Mono-methylated histones control PARP-1 in chromatin and transcription

Reviewer #2 (Public Review):

Summary:

This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

Strengths:

The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

(1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

(2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

(3) Parp1 directly binds H4K20me in vitro.

Weaknesses:

(1) The RNAseq analysis of Parp1/PR-Set7 mutants is reasonable, but there is a caveat to the author's conclusion (Line 251): "our results indicate H4K20me1 may be required for PARP-1 binding to preferentially repress metabolic genes and activate genes involved in neuron development at co-enriched genes." An alternative possibility is that many of the gene expression changes are indirect consequences of altered development induced by Parp1 or PR-Set7 mutants. For example, Parp1 could activate a transcription factor that represses metabolic genes. The authors counter this model by stating that Parp1 directly binds to "repressed" metabolic genes. While this argument supports their model, it does not rule out the competing indirect transcription factor model. Therefore, they should still mention the competing model as a possibility.

(2) The section on inducibility of heat shock genes is interesting but missing an important control that might significantly alter the author's conclusions. Hsp23 and Hsp83 (group B genes) are transcribed without heat shock, which likely explains why they have H4K20me without heat shock. The authors made the reasonable hypothesis that this H4K20me would recruit Parp-1 upon heat shock (line 270). However, they observed a decrease of H4K20me upon heat shock, which led them to conclude that "H4K20me may not be necessary for Parp1 binding/activation" (line 275). However, their RNA expression data (Fig4A) argues that both Parp1 and H40K20me are important for activation. An alternative possibility is that group B genes indeed recruit Parp1 (through H4K20me) upon heat shock, but then Parp1 promotes H3/H4 dissociation from group B genes. If Parp1 depletes H4, it will also deplete H4K20me1. To address this possibility, the authors should also do a ChIP for total H4 and plot both the raw signal of H4K20me1 and total H4 as well as the ratio of these signals. The authors could also note that Group A genes may similarly recruit Parp1 and deplete H3/H4 but with different kinetics than Group B genes because their basal state lacks H4K20me/Parp1. To test this possibility, the authors could measure Parp association, H4K20methylation, and H4 depletion at more time points after heat shock at both classes of genes.

Author Response

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

We would like to express our sincere appreciation for the invaluable comments provided by the reviewers and their constructive suggestions to enhance the quality of our manuscript. In response to their feedback, we have diligently revised and resubmitted our paper as an article, introducing five primary figures, seven supplementary figures, and two supplementary data files. Importantly, this work represents a noteworthy contribution to the field, presenting novel findings for the first time without any prior publication.

Within the enclosed document, we have provided a comprehensive response to the reviewer comments, addressing each point in a meticulous and specific manner. We extend our sincere gratitude to the reviewers for their diligent examination of our manuscript and for offering insightful recommendations.

In our latest revision, we have taken great care to respond to every reviewer's comment, ensuring that we clarify the manuscript and provide robust evidence where required. The primary focus of these revisions was to provide additional context regarding the cooperative role between PR-Set-7 and PARP-1 in the repression of metabolic genes, accompanied by a thorough description of the current state of the field. Substantial modifications and new analyses, presented in the supplemental figures, have been included to comprehensively address this concern.

Another concern raised was regarding the interaction between PARP-1 and mono-methylated active histone marks, which was not adequately described in the previous version of our manuscript. In this revised version, we have updated our Fig. 1 and Supplemental Fig. S1 and introduced Supplemental Fig. S2 to properly demonstrate that PARP-1 binds to all mono-methylated active histone marks tested. Furthermore, we extensively revised the Discussion section of our manuscript to discuss the implications of this discovery and how it fits into the broader context of PARP-1 research.

Addressing another reviewer's concern about the potential indirect regulation of transcription by PARP1 and PR-SET7, we revised the discussion section and incorporated findings from our recent study. These findings clearly demonstrate PARP1's binding to the loci of misregulated genes, suggesting a direct involvement in their regulation.

