The co-repressor Groucho limits progression through the early transcription elongation checkpoint in vivo

  1. María Lorena Martínez Quiles
  2. Barbara H Jennings

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Abstract

Promoter-proximal pausing of RNA polymerase II (RNAP II) at the early elongation checkpoint is a key regulatory step in developmental and stimulus-responsive gene expression. How pausing is established and modulated in a gene-specific manner during animal development remains unclear. The Groucho (Gro)/Transducin-like Enhancer of split (TLE) family of co-repressors is widely used by transcription factors to repress transcription, yet the mechanism of Gro-mediated repression in vivo is unresolved. Here, we combine genome-wide chromatin profiling with in vivo genetic analysis in Drosophila melanogaster to test whether Gro regulates RNAP II pausing. Analysis of ChIP-seq data across distinct cell types shows that Gro recruitment is largely cell-type specific but consistently occurs as discrete peaks within accessible, enhancer-associated chromatin. Gro occupancy frequently overlaps promoters enriched for pausing regulators, including Negative Elongation Factor (NELF), GAGA Factor (GAF), and components of Positive Transcription Elongation Factor b (P-TEFb), without excluding their recruitment. Using a sensitised wing-specific knockdown assay, we demonstrate that partial depletion of NELF subunits, GAF, and 7SK snRNP components synergistically enhances gro phenotypes beyond additive effects. These genetic interactions support a shared role in regulating transcription during development. Our findings support a model in which Gro attenuates transcription by modulating progression through the P-TEFb-dependent early elongation checkpoint in vivo.

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    Reply to the reviewers

    1. General Statements

    We thank the reviewers for their constructive evaluation of our manuscript. We are pleased by the overwhelmingly positive consensus regarding the quality and significance of our data. In particular, the reviewers highlighted that this is a "nice, clean study with interesting data" and noted that our in vivo functional genetic findings in the Drosophila wing are "clearly a strength" that "moves the paper beyond cell-culture correlations" to provide a "simple, straightforward take-home message".

    The principal critique across the reports concerns the extent of direct mechanistic evidence linking Groucho (Gro) to regulation of the early elongation checkpoint. Several reviewers suggested additional genomic experiments, including RNA-seq, PRO-seq, or Pol II ChIP approaches, to further examine transcription and pausing behaviour. However, we would like to flag up that genomic datasets addressing these questions across multiple Drosophila cell lines have already been published previously, including work from our own group and others.

    The primary objective of the current study is therefore not to replicate these existing genomic analyses, but rather to build directly upon them. We identify a consistent genomic association between Gro and pausing/elongation factors across cell types. Importantly, we extend these findings beyond genomic correlations through in vivo genetic analysis in the developing Drosophila wing.

    1. Description of the planned revisions

    Reviewer 1

    The figures and text could lay out the logic of the genetic interactions for non-Drosophila readers. For example, the comparison of single and double copies of Gro-RNAi to combinatorial knockdowns, when it is additive, and when it is interpreted as synergistic.

    The statistical analyses presented in Figure 5C, including Fisher’s exact tests comparing phenotype distributions between genotypes, were intended to address the distinction between additive and synergistic genetic interactions. However, we agree that the presentation of these comparisons could potentially be made clearer for readers less familiar with Drosophila genetic interaction assays. We would therefore be open to revising the presentation of Figure 5 and the accompanying explanatory text following editorial guidance and with consideration of the intended readership of the eventual journal.

    The statistical analysis of the phenotype distributions should be shown more clearly (Fig. 5B).

    Figure 5B is intended to present the distribution of observed phenotypic classes and does not include statistical comparisons. A similar analysis has been published for experiments looking at the phenotypes of moderate Groucho overexpression in the wing in the presence of HDAC inhibitors (Winkler et al., 2010 doi.10.1371/journal.pone.0010166). Statistical analyses of the genetic interaction experiments are presented separately in 5C. We therefore believe the current presentation of Figure 5B is appropriate for illustrating phenotype frequencies rather than statistical inference, but we will consider moving this panel to the Supplementary material.

    Minor comments

    -Figure 5 would gain clarity if the phenotype classes/panel letters were shown more clearly on the images. -The legends of the wing figures should be expanded, especially for readers outside the Drosophila field. -"in vivo" should be italicised consistently.

    We agree that clearer labelling of phenotype classes, panel annotations and expanded figure legends could improve the accessibility of Figure 5, particularly for readers less familiar with Drosophila wing phenotypes and genetic interaction assays. We would therefore be open to revising the presentation of this figure and its accompanying legends in a future revised version.

