clearly shows T-cell cluster as the main IL17A source (Explanation Figure 4 above). The dataset, however, doesn't contain phenotypic neutrophils (CEACAM (CD66b) and PGLYRP1) but monocytes, macrophages, and dendritic cells (Explanation Figure 5 above). This loss was probably due to a technical limitation given the difficulty in capturing sequencing reads from fragile neutrophils. Therefore, it is no longer possible to reanalyze IL-17 expression in the absence of neutrophils in the datapool.

2.3) Reanalysis of CXCLs in the human scRNAseq dataset (GSE173706) clarified their secretion dynamics and cellular sources under normal and psoriatic condition. In normal skin, all examined cell subsets show only low CXCLs expression. In contrast, psoriatic skin exhibits significant CXCLs upregulation with distinct cell subsets clearly showing dramatic upregulation, potentially being the major CXCLs source. CXCL1 is markedly upregulated in fibroblasts, myeloid cells, and melanocyte and nerve cells. CXCL2 is strikingly upregulated to myeloid cells, while CXCL5 is hugely increased in fibroblasts, myeloid cells, and mast cells (Explanation Figure 7). Taken together, these results suggest that CXCLs upregulation in the psoriatic skin is coordinatively executed by both stromal and immune compartments. Of note, the endothelial cells show minimal changes in CXCLs expression, even downregulate CXCL2 in psoriasis, indicating that they are unlikely to be the major contributor to CXCL-mediated neutrophil recruitment.

**Referees cross-commenting**

I agree with Reviewer 1 that the contribution of EC-derived G-CSF to circulating G-CSF levels and to emergency myelopoiesis requires additional genetic or neutralization experiments to be fully established.

Author's response: We appreciate that Reviewer#2 raised this key point. In addition to examining the serum G-CSF upon intradermal anti-G-CSF administration in point#1 from Reviewer#1 above, we will also examine the emergency myelopoiesis signs in vivo.

Minor points

1. Line 319: the text likely refers to Figure S4, not S3.

Author's response: Thank you, we will correct the nomenclature.

Line 338: "psoriatic" is misspelled.

Author's response: Thank you, we will change this to "psoriatic".

Reviewer #3

• Place the work in the context of the existing literature (provide references, where appropriate).

Psoriasis is extensively studied, a good recent reference- https://doi.org/10.1016/j.mam.2024.101306

Author's response: Thank you for Reviewer#3's suggestion. The referenced study highlights the current paradigm that largely focus on skin-restricted mechanism and overlook potential cross-organ interaction in the psoriasis inflammation. Our findings provide a new insight into the skin-bone marrow crosstalk in the disease context. In addition, the suggested reference underscores the key roles of diverse innate immune cells including neutrophils, eosinophils, dendritic cells, etc. which is fundamental for our study and might also guide future exploration of additional innate cell subsets beyond neutrophils. We will therefore include the mentioned reference to our revised manuscript.

• Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

It is all good. May add graphical-abstract.

Author's response: Thank you for the reviewer's input, we agree that a graphical-abstract will help the readers more clearly grasp the key messages of our manuscript. We will include it in the revised manuscript.

Major comments:

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

No. It is very solid.

Author's response: We appreciate the reviewer's view that the claims in our paper are solid.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Such a discovery clearly opens many options, and it is fascinating to suggest additional experiments for future studies. It is a complete study, best to publish as-is and let many to read and proceed with this new concept.

Author's response: We thank the reviewer for noting that the current experimental evidence is complete that no additional experiments are necessary at this stage. We agree that the discovery opens prospective directions for future studies.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

N/A - I suggest no additional experiments at this point. Get it published and see how many will follow this new direction!

Author's response: We thank the reviewer for recognizing that the experimental data has been sufficient to be a foundation for the future research.

• Are the data and the methods presented in such a way that they can be reproduced?

Yes.

Author's response: We thank the reviewer for recognizing that our methods are reproducible.

• Are the experiments adequately replicated, and is the statistical analysis adequate?

Yes. The data are of very high quality.

Author's response: We are grateful that the reviewer view our replication strategy and statistical analysis are of a high quality.

Minor comments:

• Specific experimental issues that are easily addressable.

None. It is good as-is. One may always suggest minor things- but this one is better published so many laboratories may rush for this new direction. I think it will be interesting studying some long-term impacts, and changes not only of neutrophils but also of other innate cells, such as DCs, Macrophages, and Eosinophils - so it is best to let laboratories that focus on these cells know of the discovery and pursue independent studies.

Author's response: We appreciate the reviewer's assessment that our paper is already well set for the community to explore the newly proposed direction.

• Are the text and figures clear and accurate?

Yes.

Author's response: We thank the reviewer's evaluation. We have ensured that the text and figures in our manuscript are clear and accurate. Once again, we thank the reviewer for the encouraging and constructive appraisal. We are pleased that the reviewer find the manuscript has already been strong and suitable for publication.

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Referee #3

Evidence, reproducibility and clarity

Summary:

Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

Study titled: "Skin-derived G-CSF activates pathological granulopoiesis upon psoriasis" by Kosasih and Takizawa. Paper show establishment of psoriasis model in C57BL/6 mice. They focus on neutrophils infiltration following the Imiquimod cream induction. Importantly, authors show that the induction of psoriasis in the skin cause a robust enhancement of granulopoiesis in the bone marrow. Mechanistically, G-CSF is produced in the skin, especially by endothelial cells. Blocking of G-CSF gained clear inhibition of psoriatic pathology. They further add human data showing that patient with psoriasis have more neutrophils and more G-CSF in their skin endothelial cells.

Parts of the study are simply in line with previous knowledge (e.g.- neutrophils infiltration into psoriatic skin, IL17a). authors show some data that largely confirm the model used. Major discovery: skin endothelial cells are secreting G-CSF that induce granulopoiesis in the bone-marrow. This is a conceptual advancement of this study: psoriatic skin not only recruit neutrophils from the blood, but also enhance the generation of new neutrophils in the bone-marrow. That a major- psoriasis at the level of the model used must not be considered as a confined-pathology. It affect systematically, and might also benefit new systemic treatments. There are plenty of follow-up experiments to pursue now, so it is critical to publish this finding and let many laboratories to know of this new direction. I expect this study to attract high interest and many citations.

Major comments:

• Are the key conclusions convincing?

Yes. The study has excellent data, with good quantification, and very solid support for the discovery and interpretations. - Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

No. It is very solid. - Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

Such a discovery clearly opens many options, and it is fascinating to suggest additional experiments for future studies. It is a complete study, best to publish as-is and let many to read and proceed with this new concept. - Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

N/A - I suggest no additional experiments at this point. Get it published and see how many will follow this new direction! - Are the data and the methods presented in such a way that they can be reproduced?

Yes. - Are the experiments adequately replicated, and is the statistical analysis adequate?

Yes. The data are of very high quality.

Minor comments:

• Specific experimental issues that are easily addressable.

None. It is good as-is. One may always suggest minor things- but this one is better published so many laboratories may rush for this new direction. I think it will be interesting studying some long-term impacts, and changes not only of neutrophils but also of other innate cells, such as DCs, Macrophages, and Eosinophils - so it is best to let laboratories that focus on these cells know of the discovery and pursue independent studies. - Are prior studies referenced appropriately?

Yes. I may suggest adding a recent review by Park and Jung, 2024, https://doi.org/10.1016/j.mam.2024.101306 to cover current concepts of innate immunity in psoriasis. - Are the text and figures clear and accurate?

Yes. - Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

It is all good. May add graphical-abstract.

Significance

• Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field.

Conceptual advancement - discovery of a major impact of psoriasis on bone-marrow granulopoiesis. Explicit finding of endothelial-cells G-CSF as a major communication moiety.

• Place the work in the context of the existing literature (provide references, where appropriate). Psoriasis is extensively studied, a good recent reference- https://doi.org/10.1016/j.mam.2024.101306

Neutrophil recruitment and IL17A are well established. G-CSF of endothelial cells brings the conceptual advancement- psoriasis at the level induced by IMQ develops local pathology, but is tightly linked to systemic changes. The impact on bone-marrow granulopoiesis may have many implications. So far, it was largely considered that chronic inflammation may affect hematopoiesis, but this study reveals an acute and specific communication between skin and bone marrow. The neutrophils are not only recruited from blood- they are made anew, so the disease is enhanced significantly! This discovery led to a novel basic understanding and suggests novel therapeutic options. - State what audience might be interested in and influenced by the reported findings.

Dermatologist, immunologist, haematologist - this one goes for a broad audience. - Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

Immunology and hematology. I am not an expert of dermatology.

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Referee #2

Evidence, reproducibility and clarity

General assessment

This is a well-written and carefully executed study that identifies skin-derived G-CSF as a key driver of pathological emergency granulopoiesis in an imiquimod-induced mouse model of psoriasis. The authors convincingly show that endothelial cells are the dominant source of G-CSF in inflamed skin, and that this cytokine mediates systemic hematopoietic skewing and neutrophil accumulation, ultimately aggravating psoriatic pathology. The eanalysis of human transcriptomic datasets strengthens the translational relevance of the findings. Overall, the conclusions are well supported by the data. However, several mechanistically relevant aspects remain underexplored, particularly regarding the functional state of psoriatic neutrophils and the human data integration. Addressing these points would substantially enhance the impact of the study.

Major points

1. Interpretation of neutrophil transcriptomic changes (Figure 2)

The RNA-seq analysis reveals substantial downregulation of several canonical proinflammatory pathways in neutrophils from psoriatic skin, including IL-6, IL-1, and type II interferon signaling. The authors should discuss the functional relevance of this unexpected transcriptional repression. For example, does this indicate a shift toward specialized effector functions rather than classical cytokine responsiveness? More importantly, the most striking transcriptional change is the upregulation of NADPH oxidase-related genes (e.g., Nox1, Nox3, Nox4, Enox2). This suggests an oxidativestress-driven pathogenic mechanism, potentially more relevant than IL-17A production. Yet this aspect is not explored in the manuscript. Assessing ROS levels or oxidative neutrophil effector functions in this model would considerably strengthen the mechanistic link.

Conversely, although IL-17A is upregulated in neutrophils, neutrophil depletion reduces total Il17a expression in skin only partially. This indicates that neutrophils are unlikely to be the dominant IL-17A source in the lesion. The authors' focus on neutrophil-derived IL17A therefore seems overstated. A more rigorous assessment-e.g., conditional deletion of Il17a specifically in neutrophils-would be required to establish its true contribution. Taken together, the data suggest that oxidative programs, rather than IL-17A production, may represent the principal pathogenic axis downstream of neutrophils, and this deserves deeper discussion. 2. Human data reanalysis (Figure 6):

The re-analysis of bulk and single-cell RNA-seq datasets is valuable but incomplete.