Furthermore, we have improved the description of the reagents and Drosophila lines used in this study to provide a more comprehensive understanding for readers. Finally, we conducted a comprehensive revision of the entire manuscript to rectify the identified typos and grammatical errors.

Enclosed, you will find a detailed, point-by-point response to each of the reviewer's comments, showcasing our commitment to addressing their concerns with precision.

We firmly believe that our revisions successfully resolve all the concerns raised by the reviewers, and we are confident that this improved version of our manuscript contributes significantly to the scientific discourse.

Reviewer #1:

The study investigates the role of PARP-1 in transcriptional regulation. Biochemical and ChIP-seq analyses demonstrate specific binding of PARP-1 to active histone marks, particularly H4K20me, in polytene chromosomes of Drosophila third instar larvae. Under heat stress conditions, PARP-1's dynamic repositioning from the Hsp70 promoter to its gene body is observed, facilitating gene activation. PARP-1, in conjunction with PR-Set7, plays a crucial role in the activation of Hsp70 and a subset of heat shock genes, coinciding with an increase in H4K20me1 levels at these gene loci. This study proposes that H4K20me1 is a key facilitator of PARP-1 binding and gene regulation. However, there are several critical concerns that are yet to be addressed. The experimental validation and demonstration of results in the main manuscript are scant. Recent developments in the area are omitted, as an important publication hasn't been discussed anywhere in the work (PMID: 36434141). The proposed mechanism operates quite selectively, and any extrapolations require intensive scientific evidence.

Major Comments:

(1) PARP1 hypomorphic mutant validation data must be provided at RNA levels as the authors have mentioned about its global reduction in RNA levels.

We sincerely appreciate Reviewer 1 for their meticulous review of our manuscript and for providing valuable insights. In response to the raised concern, we would like to highlight that the validation data for the PARP1 hypomorphic mutant at the RNA level has been previously documented in our study (PMID: 20371698), where we found that PARP1 RNA level was deeply impacted in parp1C03256. To enhance clarity, we have made corresponding modifications to the Materials and Methods section to explicitly articulate this aspect: parp-1C03256 significantly lowers the level of PARP-1 RNA and protein level (14) but also significantly diminishes the level of pADPr (11).

We hope these revisions effectively address the reviewer's suggestion and contribute to a more comprehensive understanding of our findings.

(2) The authors should provide immunoblot data for global Poly (ADP) ribosylation levels in PARP1 hypomorphic mutant condition as compared to the control. They must also provide the complete details of the mouse anti-pADPr antibody used in their immunoblot in Figure 5B.

We extend our gratitude to Reviewer 1 for drawing attention to aspects requiring further clarification. In response to the inquiry about global Poly (ADP) ribosylation levels in the PARP1 hypomorphic mutant condition, we want to emphasize that our study extensively reported on the diminished levels of pADPr in comparison to the wildtype, as documented in our previous work (PMID: 21444826). To address this, we have incorporated pertinent details in the Materials and Methods section, providing a comprehensive account of our findings. parp-1C03256 significantly lowers the level of PARP-1 RNA and protein level (14) but also significantly diminishes the level of pADPr (11).

Furthermore, in addressing the request for complete details of the mouse anti-pADPr antibody (10H) used in Figure 5B, we have taken steps to enhance transparency. The Materials and Methods section has been revised to incorporate more comprehensive information about the antibody, ensuring a clearer understanding of our experimental procedures. anti-pADPr (Mouse monoclonal, 1:500, 10H - sc-56198, Santa Cruz).

We appreciate the reviewer's diligence in ensuring the robustness of our methodology, and we believe these modifications strengthen the overall quality and transparency of our study.

(3) PR-Set7 mutant validation results should be provided in the main manuscript, as done by the authors using qRT-PCR. Also, immunoblot data for the PR-set7 null condition should be supplemented in the main manuscript as the authors have already mentioned their anti-PR-Set7 (Rabbit, 1:1000, Novus Biologicals, 44710002) antibody in the materials and methods section.