    We thank the reviewer for noting the typographical inconsistency of italics for in vivo. This will be corrected during manuscript revision and proofing.

    __Reviewer #2 __

    Reviewer #2 (Significance (Required)):

    I think this is nice little paper providing a simple, straightforward take-home message. It does not conceptually shake the world, and the evidence consists of (nice) correlations, with no direct proof put forward for the conclusions. I am not a Drosophila geneticist but probably rather an 'expert' on basic transcription mechanisms. I think the data in the paper are of high quality, if limited in scope, and that the conclusions are supported by the results, but I do not think the results or conclusions will have a big audience. Having said that, I found it interesting to learn about this group of repressors and their likely mode of action.

    On the other hand, it is worth emphasizing that proteins such as NELF and CDK9 would arguably be expected to be found at very many genes, as promoter-proximal pausing does exist at a plethora of genes, also genes that are house-keeping genes, ie not regulated by cell type or stimuli. So, lots of genes with pausing are not regulated by modulation of pausing. So, basically, the fact that knockdown of the repressor Groucho and loss of pausing is additive does not in my opinion necessarily mean that Groucho works by stabilizing pausing. Although it is admittedly a reasonably assumption, Groucho could also work by repressing transcription initiation; the genetic outcomes of 'double relief' would be the same, ie higher transcription levels. I think a brief comment to this effect might be appropriate, especially in the absence of (difficult to obtain) direct evidence that the transcription initiation step is not affected by Groucho.

    While we agree that the current study does not directly exclude possible effects of Groucho on transcription initiation, previously published work has already provided evidence arguing against repression by Groucho occurring primarily through inhibition of transcription initiation or prevention of pre-initiation complex assembly. Groucho-bound transcriptional start sites were previously shown to retain RNAP II occupancy, active chromatin features, and detectable basal transcriptional activity despite repression (Kaul et al., 2014).

    To acknowledge this possibility and explain why it is unlikely, we will add the sentence “While effects on transcription initiation cannot be completely excluded, previous work argues against Gro repressing transcription primarily through inhibition of transcription initiation. Gro-bound promoters remain accessible, overlap RNAP II occupancy, and retain active chromatin features and basal transcriptional activity” to the start of the third paragraph of the Discussion.

    Reviewer #3

    The methods section is lacking details on how ChIP-seq was performed in the BG3 cell line. The methods section does a good job of indicating how the data were processed. Information on the antibodies and conditions used is critical, as is whether spike-in controls were used.

    The generation of the ChIP-seq data from BG3 cells has already been published. __We will add the line “The production of ChIP-seq datasets for Gro binding in Kc167, S2R+ and BG3 cells has been described elsewhere (Kaul, Schuster and Jennings, 2014; Bar-Cohen et al., 2023)” in the Analysis of ChIP-seq data subsection of the Methods. __

    1. Description of analyses that authors prefer not to carry out

    __Reviewer #1 __ *Major comments

    1. The main weakness is the lack of a mechanistic link between Gro and the early elongation checkpoint. This is really the main point for this reviewer. The manuscript builds an interesting model, and the data support a functional connection between Gro and pausing-related factors, but the mechanistic link is absent. At present, the paper relies on co-localisation of ChIP peaks and genetic interaction in vivo. This is interesting and supportive, but with several possible interpretations. The title and some parts of the text are thus a bit stronger than what is directly demonstrated. Two possibilities could be proposed: either tone down the mechanistic claim or strengthen it experimentally. A more direct assay of pause release or productive elongation after Gro depletion at endogenous targets would be highly valuable. For example, Gro-KD followed by Pol II Ser2-P ChIP, or promoter vs. gene body analysis on Gro-bound genes, ideally comparing genes with Gro at TSS vs. not-TSS, would greatly support the proposed model. If the assay is established, this seems feasible in about 4 months.*

    We thank the reviewer for this thoughtful comment. We agree that the current study does not directly measure genome-wide RNAP II pause release following Gro depletion. However, several key observations linking Gro with promoter-proximal pausing have already been published and are summarised in the Introduction. Previous work demonstrated that Gro occupancy correlates with paused genes and that depletion of Gro reduces RNAP II pausing and increases elongating RNAP II at the endogenous E(spl)mbeta-HLH locus, an established target gene of Groucho-mediated repression (Kaul et al., 2014; doi.10.1371/journal.pgen.1004595). We also note that several of the experiments proposed by the reviewer have already been addressed in previous work. Specifically, Kaul et al. (2014) demonstrated that Gro depletion increases elongating RNAP II (Ser2-P) at the endogenous E(spl)mbeta-HLH locus while total promoter-associated RNAP II occupancy remains largely unchanged. Promoter versus gene body analyses in that study further supported a role for Gro in regulating progression through the early elongation checkpoint rather than transcription initiation.