Several mechanistically relevant questions could be addressed with the available data:

2.1. GM-CSF (CSF2) is also strongly upregulated in psoriatic lesions (bulk RNA-seq). It would be informative to determine whether endothelial cells also express CSF2 in the scRNA-seq dataset, as this would suggest coordinated regulation of myeloid-supporting cytokines.

2.2. Myeloid cell subsets should be examined more closely. A comparison of human myeloid transcriptomes with the mouse neutrophil RNA-seq would clarify whether similar IL-17A-related or NADPH oxidase-related signatures occur in human disease. In particular, which cell types express IL17A in human lesions?

2.3. Chemokine production should be attributed to specific cell types. Bulk RNA-seq confirms strong induction of CXCL1, CXCL2, CXCL5, but the scRNA-seq dataset allows determining whether these chemokines originate from endothelial cells or other stromal/immune populations. This information is important for defining whether endothelial cells coordinate both neutrophil recruitment and granulopoiesis. Addressing these points would make the human-mouse comparison substantially stronger.

Minor points

1. Line 319: the text likely refers to Figure S4, not S3.
2. Line 338: "psoriatic" is misspelled.

Referees cross-commenting

I agree with Reviewer 1 that the contribution of EC-derived G-CSF to circulating G-CSF levels and to emergency myelopoiesis requires additional genetic or neutralization experiments to be fully established.

Significance

The study is solid and potentially impactful, particularly for audiences working in inflammation and hematopoiesis, as it uncovers a cross-organ mechanism linking skinderived G-CSF to emergency granulopoiesis in psoriasis. My expertise lies in inflammation and hematopoiesis, and from this perspective several essential mechanistic issues remain insufficiently addressed. In particular, the neutrophil transcriptomic data highlight strong induction of NADPH oxidase-related pathways, which appears more biologically meaningful than the modest Il17a upregulation emphasized by the authors. Likewise, the human RNA-seq reanalyses leave open key questions regarding CSF2 expression, myeloid heterogeneity, and chemokine cellular sources. These issues affect the strength and interpretation of the central claims. For these reasons, I recommend major revision before the manuscript can be considered further.

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Referee #1

Evidence, reproducibility and clarity

Summary:

A role of neutrophils in psoriasis pathogenesis has been highlighted by several past studies; however, how the neutrophils are recruited to the affected skin has not been fully understood. The work by Kosasih et al. tackles a relevant question and has investigated the effect of psoriatic skin inflammation on BM myelopoiesis. Using an IMQ-induced acute psoriasis mouse model, the authors derive 3 major conclusions: (1) skin ECs secrete G-CSF into circulation in response to psoriatic stress, (2) skin EC-derived G-CSF stimulates emergency granulopoiesis, and (3) skin EC-derived G-CSF induces infiltration and accumulation of pathogenic neutrophils in the affected skin. The authors provide many pieces of interesting data, but most of them remain correlative and insufficient to support the conclusions. Many of the experiments were performed in a small number of samples or mice (mostly with n=3), leaving the story still preliminary.

Major comments:

1. First, the authors have not convincingly shown that skin cells, or more specifically skin ECs, are a major source of circulating G-CSF in the psoriasis model as stated in the title and abstract. The data in Figure 4 show selective upregulation of Csf3 gene in skin ECs and their ability to secrete G-CSF upon IMQ treatment in vitro. However, the provided data do not address to what degree the skin EC-derived G-CSF contributes to the elevated level of circulating G-CSF. Additional experiments to selectively deplete G-CSF in skin ECs, or at least in skin cells of the affected site, are warranted to support the authors' claim. Does intradermal injection of G-CSF neutralizing antibody into the psoriatic skin reduce circulating levels of G-CSF?
2. Another concern is insufficient demonstration of G-CSF-mediated emergency granulopoiesis in the psoriasis model. All data in Figure 5 were obtained from experiments with only n=3, and adding more replicates, in particular to those in Figure 5B, which show quite some variation in MPP numbers, is recommended. The relatively small reduction of BM granulocyte numbers (Figure 5C) compared to greater depletion of circulating granulocytes (Figure S5A) raises the possibility that it is the mobilization effect rather than granulopoiesis-stimulating effect that skin-derived G-CSF exerts to promote supply of circulating neutrophils that eventually infiltrate into the affected skin. This could also explain the negligible effect of IL-1blockade (Figure S4), which selectively shut off myelopoiesis-stimulating effect of IL-1 (Pietras et al. Nat Cell Biol 2016, PMID: 27111842). Are the HSPCs in the psoriasis model more cycling? Do they show myeloid-skewed differentiation when cultured ex vivo or upon transplantation?
3. The authors' claim that skin-derived G-CSF "induces" neutrophil infiltration warrants further clarification. Alternative explanation is that the upregulated neutrophil-attracting chemokines (Figure S1D) could induce infiltration, whereas G-CSF increase the number of neutrophils to circulate in the vessels near the psoriatic skin. This notion seems supported elsewhere (Moos et al. J Invest Dermatol. 2019, PMID: 30684554). Can the infiltration be inhibited by systemically injecting neutralizing antibody of their receptor, CXCR2?
4. It remains unclear how skin-derived G-CSF accumulates pathogenic neutrophils. The authors state "pathogenic granulopoiesis," but are the circulating neutrophils in the psoriatic mice already "pathogenic" or do they acquire pathogenic phenotype after cutaneous infiltration? Additional RNA-seq to compare circulating and infiltrated neutrophils would answer this question.
5. In addition, how the accumulated pathogenic neutrophils exacerbate the psoriatic changes remains obscure. Although the authors have attempted to correlate Il17a gene expression in infiltrated neutrophils with psoriatic skin changes, the data do not address to what degree it contributes to cutaneous IL-17A protein levels. The data that cutaneous neutrophil depletion leads to subtle decrease in skin IL-17A expression (Figure 2H) rather supports alternative possibilities. For instance, as indicated elsewhere, IL-17A cutaneous tone could be enhanced by neutrophil-mediated augmentation of Th17 or gamma/delta T cell function (Lambert et al. J Invest Dermatol. 2019, PMID: 30528823). Does neutrophil depletion or G-CSF neutralization alter cell numbers or function of cutaneous Th17 and gamma/delta T cells?
6. Finally, as the above conclusions rely solely on the IMQ-induced acute psoriasis model, it would be informative if they could be derived from another psoriasis model. IMQ is known to induce unintended systemic inflammation due to grooming-associated ingestion (Gangwar et al. J Invest Dermatol. 2022, PMID: 34953514), and "pathological crosstalk between skin and BM in psoriatic inflammation" could be strengthened by an intradermal injection model.

Minor comments:

1. Figure 1E shows multiple elongated Ly6G+ structures in d0-2 control and d0 IMQ skins that do not appear to be neutrophils.
2. In Figure 2C, the bottom GSEA seems to be showing type II IFN response, not type I IFN, according to the text.
3. For the BM analysis in Figures 3, 5, S3, and S5, it would be informative if BM cellularity and numbers of committed myeloid progenitors (e.g., GMPs) are shown.
4. In Figures 6B, in which cluster of human skin cells IL-17A expression would be enriched?

Significance

Although quite a few studies have reported various examples of emergency myelopoiesis (Swann et al. Nat Rev Immunol. 2024, PMID: 38467802), there is limited evidence on its occurrence and involvement in locally restricted disease, such as periodontitis (Li et al. Cell 2022, PMID: 35483374; 35483374). As an HSC biologist, I see this study is conceptually interesting as it could extend the above concept to psoriasis, a non-infectious, local inflammatory disease in the skin, and describes a potential causal link between skin-derived G-CSF and emergency myelopoiesis. That said, as detailed in the first section, the conclusions, especially that related to emergency myelopoiesis driven by skin-derived G-CSF, need to be more convincingly supported before taking its value. The findings offer additional understanding of how psoriasis is developed in concert with aberrant hematopoiesis and will be relevant to those working in the field of dermatology, immunology, and hematology.

this is a public comment

Archives leave us with fragments and silences. This line acknowledges that engaging with those silences doesn’t just reveal incompleteness in the past it produces incompleteness in us, the ones who encounter it.

Feminist theory values storytelling, embodied knowledge, and reflexivity. This line reflects the feminist method of embracing impossibility by working with fragments, silences, and erasures as sources of insight rather than treating them as dead ends.

Colonialism transformed people into commodities, corpses, or “waste.” The “afterlife of property” refers to how enslaved people were treated as property, and how that logic continues to shape Black life today. The “detritus of lives” is the residue of that commodification, lives reduced, erased, or unattended.

Official archives privilege dominant voices while silencing marginalized ones. Black counter-historical projects attempts to tell history from the perspective of the oppressed are described here as “insurgent” and “disruptive,” but they fail to become recognized as history because the archive itself is structured to exclude them.

Feminist rage and refusal as a feminist practice. This line embodies refusal by rejecting the archive’s version of events and insisting on imagining beyond it.

Feminist epistemology values oral traditions, embodied knowledge, and creative methodologies. Lowe’s “productive attention to the scene of loss” is exactly that, using imagination and alternative modes of storytelling to recover what archives cannot.

Knowledge comes from bodies, trauma, survival, and intimacy. The possibility of communication between the girls is part of that embodied knowledge; truths carried in relationships, even if undocumented.

Epistemic power highlights how power shapes what counts as legitimate knowledge. This line shows that violence itself structures the discourse, determining what can be said about slavery, and who gets to speak.

Feminist memoryscapes contrast patriarchal, linear archives with feminist memory that is emotional, embodied, and collective. This line embodies that feminist memory practice, mourning as a political act, witnessing as a way of keeping the dead present in the now.

Knowledge comes from bodies, trauma, and survival. This line recognizes that representing the girl risks reducing her body to a metaphor or lesson, rather than attending to her lived reality.

Feminist epistemology values oral traditions, embodied knowledge, and storytelling. This line underscores the impossibility of fully recovering erased lives through archival reading alone, pointing to the need for imaginative, disruptive methods.

Knowledge comes from bodies, trauma, and survival. Venus’s body is central to the story, but colonialism reduced it to an object. Representing her means confronting that violence while acknowledging its limits.

Colonialism and slavery highlight how bodies were commodified and dehumanized. “Social death” refers to being stripped of identity, kinship, and recognition; “corporeal death” is literal death. This line situates enslaved lives in the precarious space between those two forms of violence.

The archival grain emphasizes how archives erase marginalized voices. Here, the writer is grappling with how to render lives that the archive has silenced, experimenting with aesthetics to break through those erasures.

The “slave hold” represents the commodification of human beings under colonialism. The line shows how even attempts to narrate history are haunted by the violence of turning lives into cargo, bodies into waste.