We appreciate Reviewer 1's thorough examination of our manuscript and their constructive feedback. The pr-set7 null mutant has been rigorously characterized in a study conducted by Dr. Ruth Steward's laboratory (PMID: 15681608). Additionally, we employed our PR-SET7 antibody to validate the mutant, and the corresponding data can be found in Supplemental Figure 3. To enhance clarity, we have made necessary modifications to both the results and Materials and Methods sections, providing explicit details on the validation process. Result section: To validate our hypothesis, we initially confirmed that the pr-set720 mutant not only eliminated PR-SET7 RNA and protein but also abrogated H4K20me1 modification (Supplemental Fig.S3).

Material and methods section: The pr-set720 null mutant was validated in (15) and we confirmed that this mutant abolishes PR-SET7 RNA and protein level but also leads to the absence of H4K20me1 (Supplemental Fig. S3).

We believe these revisions address the reviewer's concerns and contribute to a more comprehensive presentation of our study.

(4) The authors have probably missed out on a very important recent report (PMID: 36434141), suggesting the antagonistic nature of the PARP1 and PR-SET7 association. In light of these important observations, the authors must check for the levels of PR-SET7 in PARP1 hypomorphic conditions.

We appreciate the insightful comment from Reviewer 1, drawing our attention to the recent study by Estève et al. (PMID: 36434141) highlighting the potential antagonistic relationship between PARP1 and PR-SET7. To address this important point, we have carefully examined the levels of PR-SET7 in PARP1 hypomorphic conditions.

In response to this concern, we have added two new supplemental figures, Supplemental Fig. S4 and S5, which specifically address the impact of PARP1 deficiency on PR-Set7 expression. These figures clearly demonstrate that there were no significant changes observed in PR-SET7 RNA (Fig. S4) or protein levels (Fig. S5) in the absence of Parp1. This finding supports the conclusion that Parp1 is not directly involved in the regulation of PR-SET7 in Drosophila.

Furthermore, we have updated the Results section to explicitly mention this observation:

Interestingly, in the absence of PARP-1, neither PR-SET7 RNA nor protein levels were affected (Supplemental Fig. S4-5), indicating that PARP-1 is not directly implicated in the regulation of PR-SET7.

Additionally, we have included information about the anti-H3 antibody used in Supplemental Fig. S4 in the Materials and Methods section: anti-H3 (Rabbit polyclonal, 1/1000, FL-136 sc-10809 Santa Cruz).

We believe that these modifications effectively address the raised concern and provide a more comprehensive understanding of the relationship between PARP1 and PR-SET7 in our study. We hope these clarifications enhance the overall robustness and clarity of our findings.

(5) Also, the results of the aforementioned study should be adequately discussed in the present study along with its implications in the same.

We appreciate Reviewer 1's valuable suggestion to discuss the implications of the study by Estève et al. (PMID: 36434141) within the context of our own findings. Estève et al. reported a potential antagonistic relationship between PARP1 and PR-SET7, showing that a decrease in PARP1 proteins leads to an increase in PR-SET7 protein levels. In our investigation, however, we did not observe significant changes in PR-SET7 RNA and protein levels in the parp1C03256 mutant, as demonstrated in the newly added Supplemental Fig. S3 and S4.

We acknowledge the discrepancy between our results and those of Estève et al., and we propose that this difference may be due to distinct experimental approach: Estève et al.'s study focused on mammalian cell populations and in vitro experiments, whereas our investigation employed Drosophila third-instar larvae as the whole organism model. It is plausible that regulatory mechanisms governing PR-SET7 differ between mammals and Drosophila. Another possibility is that PARP-1 may cooperate with PR-SET7 in the context of Drosophila development but could exhibit antagonistic roles against PR-SET7 in specific cell lines and under certain biological or developmental conditions.