    The aim of the current manuscript was therefore to build upon these earlier mechanistic and genomic observations by asking whether the relationship between Gro and pausing-associated factors extends across multiple cell types and whether it has functional significance in vivo. By integrating comparative genomic analyses with sensitised developmental genetic assays in the wing, we provide evidence that Gro functionally interacts with multiple regulators of the early elongation checkpoint during development.

    The bioinformatic part could be strengthened on "distinct TF repertoires" between cell types.The authors interpret the cell type-specific Gro recruitment as reflecting distinct transcription factor repertoires in BG3, Kc167 and S2R+ cells. This is interesting, but not really shown. To make this point more strongly, the author could provide a map of TF expression across different cell types, especially for the TFs corresponding to the enriched motifs they discuss. Otherwise, this remains speculative.In line, the manuscript discusses enriched motifs in BG3 and compares them to Kc167 and S2R+ cells, but this remains a bit descriptive. A clearer side-by-side comparison would strengthen the paper. This is particularly relevant to the motifs used in interpreting cell type-specific recruitment.

    The interpretation that cell type-specific Gro recruitment reflects differences in transcription factor repertoires is based on several previously established observations already described in the manuscript. BG3 cells are derived from the larval CNS, whereas Kc167 and S2R+ cells are embryonic haemocyte-like lines (Cherbas et al., 2011; doi.10.1101/gr.112961.110). Transcriptomic analyses have further shown that these Drosophila cell lines maintain stable and distinct lineage-associated transcriptional identities, including differences in transcription factor expression (Cherbas et al., 2011). Given the diversity of transcription factors known to recruit Gro, the observed cell-type-specific binding patterns and motif enrichments are consistent with the distinct lineage-associated transcriptional programmes previously described for these cell lines.

    1. Several overlap analyses could be discussed more in depth. A few statements feel too strong for the actual percentages. For example, the GAF overlap in BG3 is around 51% genome-wide and 56% at TSS, which is meaningful, but not especially high. The text already states that it is not universal, and this point could be discussed more clearly.

    We note that the manuscript already explicitly states that overlap between Gro and GAF is not universal. Given the diversity of factors known to recruit Gro and the broad genomic distribution of GAF, we consider overlap frequencies of approximately 50% to represent a substantial association, particularly at transcription start sites. Importantly, the interpretation does not rely on complete co-occupancy between these factors, but rather on the observation that Gro-bound regions show significant enrichment for multiple factors associated with promoter-proximal pausing across different cell types.

    Similarly, for the UpSet plot, the wording around the "most frequent" combination could be toned down, because this is not a dominant pattern.

    The statement that the overlap between Gro, Nelf-E, GAF, Cdk9 and RNAP II represents the “most frequent” combination refers specifically to the relative frequency of the intersection categories within the UpSet analysis. In this context, the overlap between all five factors represents the largest intersection category identified (306 of 649 Gro peaks), with the next most frequent category containing substantially fewer peaks (90 of 649). We therefore feel that the current wording accurately describes the distribution observed in the analysis.

    More generally, I think the manuscript needs a clearer quantitative breakdown of TSS versus non-TSS peaks for the overlap analyses with NELF, GAF, Cdk9 and CycT. Several interpretations depend on this distinction, and right now, this is not always clear enough.

    The overlap analyses presented in Figure 3 explicitly distinguish between TSS and non-TSS peaks, and the corresponding quantitative overlap frequencies are described in the Results section. We do not consider that additional breakdowns are required for interpretation of the current data as this distinction is already incorporated into both the analyses and figure presentation.

    The "enhancer chromatin" interpretation is interesting, but not fully integrated with the genomic distribution. The observation that Gro is enriched in open enhancer-type chromatin is interesting and supports the idea that Gro does not act mainly through classical repressed chromatin. However, Gro peaks are also enriched at promoters and introns, and this reviewer feels that the manuscript does not fully connect these observations. Where are these enhancer-type peaks located exactly? Are they often intronic? Can this be correlated with the distribution of Gro peaks? This would help the reader and also strengthen the discussion because intronic Gro peaks are present in the data, but are not well integrated into the model.