The ledger symbolizes colonial record-keeping, which reduced people to property, numbers, or commodities. The absence of friendship in those records shows how colonialism stripped away humanity and relationality.

Feminist epistemology values storytelling, oral traditions, and embodied knowledge. This line points to the need to replace degrading colonial accounts with narratives rooted in lived experience, dignity, and self-representation.

Western knowledge systems privilege written, rational accounts, often reducing lives to statistics or silence. The line reflects how feminist scholars must fashion stories to bridge past and present, because official records fail to capture marginalized realities.

Knowledge comes from lived experience, trauma, and survival. This line echoes that by insisting that the present is always entangled with embodied histories of violence and survival.

This ties into rage because it exposes injustice, this is why rage is so productive because it is a sign of growth, transforming into power. The injustice are from stories that only exist as records of domination, and feminist rage seeks to reclaim those erased voices.

Western epistemologies privilege written records and rational accounts, while oral traditions, embodied knowledge, and silence are dismissed. This line shows how enslaved women’s voices were excluded because they did not or could not fit into the dominant modes of recording knowledge.

I think this line refers to the archival grain from lecture on Sept. 22, institutions such as government records, colonial documents, such patriarchal institutions exclude or distort marginalized people. Reading against the grain questions what may be missing from official records. How did no one remember her name?

This is a really interesting point. The difference between 'preventing storage accumulation' and 'rescuing the impacts of already having storage accumulation' are definitely worth considering, and I appreciate that you chose a point that is most in line with what happens clinically.

Nice result! Do you have any idea why the sulfatase acitivity wouldn't be rescued but you can see this dramatic improvement in the downstream product levels? Is this a matter of assay sensitivity, or is something else going on?

This result is really striking! I wonder if the single condition which was not rescued (panel D) suggests maybe a specific sulfatase that still might be underperforming/underactivated?

It's intriguing that some of the sulfatase activities plateau below the point of WT, though. I'd be curious to see the different levels of the sulfatases at each expression level of the FGE as well. Perhaps there are fewer sulfatases for some reason?

Do you have a quantitative estimate of increase in expression for your lentiviral integrations vs. wild type levels? That might be interesting to see.

this type of thing sounds like what I thought wrt annotation of [[AI agents als virtueel team]]. The example prompts of questions make me think of [[Filosofische stromingen als gereedschap 20030212105451]] die al per stroming een vraagstramien bevat. Making persona's of diff thinking styles, lines of questioning. Idem for reviews, or starting a project etc.

u/capnrefsmmat reply to

u/lodger238 at https://reddit.com/r/typewriters/comments/1hslx56/huh_i_thought_wd40_was_horrible_as_a_lubricant/<br /> Agree completely and to further the point; typewriters were manufactured with low tolerance in many areas just so they wouldn't need lubricating.

• Children at a young age must take proper measures and time to understand their own language or whatever language is being taught to them.
• It establishes a groundwork to help students find their place in the educational field and society,

diddy bludd

Instructions for students Purpose: Annotating the syllabus allows you to identify questions and clarify course expectations, in addition to practicing using the annotation tool. You can review a quick-start guide for how to add annotations.

Instructions: As you review the syllabus, add at least 2 annotations. Here’s some guidance for what you might include in your annotations:

Ask a question about something that is unclear or confusing to you Paraphrase instructions in your own words (this can help your instructor understand how you’re interpreting expectations) Comment on a topic or assignment you’re looking forward to and explain why Comment on a topic or assignment that is concerning to you and explain why Comment on a topic or assignment using an outside resource, image or video that might be helpful as supplemental material (review instructions on how to add images, links or videos to annotations) Reply to a classmate with an additive annotation (add to the conversation by answering their question or extending their response). Here are some ideas on how to start an additive response to a classmate: What did you mean by … Did you consider …/ You might consider … I think you should … I connect with … It made me think … Important notes about annotating:

Make sure you hit “post” after you complete your annotation, or else your annotation will not be saved. Make sure it says “post to [this class]” and not “post to only me,” or else people won’t be able to review your annotations. If someone replies to your annotation, you will not receive a notification. Check back periodically to continue the conversation!

this, suggested by the team should work.

Via proxy deprecated by Feb 2026

The Via proxy service can be run on ones own domain as it is open source.

The hypothes.is Via proxy is being closed by Feb 2026. So the option I found to annotate on mobile using 'Share' to Via won't work anymore.

Oceans have mroe postive net radiation than land in the mid lats as they asorb more radiation Land masses during summer have low net, due to high albedo of light coloured deserts Polar regions have neggy even in summer as they're ice covered

Earths energy budget is balanced apart from outgoing radiation due to theincreate in greenhhouse gas levels But it's not for everywhere on earth, large scale variation exisits and one fundamental source of this is latitude

Net radiation is the different between incoming SR and outgoing TR

Evapotransipiration is a similar value of incoming SR directly absorbed by the atmopheres, nearly 5x that transferred by sesnisble heat and 22% ofrom surface emitted LW radiation

Global average of SR at the top of the atmosphere is 1/4 solar constant

Energy budget is determined at steady state across one or more years to account for seasonal affects

Greenhouse effect acts as a trap for energy near the surface SE which is emitted as infrared is mostly reabsorbed by the atmosphere, which radiates IR itself This is reabsorbed by other levels in the atmosphere many times over, depedning how transparent it is Radiation can be absorbed and reemitted serval times by gases at dif temps and through the atmsphereic column before escaping into space

Low altitudes the atmospehre is most dense andw arm, which intercepts and remits more radiation Higher altitudes the upward radiation escpates to space as the atmosphere is more transparentlyand colder so emits less radidation in generar The net results is that more radiation returns by being emitted downwards than escapes This recyling of radiation in the lower atmosphere means the surface receives the same energy from the sun more than wonce - first directly as solar radiation and then re-emitted infrared radiation from the atmosphere, raising its temp

-20 is the average torposphere temp, from the surface to tropoause, where LW radiation is emitted to space But gases absorb and remit radation (water/co2) meaning the surface is actually 35 waremer than blackboduy temp of earth which is the greenhosue effect. Without the atmopshere the earhts surface tempw ould be -18.

The earth isn't a single system so its hard to use the law

We can use Stefan-Boltzmann law to estiamte the temp of the Earth (steady state)

The outgoing power of Infrared radiation = the power of absorbed SR, so R = value of absrobed Radiation

Energy coming in is mostly absorbed by SW RD Energy out is infrared LW TR 1/3 SR is refelceted back, and 1/3 of whats left is absrobed by the atmospehre, so half of total radiation incident is absrobed at hte top of the atmophere in the visible region

Based on my own experiences I can positively say that AI is the ruins of this earth. I used to use AI to help me rephrase a prompt or question I didn't understand on its own. However with time I realized this only allowed my comprehension skills to worsen or more so rely on AI to further explain it to me. In the beginning I said hm maybe this is okay because it's like the teacher explaining the prompt to me in a more detailed manner, which is what I needed. But no, I became overly dependent on AI. So I stopped. It was genuinely scary.

Touching on this, I don't understand why this happens. The level of severeness is at an ultimate level, and the developers know this every time. I get that the excitement of trying to see of they can create this new thing is what blinds them but why is there no job position that specifically focuses on making sure they don't create something that will not be ethical in the long run or near future. It should be made so that it is preventable by a committee who isn't so blinded by the project and is focused on the ethics.

Actual solution missing:

• from spacy.util import filter_spans
• Before assigning to doc.ents, filter the spans to resolve overlaps with filtered_ents = filter_spans(original_ents). By default, filter_spans prioritizes longer spans.

going

a

3.2.2 The Matcher

3

It's recursion:

https://hypothes.is/a/nxL8dNRmEfCKiQNmvuH9VQ

Note that in addition to the followup that the author has added to this article (in Feb 2025), he has written a subsequent Tutorial on CWEB article.

Does it defeat the purpose, or is it a form of redundancy?

Same.

I'm amazed by the number of programmers who, having already written down how the problem gets solved, attempt to document/comment their code and don't realize that this is what should be their fundamental concern—what problem does this solve?

Reading John Smith, I realized how times have not changed. How they stated, "If he have nothing but his hands, he may...by industries quickly grow rich" People in corporate now are still the same-- they are able to grow rich through the industries. And everyone else stays beneath even if working twice as hard. It seemed like a genuine talk he had but it has become so toxic

It is important to always ask this question when choosing resources because it helps indicate if the source is credible or not.

It is important to verify that scholarly journals are credible. It is important to do that using the databases in order to verify reputation. In doing this it helps you provide stronger evidence when creating your paper.

It is important that the introduction and conclusion are tied back to the thesis. In doing this it helps to create a cohesive paper.

@chrisaldrich uses the Via proxy on mobile. Or did back in 2020. This will be deprecated by Feb 2026 however.

for - ecological civilization - chinese leaders advocated for it - because many are scientists and engineers

for - definition - ecological civilization - Eastern-based perspective of humans living in harmony with nature - Institutionalized in policy in 2012

for - paper - Earth System Science for Global Sustainability: Grand Challenges - 2009 - still valid today - Forecasting - Observing - Confining (Planetary Boundaries) - Responding (Governance) - Innovating (How we organize society)

for - anthropocene - ecological civilization from an earth system perspective - Deliang Chen

for - post-growth economy - democratic at every level

• SRG comment - democracy at every level - synchronized multi-scale, multi-dimensional wellbeing

for - anthropocene - shared struggle - post-growth and degrowth is part of it

for - anthropocene - activating shared social struggle to turn it around

• SRG comment - anthropocene struggle - TPF

for - wellbeing - claim - has regressed since hunter-gatherers -we've maximized brutality, economic power

• SRG comment
  • weigh against standard progress argument

for - planetary boundaries - postgrowth - provisioning energy - saturation - past a certain point, more energy does not create more wellbeing - decline - energy for high wellbeing has been dropping significantly over time - inefficiency - growth in prmary energy can only account for 25% of improvements in life expectancy - degrowth is technically possible - at less than half the current final energy used

for - anthropocene - provisioning - between planetary boundaries and wellbeing

for - green growth - alternative - postgrowth demand reduction

for - green growth - illusion - too little

for - growth economy - important economic decisions - people have no say

for - growth economy - anti-democratic - free-market rhetoric prevents - democratic - social - environmental - oversight

for - definition - coercive dependence - overproductive economic system imposes overconsumption via lobbying regulation, advertising - creation of resource-intensive dependence as main industrial product of our era

for - economic paradigm - beyond growth Julia Steinberger - beyond economic growth

for - anthropocene - novel entities - nano particles - micro plastic - crossing blood-brain barrier - unkjnown effects

for - anthropocene - many interpretations -SRG comment - interpretant fingerprint

for - definition - anthropocene harm

for - anthropocene - cognitive architecture

for - neuroscience - obedience - alternative - empathy to fellow perpetrators - brotherhood effect

for - neuroscience - obedience study - perpetrators - rescuers - bystanders - rescuers - higher empathy - perpetrators and bystanders - no differences detected

for - stats - genocide obedience - perpetrators vs rescuers - Rawanda 94.66% perpetrators - Nazi 87.15 %

for - neuroscience - of obedience - study - Rawanda - existing populations of perpetrators and those who refused to participate

for - comparison - neuroscience - pain center activation - following orders - activations reduced compared to free decision case

for comparison - neuroscience - guilt center activation - following orders - activations reduced compared to the free decision case

Earth surfaces also scatter light in different amounts, snow and ice for example have a higher albedo and back scatter much more than forests. All these impact how much SR is absorbed by Earth. Clouds scatter outgoing TR which can 'close' the atmospheirc IR window and keep the surface warmer.