In the Discussion section, we have incorporated this information, stating: A recent study demonstrated that in human cells overexpressing PARP-1, PR-SET7/SET8 is degraded (33). This implies that the absence of PARP-1 might lead to increased levels of PR-SET7. However, in our study involving parp-1 mutant in Drosophila third-instar larvae, we observed a slightly different scenario: we detected a minor but not significant reduction in both PR-SET7 RNA and protein levels (Supplemental Fig.S4 and S5). This outcome stands in stark contrast to the previous study's findings. The discrepancy could be due to the distinct experimental approaches used: the previous research focused on mammalian cells and in vitro experiments, whereas our study examined the functions of PARP-1 in whole Drosophila third-instar larvae during development. Consequently, while PARP-1 may cooperate with PR-SET7 in the context of Drosophila development, it could exhibit antagonistic roles against PR-SET7 in specific cell lines and under certain biological or developmental conditions.

We believe these modifications provide a comprehensive discussion of the observed discrepancies and enhance the overall interpretation of our findings. We hope that these clarifications satisfactorily address the concerns raised by Reviewer 1.

(6) Gene transcriptional activation requires open chromatin and RNA polymerase II binding to the promoter. Since, differentially expressed genes in both PR-Set7 null and PARP1 hypomorph mutants, co-enriched with PARP-1 and H4K20me1 were mainly upregulated, the authors should provide RNA polymerase II occupancy data of these genes via RNA-Pol II ChIP-seq to further attest their claims.

We appreciate the insightful comment from Reviewer 1 regarding the necessity for RNA-polymerase II (PolII) occupancy data to further support our claims on gene transcriptional activation. To address this concern, we conducted an analysis of PolII occupancy around genes co-enriched with PARP-1 and H4K20me1 that are upregulated in both pr-set720 and parp-1C03256 mutants during the third instar larvae stage. The results of this analysis have been included in the newly added supplemental Fig. S5.

Our findings reveal that these upregulated genes exhibit higher PolII occupancy compared to other genes, both at their promoter regions and gene bodies, suggesting heightened activity during third instar larval stage in wild type animals (Supplemental Fig. S6). To further validate these results, we cross-referenced publicly available RNA-seq data at the same developmental stage, confirming that, on average, these upregulated genes display a 40% higher expression compared to other genes (supplemental Fig. S6B).

Moreover, we would like to highlight the consistency of our current findings with our previous study (PMID: 38012002), where we reported the critical involvement of PARP-1 in tempering the expression of active metabolic genes at the end of the third instar larvae. The current data, suggesting a role for PR-SET7 in this regulatory process, adds another layer to our understanding of the nuanced control exerted by PARP-1 on the expression of active metabolic genes during this critical developmental transition.

In light of these results, we have modified the Results section to emphasize these findings: Intriguingly, under wild-type conditions, these genes displayed expression levels approximately 40% higher than the average and demonstrated increased RNA-Polymerase II occupancy both at their promoter regions and gene bodies compared to other genes (supplemental Fig.S6), indicating their high activity in wild type context.

Additionally, we have incorporated this information into the Discussion section to underscore the cooperative role of PARP-1 and PR-SET7 in repressing the expression of active metabolic genes: Notably, genes co-enriched with PARP-1 and H4K20me1, and are upregulated in both parp-1C03256 and pr-set720 mutants, are predominantly metabolic genes exhibiting high expression levels under wild-type conditions and a high occupancy of polymerase II both at their promoter region and gene body (Supplemental Fig. S6). In our previous study, we discovered that PARP-1 plays a crucial role in repressing highly active metabolic genes during the development of Drosophila by binding directly to their loci (34). Also, PARP-1 is required for maintaining optimum glucose and ATP levels at the third-instar larval stage (34). During Drosophila development, repression of metabolic genes is crucial for larval to pupal transition (35, 36). This repression is linked to the reduced energy requirements as the organism prepares for its sedentary pupal stage (35, 37). Notably, we observed that PARP-1 shows a high affinity for binding to the gene bodies of these metabolic genes (34).