    In the current manuscript, “enhancer chromatin” refers to chromatin states defined by combinations of enhancer-associated histone modifications, including H3K4me1, H3K27ac and H3K56ac as defined by Skalska et al.,2015 (doi.10.15252/embj.201489923), rather than exclusively to distal intergenic regulatory regions. As described in the chromatin-state analysis, these enhancer-associated chromatin signatures do occur at intronic regulatory regions, including regions classified as active intron chromatin. We therefore do not consider the enrichment of Gro peaks at promoters, enhancers and intronic regions to be mutually exclusive observations within this framework.

    Intronic enhancer localisation is common in Drosophila, where the compact organisation of the genome results in many developmental regulatory elements residing within introns (Arnold et al., 2013; doi.10.1126/science.1232542). We therefore consider the presence of Gro peaks within intronic regions to be fully consistent with the observed enrichment of Gro binding within enhancer-associated chromatin states.

    The in vivo part is a strength, but some important points need clarification.The in vivo section is a clear highlight of the manuscript. It gives functional relevance to the model and moves the paper beyond cell-culture correlations. That said, a few points need to be clearer:-RNAi efficiency is not clear for the tested genes, especially the pausing factors. This is important because the differential effects between NELF subunits could simply reflect differences in knockdown efficiency.

    While differences in RNAi efficiency could potentially contribute to variation in phenotype strength between individual knockdowns, multiple biological explanations could also account for the differing effects observed between NELF subunits, including differences in protein stability, residual complex activity, or subunit-specific functions. Importantly, the central conclusion of the manuscript does not depend on quantitative comparison of phenotype strength between individual NELF components, but rather on the observation that perturbation of multiple pausing-associated factors genetically interacts with Gro in vivo.

    If RNAi validation is possible with existing reagents, this seems realistic within 3 months.

    The manuscript focuses on the genetic interactions observed between Gro and pausing-associated factors in vivo rather than on quantitative comparison between individual RNAi lines. As no specific validation experiments were proposed, we are not currently planning additional RNAi validation analyses for the present study.

    The discussion could be expanded, especially because the mechanism is not fully shown.Since the direct mechanism is still missing, the discussion could compensate. Right now, the proposed model is interesting, but it still leaves many open questions. For example:-Is Gro affecting the recruitment or activity of elongation factors?-Could looping or enhancer-promoter communication contribute?-How should the intronic Gro peaks be interpreted in the model?-In the wing, could the phenotype be discussed more mechanistically, in light of what is already known about Gro and derepression of vein-promoting genes?For example, a model figure could help here.

    We thank the reviewer for these thoughtful suggestions.

    Several of the points raised by the reviewer are discussed in the manuscript already. For example, we discuss the possibility that Gro influences the activity or recruitment of elongation-associated factors. We agree that enhancer-promoter communication and chromatin looping are a plausible component of this mechanism. As the *Drosophila *genome is compact and intronic enhancers are highly prevalent, topological looping provides a clear physical framework for how Gro molecules distributed at non-TSS sites regulate promoter-proximal machinery. Indeed, we have previously published this model (Kaul, Schuster, and Jennings, 2015; see Figure 1C; doi.10.1080/21541264.2014.1000709). Our current in vivo and genomic findings build directly upon this model, suggesting that within these established looped configurations, Gro acts locally to interface with and stabilize the pausing machinery.

    With respect to the wing phenotypes, the Discussion focuses primarily on the interpretation of the observed genetic interactions between Gro and pausing-associated factors rather than on defining the precise downstream target genes contributing to vein phenotypes. We agree that additional mechanistic dissection of these developmental phenotypes would be interesting. However, this would require a substantial expansion of the study into the detailed developmental and signalling mechanisms underlying vein specification, which lies beyond the primary focus of the current manuscript.

    OPTIONAL: It would be interesting to know whether the same peak distribution / functional logic is observed in mammalian TLE orthologs. This is not essential for the current conclusions, but it would broaden the impact.

    Determining whether similar genomic distributions and functional relationships are conserved for mammalian TLE orthologues will be an important future project. However, relatively little comparable genome-wide TLE occupancy data are currently available, meaning that such analyses would require a substantial independent undertaking beyond the scope of the present study.

