OVO Positively Regulates Essential Maternal Pathways by Binding Near the Transcriptional Start Sites in the Drosophila Female Germline

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

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

Weaknesses:

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

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

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

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

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

Author Response

Reviewer #1 (Public Review):

Summary:

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

We fully agree with this summary.

Strengths:

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

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

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

Weaknesses:

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

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

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

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

Reviewer #2 (Public Review):

Summary:

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

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

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

Strengths:

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

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

Weaknesses:

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

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

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

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

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

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

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

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

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

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

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

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

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