Our data indicates that in both parp-1 and pr-set7 mutant animals, there was a preferential repression of metabolic genes at sites where PARP-1 and H4K20me1 are co-bound (Fig.3E), while these metabolic genes are highly active during third-instar larval stage (Supplemental Fig.S6). Thus, we propose that the presence of H4K20me1 may be essential for the binding of PARP-1 at these gene bodies, contributing to their repression. Importantly, this mechanism of gene repression has broader developmental implications. As earlier stated, mutant animals lacking functional PARP-1 and PR-SET7 undergo developmental arrest during larval to pupal transition. This arrest could be directly linked to the disruption of the normal metabolic gene repression during development. Without the repressive action of PARP-1 and PR-SET7, key metabolic processes might remain unchecked, leading to metabolic imbalances that are incompatible with the normal progression to the pupal stage.

Finaly, we have updated the Materials and Methods section to include information about the RNA-seq and PolII ChIP-seq datasets used: GSE15292 (RNA-polymerase II). In addition, we used the Developmental time-course RNA-seq dataset (54), SRP001065.

We believe that these modifications comprehensively address Reviewer 1's concern and provide a more robust foundation for our claims regarding the role of PARP-1 and PR-SET7 in the transcriptional regulation of co-enriched genes during the critical developmental transition.

(7) As discussed in Figure 4, the authors found transcriptional activation of group B genes even after a significant reduction of H3K20me1 in their gene body after heat shock. Given the dynamic equilibrium shift in epigenetic marks that regulate gene expression and their locus-specific transcriptional regulation, the authors should further look for the enrichment of other epigenetic marks and even H4K20me1 specific demethylases such as PHF8 (PMID: 20622854), and their cross-talk with PARP1 to further bridge the missing links of this tale. This will add more depth to this work.

We appreciate the thoughtful input provided by Reviewer 1 and acknowledge the importance of exploring additional epigenetic marks and potential cross-talk association with PARP1 to enhance the depth of our study. Our investigation has primarily focused on the interplay between PR-SET7/H4K20me1 and PARP-1, as evidenced by the colocalization and robust binding affinity observed between PARP-1 and H4K20me1 (Fig 1C, 2B, and 3A). This interaction is particularly noteworthy in the context of regulating specific heat shock genes, as highlighted in Figure 4A. While we recognize the potential significance of examining a broader spectrum of epigenetic marks and considering the involvement of specific demethylases, such as PHF8 (PMID: 20622854), in this regulatory network, our research strategy is intentionally tailored to leverage the unique characteristics of the PR-SET7/H4K20me1 and PARP-1 interplay in Drosophila. A key consideration is the technical advantage afforded by the fact that PR-SET7 is the exclusive methylase responsible for H4K20 in Drosophila (PMID: 15681608), allowing for specific depletion of H4K20me1 without the confounding influence of other methyltransferases.

This specificity is pivotal, especially given the similar developmental arrest patterns observed in both PR-SET7 and PARP-1 mutants. Such parallel phenotypes provide a distinct opportunity to delve deeply into the intricacies of their interaction during organismal development and in response to heat stress. Additionally, the identity of the demethylase for H4K20me1 in Drosophila remains unknown, further underscoring the rationale for our focused approach.

While we acknowledge the broader implications of exploring additional epigenetic marks, we believe that our deliberate focus on the PR-SET7/H4K20me1 and PARP-1 pathway provides a unique and valuable perspective on the regulation of gene expression in Drosophila. We hope that this clarification addresses the concerns raised by Reviewer 1 and conveys the rationale behind our chosen research strategy.

Reviewer #2:

Summary:

This study from Bamgbose et al. identifies a new and important interaction between H4K20me and Parp1 that regulates inducible genes during development and heat stress. The authors present convincing experiments that form a mostly complete manuscript that significantly contributes to our understanding of how Parp1 associates with target genes to regulate their expression.

Strengths:

The authors present 3 compelling experiments to support the interaction between Parp1 and H4K20me, including:

(1) PR-Set7 mutants remove all K4K20me and phenocopy Parp mutant developmental arrest and defective heat shock protein induction.

(2) PR-Set7 mutants have dramatically reduced Parp1 association with chromatin and reduced poly-ADP ribosylation.

(3) Parp1 directly binds H4K20me in vitro.