    Minor comments -Please explain why promoters were defined as {plus minus}250 bp from the TSS. This seems rather narrow.

    Promoters were defined as ±250 bp from annotated transcription start sites. This window size is commonly used in Drosophila genomic studies, where the compact organisation of the genome means that broader windows frequently overlap adjacent genes.

    -Please clarify why S2R+ cells are included in the comparative part but are not followed in the same way in some downstream analyses.

    S2R+ cells were included in the comparative analyses to determine which aspects of Gro recruitment were shared across multiple cell types and which were cell-type specific. Some downstream analyses focused on BG3 and Kc167 cells because these lines had the most extensive corresponding datasets available for the chromatin and pausing-factor analyses performed in the current study.

    __Reviewer #3 __ Here Martínez Quiles and Jennings investigate the role of the Groucho repressor in BG3 cells. This extends a previous study that used S2R+ cells, published previously by one of the authors, as well as Kc167 cells. They find that Gro is recruited to gene promoters in a cell-type-specific manner. Gro associates with open chromatin, is mostly associated with enhancer regions, and is primarily excluded from regions of the genome that are repressed by Polycomb. After studying its function in cell culture, the authors investigate the role of Gro in a wing-specific background. The findings here are mostly correlative, showing that loss of Gro results in stronger phenotypic defects when combined with loss of factors including NELF-B or NELF-D, LARP7, and bin3. They propose that Gro acts to attenuate gene expression during early gene expression. This claim would be greatly strengthened if the authors provided RNA-seq data in addition to the ChIP-seq data shown in this manuscript, especially to examine gene expression patterns among the different cell lines studied here. At present, this is a correlative study that does not illuminate the mechanism of Gro in directly regulating promoter-proximal pausing or RNA polymerase behavior.

    We thank the reviewer for this suggestion. However, extensive transcriptomic analyses of Drosophila cell lines, including Kc167, S2R+ and BG3-derived lines, have already been published (Cherbas et al., 2011), together with RNA-seq analyses following Gro depletion (Kaul et al., 2014). In addition, the association between Gro occupancy and paused genes has also been reported previously (Kaul et al., 2014; Chambers et al., 2017; doi. 10.1186/s12864-017-3589-6).

    While additional RNA-seq analyses could further characterise transcriptional differences between cell lines, RNA-seq alone would not directly determine whether altered transcript levels arise specifically through changes in promoter-proximal pausing, as opposed to effects on transcription initiation, transcript stability, or indirect downstream regulatory effects. We therefore do not consider additional RNA-seq analyses necessary to support the central conclusions of the present study.

    Figure 2-3: For the ChIP-seq data, scale the y-axis in the same manner to better understand enrichment between the samples.

    These ChIP-seq datasets were generated independently using different antibodies and experimental conditions, direct comparison of enrichment magnitudes across datasets would not be biologically meaningful. Accordingly, our analyses focus on significant peak calls and overlap relationships rather than relative signal intensity. Applying identical y-axis scaling across all tracks would obscure significant enrichment in several datasets and could therefore be misleading.

    RNA-seq data between different cell lines would greatly enhance the authors findings or Pro-Seq to really show a relationship with Gro binding and promoter proximal pausing.

    We note that RNA-seq datasets for Gro depletion in Kc167 and S2R+ cells have already been published previously (Kaul et al., 2014), together with evidence linking Gro occupancy to paused genes (Kaul et al., 2014; Chambers et al., 2017). We therefore do not consider that additional RNA-seq analysis would substantially strengthen the central conclusions of the current manuscript.

    Moreover, RNA-seq alone cannot distinguish if altered transcript abundance reflects changes in promoter-proximal pausing from other mechanisms influencing transcript abundance. While PRO-seq approaches could provide further mechanistic information regarding RNAPII dynamics, such experiments are beyond the scope of the present study.

    This study helps to further clarify how Gro binds DNA in different cell types and indicates that may intersect with factors involved in promoter proximal pausing. The study is highly correlative and would require additional work to show a mechanistic link between Gro and transcription attenuation due to promoter proximal pausing.

    While we agree that PRO-seq approaches could provide additional mechanistic information regarding RNAPII dynamics, establishing an appropriate experimental and analytical framework for these analyses would require a substantial extension beyond the scope of the present study. In addition, several aspects of the relationship between Gro occupancy, transcriptional repression, and promoter-proximal pausing that underpin these suggestions have already been addressed in previously published work, including RNA-seq analyses following Gro depletion (Kaul et al., 2014), evidence linking Gro occupancy with paused genes (Kaul et al., 2014; Chambers et al., 2017), and studies demonstrating that Gro-mediated repression does not occur through inhibition of pre-initiation complex assembly. The current manuscript is therefore intended to build upon these existing findings by integrating comparative genomic analyses with new in vivo genetic interaction data.

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    Referee #3

    Evidence, reproducibility and clarity

    Here Martínez Quiles and Jennings investigate the role of the Groucho repressor in BG3 cells. This extends a previous study that used S2R+ cells, published previously by one of the authors, as well as Kc167 cells. They find that Gro is recruited to gene promoters in a cell-type-specific manner. Gro associates with open chromatin, is mostly associated with enhancer regions, and is primarily excluded from regions of the genome that are repressed by Polycomb. After studying its function in cell culture, the authors investigate the role of Gro in a wing-specific background. The findings here are mostly correlative, showing that loss of Gro results in stronger phenotypic defects when combined with loss of factors including NELF-B or NELF-D, LARP7, and bin3. They propose that Gro acts to attenuate gene expression during early gene expression. This claim would be greatly strengthened if the authors provided RNA-seq data in addition to the ChIP-seq data shown in this manuscript, especially to examine gene expression patterns among the different cell lines studied here. At present, this is a correlative study that does not illuminate the mechanism of Gro in directly regulating promoter-proximal pausing or RNA polymerase behavior.

    Major comments:

    Figure 2-3: For the ChIP-seq data, scale the y-axis in the same manner to better understand enrichment between the samples.

    The methods section is lacking details on how ChIP-seq was performed in the BG3 cell line. The methods section does a good job of indicating how the data were processed. Information on the antibodies and conditions used is critical, as is whether spike-in controls were used.

    RNA-seq data between different cell lines would greatly enhance the authors findings or Pro-Seq to really show a relationship with Gro binding and promoter proximal pausing.

    Significance

    This study helps to further clarify how Gro binds DNA in different cell types and indicates that may intersect with factors involved in promoter proximal pausing. The study is highly correlative and would require additional work to show a mechanistic link between Gro and transcription attenuation due to promoter proximal pausing.

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    Referee #2

    Evidence, reproducibility and clarity

    This paper describes experiments designed to determine the mechanism of repression by the Groucho co-repressor in flies. The authors first characterize DNA binding by Groucho by ChIP-Seq analysis. This turns out to be consistent with recruitment driven by cell-type specific transcription factors. Nevertheless, its distribution across genomic features is similar across cell types, with enrichment in promoters and introns. It appears to bind in regions otherwise transcriptionally active (ie 'open chromatin'), rather than chromatin that is compacted and repressed. This suggest that Groucho regulates transcription through promoters or promoter-proximal pausing rather than by reducing chromatin accessibility. Groucho binding overlaps with NELF and GAF binding, seemingly consistent with a role in regulating pausing. However, Gro binding was also observed at promoters where P-TEFb components are detected, arguing against Gro repressing transcription P-TEFb exclusion from pausing sites. The authors next switched to investigating the consequences of Groucho kd and tested the idea that co-depletion of pausing factors might inform about the manner of gene repression, the idea being that if Groucho attenuates transcription by promoting or stabilizing promoter proximal pausing, then partial reduction of the pausing factors it affects should enhance the Groucho knock-down phenotype. Interestingly, simultaneous knock-down of Groucho and GAF resulted in enhanced patterning defects relative to Groucho knock-down alone, with the severity of the phenotypes resembling that observed upon increasing Groucho knock-down. Similarly, the knock-down of either Nelf-B or Nelf-D significantly enhanced Groucho phenotype. Finally, Kd of regulators of the pausing regulator CDK9 were tested. The 7SK snRNA complex inhibits CDK9, so any treatment leading to less 7SK will free CDK9 to positively affect pausing release. Larp kd fits that category as it directly leads to less 7SK and thus more CDK9 activity, while Bin3 kd results in less 5'-methyl capping, and thus more 7SK destabilization (less 7SK), again freeing CDK9 from inhibition - so, increasing pause release (like Nelf kd). Gratifyingly, this separate way of de-regulating/decreasing pausing again had an additive effect to Groucho depletion. Together, these genetic data thus overall support the idea that the (non-chromatin regulating) repressor Groucho works by stabilizing pausing complexes at specific genes.