This net effect is complex and the role of clouds remains a big uncertainty in climate model proejctions

Lights also scattered by larger WL, clouds which are white or grey scatter SR in all directions so have a big impact on the atmosphere

SW blue light is a clear day, but as sunrise/set the light has to pass through more atmospehre and rayleigh scattering increases, so more LW red and orange reaches us

When SR encounted agents much smaller than the WL (1/10 size) radiation is dispersed in all directions - this is called Rayleigh scattering In the atmosphere it is caused by indiviudal gas molecules and pirmarily affects SW and is effective for visible light

Another important window is at infrared WL ~8-15um, where there's low air absorption, expect just below 10 um due to ozon. This is where the radiation emitted by Earth is high and transparency in this region allows land to cool on cloudless nights as energy emited by earth is lost to space, the window is closed if clouds are present as they absrob and scatter radiation

Atmosphere is very good at absorbing long wave infrared radiation due to carbon dioxide & water vapur Because the atmosphere is mostly transpoarent to short wave radiation, but it does absorb long wave, it heats from the group up. Water vapour, espeically at the earths surface, absorbs 60% of radiation emited by earth and is the gas mainly respoinble for warmer temps inteh lower troposphere Becase the atmosphere gets most of its energy from the surface and not directly the sun it is critical for driving weather processes.

The visible and radio regions are important parts of the pectrum where the atmosphere is pretty transparent Radio & visible regions are known as windows because these wavelenghts can pass through the air without being absrobs Because the atmosphere is mostly tarnsparent to visible radiation most of it reaches the surface and doesn't warm the atmopshere.

Water vapour, oxygen and ozone are important to absorbing solar radiation Nitrogen is a poor absorber of SR Water vapour is the dominate gas which absorbs & scatters radiation across many regions of the specturm. Oxygen & Ozon are v. effective absorbing sWL, high energy radiation, less that 0.3mm reaches the earths surfaces

The image is curved but is actually lots of little lines

Peaks in gas absorption spectrum correspond to specific vibrational and rotational transitions of its molecules, with each occuring at a characteristic energy and specific WL of EMR

Gases in the atmosphere are selective, 20% of radiation that arrives at the top is abosrobed, occuring at differen regions & wavelenghts due to the properties of the gases present

White absorbs all colours and darker clours don't

Chlorophyll absorbs more blue and red so it appears green

the sky is blue because gas scxatter blue and violet more effectively than the longerwave orange lights

Albedo of earth is 30%, largely determined by clouds

Scattering - larger number of weak rays travelling in different directions as the light is bent through angles by partciles/rough surfaces mainly molecules, dust and aerosols, smoke and mist are scattering Also known as diffuse or indirect radiation Because its light travelling in different directions, areas not in a direct line from the sun can recieve light Scattering more than 90degrees is called back-scattering

Reflectin - light striking the surface bounces back at the same angle & intensity it arrived

Albedo is the proportion of radiation which is reflected

WL and size/mature of object determine SR's fate

When radiation ecounters matter, three things can happy Absorbed - molecules then vibrate faster and increase in temp Scattered - redirected or bounce off (how we see things) Transmission - when its passed through like air and water

Business owners then hiring wage laborers at predetermined rates for their work, while the owners get the excess business profits makes my blood boil. Relating this back to my experiences, working for target was my worst nightmare. It was the biggest, busiest, target in Seattle. We were being pushed to extreme workloads by higher ups like crazy. For such little pay. It was infuriating. But of course Target was making millions a day within this target.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary The manuscript by Aarts et al. explores the role of GRHL2 as a regulator of the progesterone receptor (PR) in breast cancer cells. The authors show that GRHL2 and PR interact in a hormone-independent manner and based on genomic analyses, propose that they co-regulate target genes via chromatin looping. To support this model, the study integrates both newly generated and previously published datasets, including ChIP-seq, CUT&RUN, RNA-seq, and chromatin interaction assays, in breast cancer cell models (T47DS and T47D).

Major comments: R1.1 Novelty of GRHL2 in steroid receptor biology The role of GRHL2 as a co-regulator of steroid hormone receptors has previously been described for ER (J Endocr Soc. 2021;5(Suppl 1):A819) and AR (Cancer Res. 2017;77:3417-3430). In the ER study, the authors also employed a GRHL2 ΔTAD T47D cell model. Therefore, while this manuscript extends GRHL2 involvement to PR, the contribution appears incremental rather than conceptual.

We are fully aware of the previously described role of GRHL2 as a co-regulator of steroid hormone receptors, particularly ER and AR. As acknowledged in our introduction (lines 104-108), we explicitly state: "Grainyhead-like 2 (GRHL2) has recently emerged as a potential pioneer factor in hormone receptor-positive cancers, including breast cancer21. However, nearly all studies to date have focused on GRHL2 in the context of ER and estrogen signaling, leaving its role in PR- and progesterone-mediated regulation unexplored22-26".

As for the specific publications that the reviewer refers to: The first refers to an abstract from an annual meeting of the Endocrine Society. As we have been unable to assess the original data underpinning the abstract - including the mentioned GRHL2 DTAD model - we prefer not to cite this particular reference. We do cite other work by the same authors (Reese et al. 2022, our ref. 25). We also cite the AR study mentioned by the reviewer (our ref. 55) in our discussion. As such, we think we do give credit to prior work done in this area.

By characterizing GRHL2 as a co-regulator of the progesterone receptor (PR), we expand on the current understanding of GRHL2 as a common transcriptional regulator within the broader context of steroid hormone receptor biology. Given that ER and PR are frequently co-expressed and active within the same breast cancer cells, our findings raise the important possibility that GRHL2 may actively coordinate or modulate the balance between ER- and PR-driven transcriptional programs, as postulated in the discussion paragraph.

Importantly, we also functionally link PR/GRHL2-bound enhancers to their target genes (Fig5), providing novel insights into the downstream regulatory networks influenced by this interaction. These results not only offer a deeper mechanistic understanding of PR signaling in breast cancer but also lay the groundwork for future comparative analyses between GRHL2's role in ER-, AR-, and PR-mediated gene regulation.

As such, we respectfully suggest that our work offers more than an incremental advance in our knowledge and understanding of GRHL2 and steroid hormone receptor biology.

R1.2 Mechanistic depth The study provides limited mechanistic insight into how GRHL2 functions as a PR co-regulator. Key mechanistic questions remain unaddressed, such as whether GRHL2 modulates PR activation, the sequential recruitment of co-activators/co-repressors, engages chromatin remodelers, or alters PR DNA-binding dynamics. Incorporating these analyses would considerably strengthen the mechanistic conclusions.

Although our RNA-seq data demonstrate that GRHL2 modulates the expression of PR target genes, and our CUT&RUN experiments show that GRHL2 chromatin binding is reshaped upon R5020 exposure, we acknowledge that we have not further dissected the molecular mechanisms by which GRHL2 functions as a PR co-regulator.

We did consider several follow-up experiments to address this, including PR CUT&RUN in GRHL2 knockdown cells, CUT&RUN for known co-activators such as KMT2C/D and P300, as well as functional studies involving GRHL2 TAD and DBD mutants. However, due to technical and logistical challenges, we were unable to carry out these experiments within the timeframe of this study.

That said, we fully recognize that such approaches would provide deeper mechanistic insight into the interplay between PR and GRHL2. We have therefore explicitly acknowledged this limitation in our limitations of the study section (line 502-507) and mention this as an important avenue for future investigation.

R1.3 Definition of GRHL2-PR regulatory regions (Figure 2) The 6,335 loci defined as GRHL2-PR co-regulatory regions are derived from a PR ChIP-seq performed in the presence of hormone and a GRHL2 ChIP-seq performed in its absence. This approach raises doubts about whether GRHL2 and PR actually co-occupy these regions under ligand stimulation. GRHL2 ChIP-seq experiments in both hormone-treated and untreated conditions are necessary to provide stronger support for this conclusion.

Although bulk ChIP-seq cannot definitively demonstrate simultaneous binding of PR and GRHL2 at the same genomic regions, we agree that the ChIP-seq experiments we present do not provide a definitive answer on if GRHL2 and PR co-occupy these regions under ligand stimulation. As a first step to address this, we performed CUT&RUN experiments for both GRHL2 and PR under untreated and R5020-treated conditions. These experiments revealed a subset of overlapping PR and GRHL2 binding sites (approximately {plus minus}5% of the identified PR peaks under ligand stimulation).

We specifically chose CUT&RUN to minimize artifacts from crosslinking and sonication, thereby reducing background and enabling the mapping of high-confidence direct DNA-binding events: Given that a fraction of GRHL2 physically interacts with PR (Fig1D), it is possible that ChIP-seq detects indirect binding of GRHL2 at PR-bound sites and vice versa. CUT&RUN, by contrast, allows us to identify direct binding sites with higher confidence.

Nonetheless, although outside the scope of the current manuscript, we agree that a dedicated GRHL2 ChIP with and without ligand stimulation would provide additional insight, and we have accordingly added this suggestion to the discussion (line 502-507).

R1.4 Cell model considerations The manuscript relies heavily on the T47DS subclone, which expresses markedly higher PR levels than parental T47D cells (Aarts et al., J Mammary Gland Biol Neoplasia 2023; Kalkhoven et al., Int J Cancer 1995). This raises concerns about physiological relevance. Key findings, including co-IP and qPCR-ChIP experiments, should be validated in additional breast cancer models such as parental T47D, BT474, and MCF-7 cells to generalize the conclusions. Furthermore, data obtained from T47D (PR ChIP-seq, HiChIP, CTCF and Rad21 ChIP-seq) and T47DS (RNA-seq, CUT&RUN) are combined along the manuscript. Given the substantial differences in PR expression between these cell lines, this approach is problematic and should be reconsidered.