Weaknesses:

(1) The histone array experiment in Fig1 strongly suggests that PARP binds to all mono-methylated histone residues (including H3K27, which is not discussed). Phosphorylation of nearby residues sometimes blocks this binding (S10 and T11 modifications block binding to K9me1, and S28P blocks binding to K27me1). However, H3S3P did not block H3K4me1, which may be worth highlighting. The H3K9me2/3 "blocking effect" is not nearly as strong as some of these other modifications, yet the authors chose to focus on it. Rather than focusing on subtle effects and the possibility that PARP "reads" a "histone code," the authors should consider focusing on the simple but dramatic observation that PARP binds pretty much all mono-methylated histone residues. This result is interesting because nucleosome mono-methylation is normally found on nucleosomes with high turnover rates (Chory et al. Mol Cell 2019)- which mostly occurs at promoters and highly transcribed genes. The author's binding experiments could help to partially explain this correlation because PARP could both bind mono-methylated nucleosomes and then further promote their turnover and lower methylation state.

We appreciate the comprehensive review and valuable insights provided. In response to the comments, we have made substantial revisions to address the concerns and enhance the clarity of our findings. In Figure 1B, C, D, F, and G, we have expanded our data presentation to demonstrate PARP-1's binding affinity for H3K27me1. This addition is now incorporated into the revised results section. Additionally, we have updated Supplemental Fig.S1 and introduced new supplemental data (Supplemental Fig.S2) to illustrate the inhibition of PARP-1 binding by H3S10P, H3S28P, and H3T11P. The comprehensive exploration of PARP-1's interaction with mono-methylated histones, as suggested by the reviewer, is now more robustly documented in our revised figures and supplementary materials.

Our Discussion section has been refined to articulate more clearly how PARP-1 may be selectively recruited to active chromatin domains through its interaction with mono-methylated histone marks. We have proposed a model where PARP-1 actively participates in the turnover process, contributing to the maintenance of an active chromatin environment. This proposed mechanism involves PARP-1 selectively binding to mono-methylated active histone marks associated with highly transcribed genes. Upon activation, PARP-1 undergoes automodification, leading to its release from chromatin and facilitating the reassembly of nucleosomes carrying the mono-methylated marks. The enzymatic action of Poly(ADP)-ribose glycohydrolase (PARG) subsequently cleaves pADPr, allowing for the restoration of PARP-1's binding affinity to mono-methylated active histone marks. This proposed hypothesis is consistent with existing research across various model organisms and aligns with the known association of PARP-1 with highly expressed genes, as well as its role in mediating nucleosome dynamics and assembly.

Our Discussion section is modified a followed: Finaly, highly transcribed genes have been reported to present a high turnover of mono-methylated modifications, maintaining a state of low methylation (50). Then, our findings suggest that PARP-1 might actively participate in the turnover process to uphold an active chromatin environment. The proposed mechanism unfolds as follows: 1) PARP-1 selectively binds to mono-methylated active histone marks associated with highly transcribed genes. 2) Upon activation, PARP-1 undergoes automodification and is subsequently released from chromatin, facilitating the reassembly of nucleosomes carrying the mono-methylated marks. 3) The enzymatic action of Poly(ADP)-ribose glycohydrolase (PARG) cleaves pADPr, allowing for the restoration of PARP-1's binding affinity to mono-methylated active histone marks. This proposed hypothesis aligns cohesively with existing research conducted across various model organisms, including mice, Drosophila, and Humans (7, 23, 29, 51-53). Notably, previous studies have consistently demonstrated that PARP-1 predominantly associates with highly expressed genes and plays a crucial role in mediating nucleosome dynamics and assembly. Thus, our proposed model provides a molecular framework that may contribute to understanding the relationship between PARP-1 and the epigenetic regulation of gene expression. Further experimental validation is warranted to elucidate the precise details of this proposed mechanism and its implications in the broader context of chromatin dynamics and transcriptional control.

We hope that these revisions address the reviewer's concerns and contribute to the overall strength and clarity of our manuscript.