    Significance

    I think this is nice little paper providing a simple, straightforward take-home message. It does not conceptually shake the world, and the evidence consists of (nice) correlations, with no direct proof put forward for the conclusions. I am not a Drosophila geneticist but probably rather an 'expert' on basic transcription mechanisms. I think the data in the paper are of high quality, if limited in scope, and that the conclusions are supported by the results, but I do not think the results or conclusions will have a big audience. Having said that, I found it interesting to learn about this group of repressors and their likely mode of action.

    On the other hand, it is worth emphasizing that proteins such as NELF and CDK9 would arguably be expected to be found at very many genes, as promoter-proximal pausing does exist at a plethora of genes, also genes that are house-keeping genes, ie not regulated by cell type or stimuli. So, lots of genes with pausing are not regulated by modulation of pausing. So, basically, the fact that knockdown of the repressor Groucho and loss of pausing is additive does not in my opinion necessarily mean that Groucho works by stabilizing pausing. Although it is admittedly a reasonably assumption, Grouch could also work by repressing transcription initiation; the genetic outcomes of 'double relief' would be the same, ie higher transcription levels. I think a brief comment to this effect might be appropriate, especially in the absence of (difficult to obtain) direct evidence that the transcription initiation step is not affected by Groucho.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In this manuscript entitled "the co-repressor Groucho limits progression through the early transcription elongation checkpoint in vivo", the authors study how the co-repressor Groucho (Gro) may repress transcription in Drosophila. They combine Gro ChIP-seq analysis in BG3 cells with published data from Kc167 and S2R+ cells, chromatin-state and overlap analyses with pausing/elongation factors, and functionally link these interactions in vivo by genetic interaction assays in the wing. The manuscript shows that Gro recruitment is largely cell type-specific, while Gro binding is detected as discrete peaks with similar genomic distribution across cell types. Gro peaks are enriched in open enhancer-type chromatin and overlap with factors linked to promoter-proximal pausing. In vivo, knock-down (KD) of several pausing-related factors enhances the gro RNAi phenotype in the wing. Overall, this is a nice, clean study with interesting data, and the in vivo findings are clearly a strength. However, the mechanistic link between Gro and the early elongation checkpoint remains unclear, and several bioinformatics and presentation points could be strengthened.

    Major comments

    1. The main weakness is the lack of a mechanistic link between Gro and the early elongation checkpoint. This is really the main point for this reviewer. The manuscript builds an interesting model, and the data support a functional connection between Gro and pausing-related factors, but the mechanistic link is absent. At present, the paper relies on co-localisation of ChIP peaks and genetic interaction in vivo. This is interesting and supportive, but with several possible interpretations. The title and some parts of the text are thus a bit stronger than what is directly demonstrated. Two possibilities could be proposed: either tone down the mechanistic claim or strengthen it experimentally. A more direct assay of pause release or productive elongation after Gro depletion at endogenous targets would be highly valuable. For example, Gro-KD followed by Pol II Ser2-P ChIP, or promoter vs. gene body analysis on Gro-bound genes, ideally comparing genes with Gro at TSS vs. not-TSS, would greatly support the proposed model. If the assay is established, this seems feasible in about 4 months.
    2. The bioinformatic part could be strengthened on "distinct TF repertoires" between cell types. The authors interpret the cell type-specific Gro recruitment as reflecting distinct transcription factor repertoires in BG3, Kc167 and S2R+ cells. This is interesting, but not really shown. To make this point more strongly, the author could provide a map of TF expression across different cell types, especially for the TFs corresponding to the enriched motifs they discuss. Otherwise, this remains speculative. In line, the manuscript discusses enriched motifs in BG3 and compares them to Kc167 and S2R+ cells, but this remains a bit descriptive. A clearer side-by-side comparison would strengthen the paper. This is particularly relevant to the motifs used in interpreting cell type-specific recruitment.
    3. Several overlap analyses could be discussed more in depth. A few statements feel too strong for the actual percentages. For example, the GAF overlap in BG3 is around 51% genome-wide and 56% at TSS, which is meaningful, but not especially high. The text already states that it is not universal, and this point could be discussed more clearly. Similarly, for the UpSet plot, the wording around the "most frequent" combination could be toned down, because this is not a dominant pattern. More generally, I think the manuscript needs a clearer quantitative breakdown of TSS versus non-TSS peaks for the overlap analyses with NELF, GAF, Cdk9 and CycT. Several interpretations depend on this distinction, and right now, this is not always clear enough.
    4. The "enhancer chromatin" interpretation is interesting, but not fully integrated with the genomic distribution. The observation that Gro is enriched in open enhancer-type chromatin is interesting and supports the idea that Gro does not act mainly through classical repressed chromatin. However, Gro peaks are also enriched at promoters and introns, and this reviewer feels that the manuscript does not fully connect these observations. Where are these enhancer-type peaks located exactly? Are they often intronic? Can this be correlated with the distribution of Gro peaks? This would help the reader and also strengthen the discussion because intronic Gro peaks are present in the data, but are not well integrated into the model.
    5. The in vivo part is a strength, but some important points need clarification. The in vivo section is a clear highlight of the manuscript. It gives functional relevance to the model and moves the paper beyond cell-culture correlations. That said, a few points need to be clearer:
      • RNAi efficiency is not clear for the tested genes, especially the pausing factors. This is important because the differential effects between NELF subunits could simply reflect differences in knockdown efficiency.
      • The figures and text could lay out the logic of the genetic interactions for non-Drosophila readers. For example, the comparison of single and double copies of Gro-RNAi to combinatorial knockdowns, when it is additive, and when it is interpreted as synergistic.
      • The statistical analysis of the phenotype distributions should be shown more clearly (Fig. 5B). If RNAi validation is possible with existing reagents, this seems realistic within 3 months.
    6. The discussion could be expanded, especially because the mechanism is not fully shown. Since the direct mechanism is still missing, the discussion could compensate. Right now, the proposed model is interesting, but it still leaves many open questions. For example:
      • Is Gro affecting the recruitment or activity of elongation factors?
      • Could looping or enhancer-promoter communication contribute?
      • How should the intronic Gro peaks be interpreted in the model?
      • In the wing, could the phenotype be discussed more mechanistically, in light of what is already known about Gro and derepression of vein-promoting genes? For example, a model figure could help here.

    OPTIONAL:

    It would be interesting to know whether the same peak distribution / functional logic is observed in mammalian TLE orthologs. This is not essential for the current conclusions, but it would broaden the impact.

    Minor comments

    • Please explain why promoters were defined as {plus minus}250 bp from the TSS. This seems rather narrow.
    • Please clarify why S2R+ cells are included in the comparative part but are not followed in the same way in some downstream analyses.
    • Figure 5 would gain clarity if the phenotype classes/panel letters were shown more clearly on the images.
    • The legends of the wing figures should be expanded, especially for readers outside the Drosophila field.
    • "in vivo" should be italicised consistently.

    Referee cross-commenting

    My main concerns are broadly echoed by Reviewer 2, notably regarding the need to clarify the level of mechanistic support for the proposed model. Reviewer 3 also raises related points about the correlative nature of the evidence. Overall, I think the reports converge on the need to better align the conclusions with the current data, while recognising the value of the functional in vivo results, though with different levels of requested additional analysis.

    Significance

    General assessment

    This is a nice paper, with clean data and an interesting model. The strongest point is the attempt to connect the Gro genomic localisation with functional interaction in a developmental context. The observation that Gro is found in open enhancer-type chromatin, together with the in vivo genetic interactions, makes the study significant. The main limitation is that the mechanistic link is still missing. Overall, this reviewer finds the study convincing as a functional and descriptive paper but less convincing as a mechanistic one.

    Advance

    The study extends previous work on Gro by comparing several cell types and by adding in vivo genetic data in the wing. The main advance is thus conceptual and functional: it supports the idea that Gro acts in concert with the pausing/elongation machinery rather than simply through repressed chromatin. However, the mechanistic advance remains limited because a direct link to the early elongation checkpoint has not yet been demonstrated. This is the main thing preventing the paper from being stronger.

    Audience

    This reviewer feels that the manuscript will mainly interest a specialised basic research audience: scientists working on transcriptional regulation, co-repressors, RNA polymerase II pausing, chromatin regulation, and Drosophila developmental genetics. It can also be relevant to those broadly interested in Gro/TLE biology.

    Expertise

    This reviewer's expertise includes gene regulation and its nuclear organisation, transcriptional/co-transcriptional and post-transcriptional regulations, transcription factors biology, and Drosophila genetics. This reviewer is comfortable evaluating the developmental genetics, the conceptual aspect, and the interpretation of genomic analyses, but has less competence in evaluating bioinformatic ChIP-seq processing pipelines.

  5. Version published to 10.1101/2022.09.17.508372 on bioRxiv