We agree that physiological relevance is important to consider. Here, all existing model systems have some limitations. In our experience, it is technically challenging to robustly measure gene expression changes in parental T47D cells (or MCF7 cells, for that matter) in response to progesterone stimulation (Aarts et al., J Mammary Gland Biol Neoplasia 2023). As we set out to integrate PR and GRHL2 binding to downstream target gene induction, we therefore opted for the most progesterone responsive model system (T47DS cells). We agree that observations made in T47D and T47DS cells should not be overinterpreted and require further validation. We have now explicitly acknowledged this and added it to the discussion (line 507-509).

As for the reviewer's suggestion to use MCF7 cells: apart from its suboptimal PR-responsiveness, this cell line is also known to harbor GRHL2 amplification, resulting in elevated GRHL2 levels (Reese et al., Endocrinology2019). By that line of reasoning, the use of MCF7 cells would also introduce concerns about physiological relevance. That being said, and as noted in the discussion (line 390-391), the study by Mohammed et al. which identified GRHL2 as a PR interactor using RIME, was performed in both MCF7 and T47D cells. This further supports the notion that the PR-GRHL2 interaction is not limited to a single cell line.

R1.5 CUT&RUN vs ChIP-seq data The CUT&RUN experiments identify fewer than 10% of the PR binding sites reported in the ChIP-seq datasets. This discrepancy likely results from methodological differences (e.g., absence of crosslinking, potential loss of weaker binding events). The overlap of only 158 sites between PR and GRHL2 under hormone treatment (Figure 3B) provides limited support for the proposed model and should be interpreted with greater caution.

We acknowledge the discrepancy between the number of binding sites between ChIP-seq and CUT&RUN. Indeed, methodological differences likely contribute to the differences in PR binding sites reported between the ChIP-seq and CUT&RUN datasets. As the reviewer correctly notes, the absence of crosslinking and sonication in CUT&RUN reduces detection of weaker binding events. However, it also reduces the detection of indirect binding events which could increase the reported number of peaks in ChIPseq data (e.g. the common presence of "shadow peaks").

As also discussed in our response to R1.3, we deliberately chose the CUT&RUN approach to enable the identification of high-confidence direct DNA-binding events. Since GRHL2 physically interacts with PR, ChIP-seq could potentially capture indirect binding of GRHL2 at PR-bound sites, and vice versa. By contrast, CUT&RUN primarily captures direct DNA-protein interactions, offering a more specific binding profile. Thus, while the number of CUT&RUN binding sites is much smaller than previously reported by ChIP-seq, we are confident that they represent true, direct binding events.

We would also like to emphasize that the model presented in figure 6 does not represent a generic or random gene, but rather a specific gene that is co-regulated by both GRHL2 and PR. In this specific case, regulation is proposed to occur via looping interactions from either individual TF-bound sites (e.g., PR-only or GRHL2-only) or shared GRHL2/PR sites. We do not propose that only shared sites are functionally relevant, nor do we assume that GRHL2 and PR must both be directly bound to DNA at these shared sites. Therefore, overlapping sites identified by ChIP-seq-potentially reflecting indirect binding events-could indeed be missed by CUT&RUN, yet still contribute to gene regulation. To clarify this, we have revised the main text (line 331-334) and the legend of Figure 6 to explicitly state that the model refers to a gene with established co-regulation by both GRHL2 and PR.

R1.6 Gene expression analyses (Figure 4) The RNA-seq analysis after 24 hours of hormone treatment likely captures indirect or secondary effects rather than the direct PR-GRHL2 regulatory program. Including earlier time points (e.g., 4-hour induction) in the analysis would better capture primary transcriptional responses. The criteria used to define PR-GRHL2 co-regulated genes are not convincing and may not reflect the regulatory interactions proposed in the model. Strong basal expression changes in GRHL2-depleted cells suggest that much of the transcriptional response is PR-independent, conflicting with the model (Figure 6). A more straightforward approach would be to define hormone-regulated genes in shControl cells and then examine their response in GRHL2-depleted cells. Finally, integrating chromatin accessibility and histone modification datasets (e.g., ATAC-seq, H3K27ac ChIP-seq) would help establish whether PR-GRHL2-bound regions correspond to active enhancers, providing stronger functional support for the proposed regulatory model.

We thank the reviewer for pointing this out. We now recognize that our criteria for selecting PR/GRHL2 co-regulated genes were not clearly described. To address this, we have revised our approach as per the reviewer's suggestion: we first identified early and sustained PR target genes based on their response at 4 and 24 hours of induction and subsequently overlaid this list with the gene expression changes observed in GRHL2-depleted cells. This revised approach reduced the amount of PR-responsive, GRHL2 regulated target genes from 549 to 298 (46% reduction). We consequently updated all following analyses, resulting in revised figures 4 and 5 and supplementary figures 2,3 and 4. As a result of this revised approach, the number of genes that are transcriptionally regulated by GRHL2 and PR (RNAseq data) that also harbor a PR loop anchor at or near their TSS after 30 minutes of progesterone stimulation (PR HiChIP data) dropped from 114 to 79 (30% reduction). We thank the reviewer for suggesting this more straightforward approach and want to emphasize that our overall conclusions remain unaltered.

As above in our response to R1.3, we want to emphasize that the model presented in figure 6 does not depict a generic or randomly chosen gene, but a gene that is specifically co-regulated by both GRHL2 and PR. We also want to emphasize that the majority of GRHL2's transcriptional activity is PR-independent. This is consistent with the limited fraction of GRHL2 that co-immunoprecipitated with PR (Figure 1D), and with the well-established roles of GRHL2 beyond steroid receptor signaling. In fact, the overall importance of GRHL2 for cell viability in T47D(S) cells is underscored by our inability to generate a full knockout (multiple failed attempts of CRISPR/Cas mediated GRHL2 deletion in T47D(S) and MCF7 cells), and by the strong selection we observed against high-level GRHL2 knockdown using shRNA.

As for the reviewer's suggestion to assess whether GRHL2/PR co-bound regions correspond to active enhancers by integrating H3K27ac and ATAC-seq data: We have re-analyzed publicly available H3K27ac and ATAC-seq datasets from T47D cells (references 42 and 43). These analyses are now added to figure 2 (F and G). The H3K27Ac profile suggests that GRHL2-PR overlapping sites indeed correspond to more active enhancers (Figure 2F), with a proposed role for GRHL2 since siGRHL2 affects the accessibility of these sites (Figure 2G).

Minor comments Page 19: The statement that "PR and GRHL2 trigger extensive chromatin reorganization" is not experimentally supported. ATAC-seq would be an appropriate method to test this directly.

We agree with the reviewer and have removed this sentence, as it does not contribute meaningfully to the flow of the manuscript.

Prior literature on GRHL2 as a steroid receptor co-regulator should be discussed more thoroughly.

We now added additional literature on GRHL2 as a steroid hormone receptor co-regulator in the discussion (line 397-401) and we cite the papers suggested by R1 in R1.1 (references 25 and 54).

Reviewer #1 (Significance (Required)):

The identification of novel PR co-regulators is an important objective, as the mechanistic basis of PR signaling in breast cancer remains incompletely understood. The main strength of this study lies in highlighting GRHL2 as a factor influencing PR genomic binding and transcriptional regulation, thereby expanding the repertoire of regulators implicated in PR biology.

That said, the novelty is limited, given the established roles of GRHL2 in ER and AR regulation. Mechanistic insight is underdeveloped, and the reliance on an engineered T47DS model with supra-physiological PR levels reduces the general impact. Without validation in physiologically relevant breast cancer models and clearer separation of direct versus indirect effects, the overall advance remains modest.

The manuscript will be of interest to a specialized audience in the fields of nuclear receptor signaling, breast cancer genomics, and transcriptional regulation. Broader appeal, including translational or clinical relevance, is limited in its current form.

We have addressed all of these points in our response above and agree that with our implemented changes, this study should reach (and appeal to) an audience interested in transcriptional regulation, chromatin biology, hormone receptor signaling and breast cancer.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors present a study investigating the role of GRHL2 in hormone receptor signaling. Previous research has primarily focused on GRHL2 interaction with estrogen receptor (ER) and androgen receptor (AR). In breast cancer, GRHL2 has been extensively studied in relation to ER, while its potential involvement with the progesterone receptor (PR) remains largely unexplored. This is the rationale of this study to investigate the relation between PR and GRHL2. The authors demonstrate an interaction between GRHL2 and PR and further explore this relationship at the level of genomic binding sites. They also perform GRHL2 knockdown experiments to identify target genes and link these transcriptional changes back to GRHL2-PR chromatin occupancy. However, several conceptual and technical aspects of the study require clarification to fully support the authors' conclusions.

R2.1 Given the high sequence similarity among GRHL family members, this raises questions about the specificity of the antibody used for GRHL2 RIME. The authors should address whether the antibody cross-reacts with GRHL1 or GRHL3. For example, GRHL1 shows a higher log fold change than GRHL2 in the RIME data.

Indeed, GRHL1, GRHL2, and GRHL3 are structurally related. They share a similar domain organization and are all {plus minus}70kDa in size. Sequence similarity is primarily confined to the DNA-binding domain, with GRHL2 and GRHL3 showing 81% similarity in this region, and GRHL1 showing 63% similarity to GRHL2/3 (Ming, Nucleic Acids Res 2018).

The antibody used, sourced from the Human Protein Atlas, is widely used in the field. It targets an epitope within the transactivation domain (TAD) of GRHL2-a region with relatively low sequence similarity to the corresponding domains in GRHL1 and GRHL3.

We assessed the specificity of the antibody using western blotting (Supplementary Figure 2A) in T47DS wild-type and GRHL2 knockdown cells. As expected, GRHL2 protein levels were reduced in the knockdown cells providing convincing evidence that the antibody recognizes GRHL2. The remaining signal in shGRHL2 knockdown cells could either be due to remaining GRHL2 protein or due to the antibody detecting GRHL1/3. Furthermore, the observed high log-fold enrichment of GRHL1 in our RIME may reflect known heterodimer formation between GRHL1 and GRHL2, rather dan antibody cross-reactivity. As such, we cannot formally rule out cross-reactivity and have mentioned this in the limitations section (line 497-501).

R2.2 In addition, in RIME experiments, one would typically expect the bait protein to be among the most highly enriched proteins compared to control samples. If this is not the case, it raises questions about the efficiency of the pulldown, antibody specificity, or potential technical issues. The authors should comment on the enrichment level of the bait protein in their data to reassure readers about the quality of the experiment.

We agree with the reviewer that this information is crucial for assessing the quality of the experiment. We have therefore added the enrichment levels (log₂ fold change of IgG control over pulldown) to the methods section (line 592).

As the reviewer notes, GRHL2 was not among the top enriched proteins in our dataset. This is due to unexpectedly high background binding of GRHL2 to the IgG control antibody/beads, for which we currently have no explanation. As a result, although we detected many unique GRHL2 peptides, observed high sequence coverage (>70%), and GRHL2 ranked among the highest in both iBAQ and LFQ values, its relative enrichment was reduced due to the elevated background. During our RIME optimization, Coomassie blue staining of input and IP samples revealed a band at the expected molecular weight of GRHL2 in the pull down samples that was absent in the IgG control (see figure 1 for the reviewer below, 4 right lanes), supporting the conclusion that GRHL2 is specifically enriched in our GRHL2 RIME samples. Combined with enrichment of some of the expected interacting proteins (e.g. KMT2C and KMT2D), we are convinced that the experiment of sufficient quality to support our conclusions.

Figure 1 for reviewer: Coomassie blue staining of input and IP GRHL2 and IgG RIME samples. NT = non-treated, T = treated.

R2.3 The authors report log2 fold changes calculated using iBAQ values for the bait versus IgG control pulldown. While iBAQ provides an estimate of protein abundance within samples, it is not specifically designed for quantitative comparison between samples without appropriate normalization. It would be helpful to clarify the normalization strategy applied and consider using LFQ intensities.

We understand the reviewer's concern. Due to the high background observed in the IgG control sample (see R2.2), the LFQ-based normalization did not accurately reflect the enrichment of GRHL2, which was clearly supported by other parameters such as the number of unique peptides (see rebuttal Table 1). After discussions with our Mass Spectrometry facility, we decided to consider the iBAQ values-which reflect the absolute protein abundance within each sample-as a valid and informative measure of enrichment. In the context of elevated background levels, iBAQ provides an alternative and reliable metric for assessing protein enrichment and was therefore used for our interactor analysis.

Unique peptides

IBAQ GRHL2

IBAQ IgG

LFQ GRHL2

LFQ IgG

GRHL2

52

1753400.00

155355.67

5948666.67

3085700.00

GRHL1

23

56988.33

199.03

334373.33

847.23

*Table 1. Unique peptide, IBAQ and LFQ values of the GRHL2 and IgG pulldowns for GRHL2 and GRHL1 *

R2.4 Other studies have reported PR RIME, which could be a valuable source to investigate whether GRHL proteins were detected.

We thank the reviewer for pointing this out. We are aware of the PR RIME, generated by Mohammed et al., which we refer to in the discussion (lines 390-391). This study indeed identified GRHL2 as a PR-interacting protein in MCF7 and T47D cells. Although they do not mention this interaction in the text, the interaction is clearly indicated in one of the figures from their paper, which supports our findings. To our knowledge, no other PR RIME datasets in MCF7 or T47D cells have been published to date.

R2.5 In line 137, the term "protein score" is mentioned. Could the authors please clarify what this means and how it was calculated.

We agree that this point was not clearly explained in the original text. The scores presented reflect the MaxQuant protein identification confidence, specifically the sum of peptide-level scores (from Andromeda), which indicates the relative confidence in protein detection. We have now added this clarification to line 137 and to the legend of Figure 1.

R2.6 In line 140-141. The fact that GRHL2 interacts with chromatin remodelers does not by itself prove that GRHL2 acts as a pioneer factor or chromatin modulator. Demonstrating pioneer function typically requires direct evidence of chromatin opening or binding to closed chromatin regions (e.g., ATAC-seq, nucleosome occupancy assays). I recommend revising this statement or providing supporting evidence.

We agree that the fact that GRHL2 interacts with chromatin remodelers does not by itself prove that GRHL2 acts as a pioneer factor or chromatin modulator. However, a previous study (Jacobs et al, Nature genetics, 2018) has shown directly that the GRHL family members (including GRHL2) have pioneering function and regulate the accessibility of enhancers. We adapted line 140-141 to state this more clearly. In addition, our newly added data in Figure 2G also support the fact that GRHL2 has a role in regulating chromatin accessibility in T47D cells.

R2.7 The pulldown Western blot lacks an IgG control in panel D.

This is correct. As the co-IP in Figure 1D served as a validation of the RIME and was specifically aimed at determining the effect of hormone treatment on the observed PR/GRHL2 interaction, we did not perform this control given the scale of the experiment. However, during RIME optimization, we performed GRHL2 staining of the IgG controls by western blot, shown in figure 2 for the reviewer below. As stated above, some background GRHL2 signal was observed in the IgG samples, but a clear enrichment is seen in the GRHL2 IP.

Taken together, we believe that the well-controlled RIME, combined with the co-IP presented, provides strong evidence that the observed signal reflects a genuine GRHL-PR interaction.

Figure 2 for reviewer: WB of input and IP GRHL2 and IgG RIME samples stained for GRHL2. NT = non-treated, T = treated

R2.8 Depending on the journal and target audience, it may be helpful to briefly explain what R5020 is at its first mention (line 146).

Thank you. We have adapted this accordingly.

R2.9 The authors state that three technical replicates were performed for each experimental condition. It would be helpful to clarify the expected level of overlap between biological replicates of RIME experiments. This clarification is necessary, especially given the focus on uniquely enriched proteins in untreated versus treated cells, and the observation that some identified proteins in specific conditions are not chromatin-associated. Replicates or validations would strengthen the findings.

We use the term technical rather than biological replicates because for cell lines, defining true biological replicates is challenging, as most variability arises from experimental rather than biological differences. To introduce some variation, we split our T47DS cells into three parallel dishes 5 days prior to starting the treatment. We purposely did this, to minimize to minimize the likelihood that proteins identified as uniquely enriched are artifacts. Each of the three technical replicates comes from one of these three parallel splits (so equal passage numbers but propagated in parallel dishes for 5 days before the start of the experiment).

To generate the three technical replicates for our RIME, we plated cells from the parallel grown splits. Treatments for the three replicates were performed per replicate. Samples were crosslinked, harvested and lysed for subsequent RIME analysis, the three replicates were processed in parallel, for technical and logistical reasons. To clarify the experimental setup, we have updated the methods section accordingly (lines 566-568).

As for the detection of non-chromatin-associated proteins: We cannot rule out that these are artifacts, as they may arise from residual cytosolic lysate during nuclear extraction. Alternatively, they could reflect a more dynamic subcellular localization of these proteins than currently annotated or appreciated.

R2.10 The volcano plot for the RIME experiment appears to show three distinct clusters of proteins on the right, which is unusual for this type of analysis. The presence of these apparent groupings may suggest an artifact from the data processing, such as imputation. Can the authors clarify the origin of these groupings? If it is due to imputation or missing values, I recommend applying a stricter threshold, such as requiring detection in all three replicates (3/3) to improve the robustness of the enrichment analysis and increase confidence in the identified interactors.

We thank the reviewer for pointing this out. As suggested, we re-evaluated the imputation and applied a stricter threshold, requiring detection in all three replicates. Indeed, the separate clusters were due to missing values, therefore we now revised the imputation method by imputing values based on the normal distribution. Using this revised analysis, we identify 2352 GRHL2 interactors instead of 1140, but the number of interacting proteins annotated as transcription factors or chromatin-associated/modifying proteins was still 103. Figure 1B, 1E, and Supplementary Figure 4A have been updated accordingly. We also revised the methods section to reflect this change. We think this suggestion has improved our analysis of the data and we thank the reviewer for pointing this out.

R2.11 The statement that "PR and GRHL2 frequently overlap" may be overstated given that only ~700 overlapping sites are reported (cut&run).

We have replaced "frequently overlap" by "can overlap" (line 229-230).

R2.12 The model in Figure 6 suggests limited chromatin occupancy of PR and GRHL2 in hormone-depleted conditions, consistent with the known requirement of ligand for stable PR-DNA binding. However, Figure 1 shows no major difference in GRHL2-PR interaction between untreated and hormone-treated cells. This raises questions about where and how this interaction occurs in the absence of hormone. Since PR binding to chromatin is typically minimal without ligand, can the authors clarify this given that RIME data reflect chromatin-bound interactions.

Indeed, the model in figure 6 suggests limited chromatin occupancy of PR and GRHL2 under hormone-depleted conditions. It is, however, important to note that the locus shown represents a gene regulated by both PR and GRHL2 - and not just any gene. We recognize that this was not sufficiently clear in the original version, and we have now clarified this in both the main text (line 331-334) and the figure legend.

We propose that PR and GRHL2 bind or become enriched at enhancer sites associated with their target genes upon ligand stimulation. This is consistent with the known requirement of ligand for stable PR-DNA binding and with our observation that PR/GRHL2 overlapping peaks are detected only in the ligand-treated condition of the CUT&RUN experiment. Given the broader role of GRHL2, it also binds chromatin independently of progesterone and the progesterone receptor, which is why we included-but did not focus on-GRHL2-only binding events in our model.

We would also like to clarify that, although RIME includes a nuclear enrichment step that enriches for chromatin-associated proteins, the pulldown is performed on nuclear lysates. Therefore, it captures both chromatin-bound protein complexes and freely soluble nuclear complexes, which unfortunately cannot be distinguished. GRHL2 is well established as a nuclear protein (Zeng et al., Cancers 2024; Riethdorf et al., International Journal of Cancer 2015), and although PR is classically described as translocating to the nucleus upon hormone stimulation, several studies-including our own-have shown that PR is continuously present in the nucleus (Aarts et al., J Mammary Gland Biol Neoplasia 2023; Frigo et al., Essays Biochem. 2021).

We therefore propose that PR and GRHL2 may already interact in the nucleus without directly binding to chromatin. Given our observation that GRHL2 binding sites on the chromatin are redistributed upon R5020 mediated signaling activation, we hypothesize that such pre-formed PR-GRHL2 nuclear complexes may assist the rapid recruitment of GRHL2 to progesterone-responsive chromatin regions.

We have expanded the discussion to include a dedicated section addressing this point (line 376-388).

R2.13 It would be of interest to assess the overlap between the proteins identified in the RIME experiment and the motif analysis results.

In the discussion section of our original manuscript, we highlighted some overlapping proteins in the RIME and motif analysis, including STAT6 and FOXA1. However, we had not yet systematically analyzed overlap in both analyses. To address this, we now compared all enriched motifs (so not only the top 5 as displayed in our figures) under GRHL2, PR, and GRHL2/PR shared sites from both the CUT&RUN and ChIP-seq datasets with the proteins identified as GRHL2 interactors in our RIME. Although we identified numerous GRHL2-associated proteins, relatively few of them were transcription factors whose binding motifs were also enriched under GRHL2 peaks.

In our revised manuscript we have added a section in the discussion highlighting our systematic overlap of the results of our RIME experiment and the motif enrichment of the ChIP-seq and CUT&RUN analysis (line 415-436).

R2.14 The authors chose CUT&RUN to assess chromatin binding of PR and GRHL2. Given that RIME is also based on chromatin immunoprecipitation - ChIP protocol, it would be helpful to clarify why CUT&RUN was selected over ChIP-seq for the DNA-binding assays. What is the overlap with published data?

As also mentioned in our response to R1.3 and R1.5, we deliberately chose the CUT&RUN approach to minimize artifacts introduced by crosslinking and sonication, thereby reducing background and allowing the identification of high-confidence, direct DNA-binding events. Since GRHL2 physically interacts with PR, ChIP-seq could potentially capture indirect binding of GRHL2 at PR-bound sites (and vice versa). In contrast, CUT&RUN primarily detects direct DNA-protein interactions, providing a more specific and accurate binding profile. Additionally, CUT&RUN serves as an independent validation method for data obtained using ChIP-like protocols.

Since CUT&RUN, similar to ChIP, can show limited reproducibility (Nordin et al., Nucleic Acids Research, 2024), and to our knowledge few PR CUT&RUN and no GRHL2 CUT&RUN datasets are currently available, it is challenging to directly compare our data with published datasets. Nevertheless, studies performing PR or ER CUT&RUN (Gillis et al., Cancer Research, 2024; Reese et al., Molecular and Cellular Biology, 2022) report a comparable number of peaks-in the same range of thousands-as observed in our data. This suggests that a single CUT&RUN experiment in general may detect fewer events than a single ChIP-seq experiment, but that the peaks that are found are likely to reflect direct binding events.

Reviewer #2 (Significance (Required)):

General Assessment: This study investigates the role of the transcription factor GRHL2 in modulating PR function, using RIME and CUT&RUN to explore protein-protein and protein-chromatin interactions. GRHL2 have been implicated in epithelial biology and transcriptional regulation and interaction with steroid hormone receptors has been reported. This study extends the field by showing a functional link between GRHL2 and PR, which has implications for understanding hormone-dependent gene regulation.

The research will primarily interest a specialized audience in transcriptional regulation, chromatin biology, and hormone receptor signaling.

Key words for this reviewer: chromatin biology, transcription factor function, epigenomics, and proteomics.

We agree that with our implemented changes, this study should reach (and appeal to) an audience interested in transcriptional regulation, chromatin biology, hormone receptor signaling and breast cancer.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This study explores the important transcriptional coordination role of Grainyhead-like 2 (GRHL2) on the transcriptional regulatory function of progesterone receptor (PR). In this paper, the authors start with their recruitment characteristics, take into account their regulatory effects on downstream genes and their effects on the occurrence and development of breast cancer, and further clarify the coordination between them in three-dimensional space. The interaction between GRHL2 and PR, and the subsequent important influence on the co-regulated genes by GRHL2 and PR are analyzed. The overall framework of this study is mainly by RNA seq and CUT-TAG analysis, the molecular mechanism underlying the association between GRHL2 and PR and regulation function of two proteins in breast cancer is not clearly clarified. Some details need to be further improved：

Major comments: R3.1 For Fig.1D, the molecular weight of each protein should be marked in the diagram, and the expression of GRHL2 in the input group should be supplemented.

We apologize for not including molecular weights in our initial submission. We are not entirely clear what the reviewer means with their statement that "the expression of GRHL2 in the input group should be supplemented". The blot depicted in Figure 1D shows both the input signal and the IP. For the reviewer's information, the full Western blot is depicted below.

Figure 3 for reviewer: Full WBs of input and IP GRHL2 samples stained for GRHL2 or PR. NT = non-treated, T = treated

R3.2 In Fig.2B and Fig 5C, it should be describe well whether GRHL2 recruitment is in the absence or presence of R5020? How about the co-occupancy of PR and GRHL2 region, Promoter or enhancer region? It would be better to show histone marks such as H3K27ac and H3K4me1 to annotate the enhancer region.

As also stated in our response to R1.3, we acknowledge that the ChIP-seq experiments cannot definitively determine whether GRHL2 and PR co-occupy genomic regions under ligand-stimulated conditions, since the GRHL2 dataset was generated in the absence of progesterone stimulation (as indicated in lines 167-169). To clarify this, we have now specified this detail in the legend of figure 2 by noting "untreated GRHL2 ChIP." To directly assess GRHL2 chromatin binding under progesterone-stimulated conditions, we performed CUT&RUN experiments for both GRHL2 and PR under untreated and R5020-treated conditions. These experiments revealed a subset of overlapping PR and GRHL2 binding sites (approximately 5% of all identified PR peaks.

In our original manuscript, we performed genomic annotation of the GRHL2, PR, and GRHL2/PR overlapping peaks (Figure 2E) and found that most of these sites were located in intergenic regions, where enhancers are typically found, with ~20% located in promoter regions. We appreciate the reviewer's suggestion to further overlap the ChIP-seq peaks with histone marks such as H3K27ac and H3K4me1. We have now incorporated publicly available ATAC-seq and H3K27ac ChIP datasets in our revised manuscript (as also suggested by Reviewer 1) and find that shared GRHL2/PR sites are indeed located in active enhancer regions marked by H3K27ac (see Figure 2F). Additionally, as expected, we find that GRHL2/PR overlapping sites are enriched at open chromatin (Figure 2G).

R3.3 What is the biological function analysis by KEGG or GO analysis for the overlapping genes from VN plots of RNA-seq with CUT-TAG peaks. The genes co-regulated by GRH2L and PR are further determined.

For us, it is not entirely clear what reviewer 3 is asking here, but we can explain the following: as it is challenging to integrate HiChIP with CUT&RUN, due to the fundamentally different nature of the two techniques, we chose not to directly assign genes to CUT&RUN peaks. However, we did carefully link the GRHL2, PR, and GRHL2/PR ChIP-seq peaks to their target genes by integrating chromatin looping data from a PR HiChIP analysis. The result from this analysis is depicted in Figure 4B.

As suggested by this reviewer, we also performed a GO-term analysis on the 79 genes that are regulated by both GRHL2 and PR (we now have 79 genes after the re-analysis as suggested in R1.6). The corresponding results are provided for the reviewer in figure 3 of this rebuttal (below). As this additional analysis does not provide further biological insight beyond what is already presented in Figure 4C, we decided to not include this figure in the manuscript.

Figure 4 for reviewer: GO-term analysis on the 79 GRHL2-PR co-regulated genes that are transcriptionally regulated by GRHL2 and PR and that also harbor a PR HiChIP loop anchor at or near their TSS

R3.4 Western blotting should be performed to determine the protein levels of downstream genes co-regulated genes by GRH2L and PR in the absence or presence of R5020.

We agree that determining the response of co-regulated is important. Therefore, in Figure 4D, we present three representative examples of genes that are directly co-regulated by GRHL2 and PR-specifically, genes that are differentially expressed after 4 hours of R5020 exposure. Although protein levels of the targets are of functional importance, GRHL2 and PR are of transcription factors whose immediate effects are primarily exerted at the level of gene transcription. Therefore, in our opinion, changes in mRNA abundance provide the most direct and mechanistically relevant readout of their regulatory activity.

R3.5 The author mentioned that this study positions that GRHL2 acts as a crucial modulator of steroid hormone receptor function, while the authors do not provide the evidences that how does GRHL2 regulate PR-mediated transactivation, and how about these two proteins subcellular distribution in breast cancer cells.

We agree that while our RNA-seq data demonstrate that GRHL2 modulates the expression of PR target genes, and our CUT&RUN experiments show that GRHL2 chromatin binding is reshaped upon R5020 exposure, we have not yet further dissected the molecular mechanism by which GRHL2 functions as a PR co-regulator.

As also mentioned in our response to R1.2, we did consider several follow-up experiments to address this, including PR CUT&RUN in GRHL2 knockdown cells, CUT&RUN for known co-activators such as KMT2C/D and P300, as well as functional studies involving GRHL2 TAD and DBD mutants. However, due to technical and logistical challenges, we were unable to carry out these experiments within the timeframe of this study.

That said, we fully recognize that such approaches would provide deeper mechanistic insight into the interplay between PR and GRHL2. We have therefore explicitly acknowledged this limitation in our limitations of the study section (lines 502-507) and consider it an important avenue for future investigation.

Regarding the subcellular distribution in breast cancer cells: As also mentioned in our response to R2.12, GRHL2 is well established as a nuclear protein (Zeng et al., Cancers 2024; Riethdorf et al., International Journal of Cancer 2015), and although PR is classically described as translocating to the nucleus upon hormone stimulation, several studies-including our own-have shown that PR is continuously present in the nucleus (Aarts et al., J Mammary Gland Biol Neoplasia 2023; Frigo et al., Essays Biochem. 2021). Thus, both proteins mostly reside in the nucleus in breast (cancer) cells both in the absence and presence of hormone stimulation, but dynamic subcellular shuttling is likely to occur.

Minor comments: Please describe in more detail the relationship between PR and GRHL2 binding independent of the hormone in the discussion section.

As also mentioned in our response to R2.12, we have expanded the discussion to include a dedicated section addressing this point (lines 376-388).

Reviewer #3 (Significance (Required)):

Advance: Compare the study to existing published knowledge, it fills a gap. The authors provide RNA seq and CUT-TAG sequence analysis to show the recruitment of GRHL2 and PR and the co-regulated genes in the absence or presence of progesterone.

Audience: breast surgery will be interested, and the audiences will cover clinical and basic research.

My expertise is focused on the epigenetic modulation of steroid hormone receptors in the related cancers, such as breast cancer, prostate cancer, and endometrial carcinoma.

We agree that with our implemented changes, this study should reach (and appeal to) an audience interested in transcriptional regulation, chromatin biology, hormone receptor signaling and breast cancer.

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

Referee #3

Evidence, reproducibility and clarity

This study explores the important transcriptional coordination role of Grainyhead-like 2 (GRHL2) on the transcriptional regulatory function of progesterone receptor (PR). In this paper, the authors start with their recruitment characteristics, take into account their regulatory effects on downstream genes and their effects on the occurrence and development of breast cancer, and further clarify the coordination between them in three-dimensional space. The interaction between GRHL2 and PR, and the subsequent important influence on the co-regulated genes by GRHL2 and PR are analyzed. The overall framework of this study is mainly by RNA seq and CUT-TAG analysis, the molecular mechanism underlying the association between GRHL2 and PR and regulation function of two proteins in breast cancer is not clearly clarified. Some details need to be further improved：

Major comments:

1. For Fig.1D, the molecular weight of each protein should be marked in the diagram, and the expression of GRHL2 in the input group should be supplemented.
2. In Fig.2B and Fig 5C, it should be describe well whether GRHL2 recruitment is in the absence or presence of R5020? How about the co-occupancy of PR and GRHL2 region, Promoter or enhancer region? It would be better to show histone marks such as H3K27ac and H3K4me1 to annotate the enhancer region.
3. What is the biological function analysis by KEGG or GO analysis for the overlapping genes from VN plots of RNA-seq with CUT-TAG peaks. The genes co-regulated by GRH2L and PR are further determined.
4. Western blotting should be performed to determine the protein levels of downstream genes co-regulated genes by GRH2L and PR in the absence or presence of R5020.
5. The author mentioned that this study positions that GRHL2 acts as a crucial modulator of steroid hormone receptor function, while the authors do not provide the evidences that how does GRHL2 regulate PR-mediated transactivation, and how about these two proteins subcellular distribution in breast cancer cells.