(2) The RNAseq analysis of Parp1/PR-Set7 mutants is reasonable, but there is a caveat to the author's conclusion (Line 251): "our results indicate H4K20me1 may be required for PARP-1 binding to preferentially repress metabolic genes and activate genes involved in neuron development at co-enriched genes." An alternative possibility is that many of the gene expression changes are indirect consequences of altered development induced by Parp1 or PR-Set7 mutants. For example, Parp1 could activate a transcription factor that represses the metabolic genes that they mention. The authors should consider discussing this possibility.

We hope that these revisions address the reviewer's concerns and contribute to the overall strength and clarity of our manuscript.

We extend our gratitude to Reviewer 2 for their thoughtful consideration of our manuscript and the insightful suggestion. In response to the raised concern regarding the conclusion on Line 251, where we proposed that "our results indicate H4K20me1 may be required for PARP-1 binding to preferentially repress metabolic genes and activate genes involved in neuron development at co-enriched genes," we acknowledge the alternative possibility suggested by the reviewer. It is plausible that many of the observed gene expression changes are indirect consequences of altered development induced in parp-1 or pr-set7 mutants. For example, PARP-1 could activates a transcription factor that represses the mentioned metabolic genes.

To address this concern, we have revisited our data and incorporated relevant findings from one of our recent studies that utilized a ChIP-seq approach. The results from this study suggest a direct binding of PARP-1 to the loci of metabolic genes, providing support for the notion that PARP-1 may indeed directly regulate their expression (PMID: 37347109). We have updated the Discussion section to reflect this information, aiming to provide a more comprehensive perspective on the potential mechanisms underlying the observed gene expression changes: In our previous study, we discovered that PARP-1 plays a crucial role in repressing highly active metabolic genes during the development of Drosophila by binding directly to their loci (34). Also, PARP-1 is required for maintaining optimum glucose and ATP levels at the third-instar larval stage (34). During Drosophila development, repression of metabolic genes is crucial for larval to pupal transition (35, 36). This repression is linked to the reduced energy requirements as the organism prepares for its sedentary pupal stage (35, 37). Notably, we observed that PARP-1 shows a high affinity for binding to the gene bodies of these metabolic genes (34).

We believe these modifications contribute to a more informed interpretation of our findings.

(3) The section on the inducibility of heat shock genes is interesting but missing an important control that might significantly alter the author's conclusions. Hsp23 and Hsp83 (group B genes) are transcribed without heat shock, which likely explains why they have H4K20me without heat shock. The authors made the reasonable hypothesis that this H4K20me would recruit Parp-1 upon heat shock (line 270). However, they observed a decrease of H4K20me upon heat shock, which led them to conclude that "H4K20me may not be necessary for Parp1 binding/activation" (line 275). However, their RNA expression data (Fig4A) argues that both Parp1 and H40K20me are important for activation. An alternative possibility is that group B genes indeed recruit Parp1 (through H4K20me) upon heat shock, but then Parp1 promotes H3/H4 dissociation from group B genes. If Parp1 depletes H4, it will also deplete H4K20me1. To address this possibility, the authors should also do a ChIP for total H4 and plot both the raw signal of H4K20me1 and total H4 as well as the ratio of these signals. The authors could also note that Group A genes may similarly recruit Parp1 and deplete H3/H4 but with different kinetics than Group B genes because their basal state lacks H4K20me/Parp1. To test this possibility, the authors could measure Parp association, H4K20methylation, and H4 depletion at more time points after heat shock at both classes of genes.

We thank Reviewer 2 for their valuable comment on our manuscript. We acknowledge your hypothesis suggesting that PARP-1 may induce H3/H4 dissociation from group B genes, potentially leading to a reduction in H4K20me1. However, our findings support a different interpretation.

Our data indicate that while H4K20me1 is present under normal conditions at group B genes, its reduction following heat shock does not appear to hinder PARP-1's role in transcriptional activation (Fig 4A, C and E). We propose that the observed decrease in H4K20me1 might reflect a regulatory shift in chromatin structure that is conducive to transcriptional activation during heat shock, facilitated by PARP-1 independently of sustained H4K20me1 levels at group B genes. