- Jun 2024
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www.biorxiv.org www.biorxiv.org
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Whether RTN4IP1 functions through interactions with other partners or by facilitating posttranslational modifications during CI assembly remains unanswered and warrants further investigation
Do you have thoughts on how you would further experimentally decouple the Complex I assembly and CoQ biosynthesis functions of RTN4IP1?
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Pathway analysis revealed that the affected biological processes of upregulated proteins were related to cell-substrate adhesion and extracellular organization
Did you look into why RTN4IP1 upregulated cell adhesion and extracellular organization factors? This is surprising to me given the clear role you have demonstrated in Complex I assembly.
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This phenomenon was particularly evident in the context of CI, where the identification of assembly factors increased nearly threefold between 24 hours and 1 week
Do you have a hypothesis as to why the galactose growth phenotype is particularly pronounced for Complex I assembly factors? Is complex I especially reliant on assembly factors to adopt its active configuration?
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Dynamics and surveillance, along with the Signaling pathway, exhibited notable enrichment in genes with no discernible phenotype, implying that genes within these categories may be dispensable for proliferation in galactose, likely owing to their limited influence on OXPHOS activity
Is it possible to use an alternative functional genomics approach to study genes involved in dynamics and signaling? How would you set up such a screen?
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Even though we now possess a very detailed picture of the core proteins that comprise the OXPHOS complexes, the list of assembly factors and regulatory components is far from being completed
You mention that many nuclear-encoded genes important in mitochondrial function have not yet been identified. Is it possible to adapt your screening approach to identify these genes without having to use a genome-wide library?
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- May 2024
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www.ncbi.nlm.nih.gov www.ncbi.nlm.nih.gov
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Using Enformer87, a transformer-based neural network, we created a combined score of variant effects on chromatin accessibility and TF occupancy
What mechanisms besides chromatin accessibility and interactions with specific transcription factors could potentially be at play mediating the effects of cis-regulatory elements on gene expression?
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Ultimately, our results provide support for a model where trait associated variants reside in CREs that can be either cell-type specific or cell-type agnostic, the latter being an underappreciated mechanism of causal variants
I think it’s exciting that you can identify cell-type agnostic as well as cell-type specific transcriptional changes. While we often think about cell-type differences in gene expression in the context of disease, I’m looking forward to seeing how this work will improve our ability to consider system-wide changes that are nonetheless more or less likely to manifest pathology in specific tissues.
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the magnitude of cell-type agnostic transcriptional activation in our assay correlates well with quantitative measures (maximum) of the chromatin accessibility of DNA across 438 cell-types
I’m curious about this. Why does chromatin accessibility correlate with transcriptional activation for a non-integrated plasmid?
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with the majority enhancing rather than repressing transcription (96.6%), reflecting the design of our reporter assay
Why does the design of the reporter assay make it more likely to identify enhancers? Have you considered an assay design that enables discovery of more suppressors? Are enhancers and suppressors equally likely in human polymorphisms, or is there a biological reason why more of one or the other would be able to persist in the population?
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Libraries of MPRA constructs were transfected into four diverse cell-types
This is a very smart approach to start cataloguing the effects of different non-coding variants, but the use of plasmid DNA necessarily removes the sequence of interest from its true genomic context. Is there a way to evaluate or predict the impact of this effect computationally or in a high-throughput experiment? Are you thinking at all about how version 2.0 of this technology might be able to close this gap?
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- Apr 2024
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www.biorxiv.org www.biorxiv.org
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In contrast, excess high mannose epitopes can over-activate complement pathways through interaction with mannose-binding lectin 7,8 and promote cancer metastasis
It would be very interesting to see whether changes in the presentation of the high-mannose glycans mediated by knockdown of either TM9SF3 or CCDC22 can lead to immune lectin engagement or immune cell activation. Have you thought at all about doing some sort of immune receptor binding assay (i.e. via flow cytometry with an immune lectin-Fc fusion) or an immune activation assay with macrophages or dendritic cells?
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Interestingly, these scattered, smaller Golgi structures observed in our TM9SF3 and CCDC22 knockdown cells are reminiscent of Golgi satellites or outposts in dendrites of neurons, where localized glycosylation events can occur in response to neuronal excitation26,38, suggesting that such regulation of Golgi morphology and function may be a general mechanism by which cells control glycosylation.
I was curious about tissue expression of TM9SF3 and CCDC22 because you mentioned that the Golgi structures you observed in your imaging experiments was reminiscent of Golgi satellites observed in neurons. Interestingly, it seems that TM9SF3 levels are lower in brain tissue than most others in the body, but this is not the case for CCDC22. I’d be curious to know whether this corresponds to glycomic profile changes that match the data you present for A549s.
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Together, these results suggest that the fragmented cis- and medial-Golgi and the reduction in TGN in TM9SF3 knockdown cells may impede the trafficking of glycoproteins through Golgi compartments for glycan remodeling, resulting in a glycan repertoire enriched in high and oligomannose structures
I’m very curious about the hypothesis that TM9SF3 leads to increased high-mannose presentation via TGN bypass. Have you looked at colocalization of high-mannose structures (i.e. using fluorescent GNA or HHL) with the TGN to see if it is reduced in TM9SF3 KD cells?
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The CCC complex negatively regulates Golgi function and complex glycan formation
I found it really interesting how a similar Golgi effect can produce very divergent outcomes on the presentation of N-glycans! I was especially interested in CCDC22’s effect, since it is not annotated to have any Golgi localization. Have you explored whether CCDC22 or any of its interacting partners localize to the Golgi under any circumstances, or else how its knockdown affects Golgi structure and glycosylation pathways?
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