Minor comments:

Please describe in more detail the relationship between PR and GRHL2 binding independent of the hormone in the discussion section.

Significance

Advance: Compare the study to existing published knowledge, it fills a gap. The authors provide RNA seq and CUT-TAG sequence analysis to show the recruitment of GRHL2 and PR and the co-regulated genes in the absence or presence of progesterone.

Audience: breast surgery will be interested, and the audiences will cover clinical and basic research.

My expertise is focused on the epigenetic modulation of steroid hormone receptors in the related cancers, such as breast cancer, prostate cancer, and endometrial carcinoma.

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

Referee #2

Evidence, reproducibility and clarity

The authors present a study investigating the role of GRHL2 in hormone receptor signaling. Previous research has primarily focused on GRHL2 interaction with estrogen receptor (ER) and androgen receptor (AR). In breast cancer, GRHL2 has been extensively studied in relation to ER, while its potential involvement with the progesterone receptor (PR) remains largely unexplored. This is the rational of this study to investigate the relation between PR and GRHL2. The authors demonstrate an interaction between GRHL2 and PR and further explore this relationship at the level of genomic binding sites. They also perform GRHL2 knockdown experiments to identify target genes and link these transcriptional changes back to GRHL2-PR chromatin occupancy. However, several conceptual and technical aspects of the study require clarification to fully support the authors' conclusions.

1. Given the high sequence similarity among GRHL family members, this raises questions about the specificity of the antibody used for GRHL2 RIME. The authors should address whether the antibody cross-reacts with GRHL1 or GRHL3. For example, GRHL1 shows a higher log fold change than GRHL2 in the RIME data.
2. In addition, in RIME experiments, one would typically expect the bait protein to be among the most highly enriched proteins compared to control samples. If this is not the case, it raises questions about the efficiency of the pulldown, antibody specificity, or potential technical issues. The authors should comment on the enrichment level of the bait protein in their data to reassure readers about the quality of the experiment.
3. The authors report log2 fold changes calculated using iBAQ values for the bait versus IgG control pulldown. While iBAQ provides an estimate of protein abundance within samples, it is not specifically designed for quantitative comparison between samples without appropriate normalization. It would be helpful to clarify the normalization strategy applied and consider using LFQ intensities.
4. Other studies have reported PR RIME, which could be a valuable source to investigate whether GRHL proteins were detected.
5. In line 137, the term "protein score" is mentioned. Could the authors please clarify what this means and how it was calculated.
6. In line 140-141. The fact that GRHL2 interacts with chromatin remodelers does not by itself prove that GRHL2 acts as a pioneer factor or chromatin modulator. Demonstrating pioneer function typically requires direct evidence of chromatin opening or binding to closed chromatin regions (e.g., ATAC-seq, nucleosome occupancy assays). I recommend revising this statement or providing supporting evidence.
7. The pulldown Western blot lacks an IgG control in panel D.
8. Depending on the journal and target audience, it may be helpful to briefly explain what R5020 is at its first mention (line 146).
9. The authors state that three technical replicates were performed for each experimental condition. It would be helpful to clarify the expected level of overlap between biological replicates of RIME experiments. This clarification is necessary, especially given the focus on uniquely enriched proteins in untreated versus treated cells, and the observation that some identified proteins in specific conditions are not chromatin-associated. Replicates or validations would strengthen the findings.
10. The volcano plot for the RIME experiment appears to show three distinct clusters of proteins on the right, which is unusual for this type of analysis. The presence of these apparent groupings may suggest an artifact from the data processing, such as imputation. Can the authors clarify the origin of these groupings? If it is due to imputation or missing values, I recommend applying a stricter threshold, such as requiring detection in all three replicates (3/3) to improve the robustness of the enrichment analysis and increase confidence in the identified interactors.
11. The statement that "PR and GRHL2 frequently overlap" may be overstated given that only ~700 overlapping sites are reported (cut&run).
12. The model in Figure 6 suggests limited chromatin occupancy of PR and GRHL2 in hormone-depleted conditions, consistent with the known requirement of ligand for stable PR-DNA binding. However, Figure 1 shows no major difference in GRHL2-PR interaction between untreated and hormone-treated cells. This raises questions about where and how this interaction occurs in the absence of hormone. Since PR binding to chromatin is typically minimal without ligand, can the authors clarify this given that RIME data reflect chromatin-bound interactions.
13. It would be of interest to assess the overlap between the proteins identified in the RIME experiment and the motif analysis results.
14. The authors chose CUT&RUN to assess chromatin binding of PR and GRHL2. Given that RIME is also based on chromatin immunoprecipitation - ChIP protocol, it would be helpful to clarify why CUT&RUN was selected over ChIP-seq for the DNA-binding assays. What is the overlap with published data?

Significance

General Assessment:

This study investigates the role of the transcription factor GRHL2 in modulating PR function, using RIME and CUT&RUN to explore protein-protein and protein-chromatin interactions. GRHL2 have been implicated in epithelial biology and transcriptional regulation and interaction with steroid hormone receptors has been reported. This study extends the field by showing a functional link between GRHL2 and PR, which has implications for understanding hormone-dependent gene regulation.

The research will primarily interest a specialized audience in transcriptional regulation, chromatin biology, and hormone receptor signaling.

Key words for this reviewer: chromatin biology, transcription factor function, epigenomics, and proteomics.

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

Referee #1

Evidence, reproducibility and clarity

Summary

The manuscript by Aarts et al. explores the role of GRHL2 as a regulator of the progesterone receptor (PR) in breast cancer cells. The authors show that GRHL2 and PR interact in a hormone-independent manner and, based on genomic analyses, propose that they co-regulate target genes via chromatin looping. To support this model, the study integrates both newly generated and previously published datasets, including ChIP-seq, CUT&RUN, RNA-seq, and chromatin interaction assays, in breast cancer cell models (T47DS and T47D).

Major comments:

1. Novelty of GRHL2 in steroid receptor biology The role of GRHL2 as a co-regulator of steroid hormone receptors has previously been described for ER (J Endocr Soc. 2021;5(Suppl 1):A819) and AR (Cancer Res. 2017;77:3417-3430). In the ER study, the authors also employed a GRHL2 ΔTAD T47D cell model. Therefore, while this manuscript extends GRHL2 involvement to PR, the contribution appears incremental rather than conceptual.
2. Mechanistic depth The study provides limited mechanistic insight into how GRHL2 functions as a PR co-regulator. Key mechanistic questions remain unaddressed, such as whether GRHL2 modulates PR activation, the sequential recruitment of co-activators/co-repressors, engages chromatin remodelers, or alters PR DNA-binding dynamics. Incorporating these analyses would considerably strengthen the mechanistic conclusions.
3. Definition of GRHL2-PR regulatory regions (Figure 2) The 6,335 loci defined as GRHL2-PR co-regulatory regions are derived from a PR ChIP-seq performed in the presence of hormone and a GRHL2 ChIP-seq performed in its absence. This approach raises doubts about whether GRHL2 and PR actually co-occupy these regions under ligand stimulation. GRHL2 ChIP-seq experiments in both hormone-treated and untreated conditions are necessary to provide stronger support for this conclusion.
4. Cell model considerations The manuscript relies heavily on the T47DS subclone, which expresses markedly higher PR levels than parental T47D cells (Aarts et al., J Mammary Gland Biol Neoplasia 2023; Kalkhoven et al., Int J Cancer 1995). This raises concerns about physiological relevance. Key findings, including co-IP and qPCR-ChIP experiments, should be validated in additional breast cancer models such as parental T47D, BT474, and MCF-7 cells to generalize the conclusions. Furthermore, data obtained from T47D (PR ChIP-seq, HiChIP, CTCF and Rad21 ChIP-seq) and T47DS (RNA-seq, CUT&RUN) are combined along the manuscript. Given the substantial differences in PR expression between these cell lines, this approach is problematic and should be reconsidered.
5. CUT&RUN vs ChIP-seq data The CUT&RUN experiments identify fewer than 10% of the PR binding sites reported in the ChIP-seq datasets. This discrepancy likely results from methodological differences (e.g., absence of crosslinking, potential loss of weaker binding events). The overlap of only 158 sites between PR and GRHL2 under hormone treatment (Figure 3B) provides limited support for the proposed model and should be interpreted with greater caution.
6. Gene expression analyses (Figure 4) The RNA-seq analysis after 24 hours of hormone treatment likely captures indirect or secondary effects rather than the direct PR-GRHL2 regulatory program. Including earlier time points (e.g., 4-hour induction) in the analysis would better capture primary transcriptional responses. The criteria used to define PR-GRHL2 co-regulated genes are not convincing and may not reflect the regulatory interactions proposed in the model. Strong basal expression changes in GRHL2-depleted cells suggest that much of the transcriptional response is PR-independent, conflicting with the model (Figure 6). A more straightforward approach would be to define hormone-regulated genes in shControl cells and then examine their response in GRHL2-depleted cells. Finally, integrating chromatin accessibility and histone modification datasets (e.g., ATAC-seq, H3K27ac ChIP-seq) would help establish whether PR-GRHL2-bound regions correspond to active enhancers, providing stronger functional support for the proposed regulatory model.

Minor comments

Page 19: The statement that "PR and GRHL2 trigger extensive chromatin reorganization" is not experimentally supported. ATAC-seq would be an appropriate method to test this directly.

Prior literature on GRHL2 as a steroid receptor co-regulator should be discussed more thoroughly.

Significance

The identification of novel PR co-regulators is an important objective, as the mechanistic basis of PR signaling in breast cancer remains incompletely understood. The main strength of this study lies in highlighting GRHL2 as a factor influencing PR genomic binding and transcriptional regulation, thereby expanding the repertoire of regulators implicated in PR biology. That said, the novelty is limited, given the established roles of GRHL2 in ER and AR regulation. Mechanistic insight is underdeveloped, and the reliance on an engineered T47DS model with supra-physiological PR levels reduces the general impact. Without validation in physiologically relevant breast cancer models and clearer separation of direct versus indirect effects, the overall advance remains modest.

The manuscript will be of interest to a specialized audience in the fields of nuclear receptor signaling, breast cancer genomics, and transcriptional regulation. Broader appeal, including translational or clinical relevance, is limited in its current form.

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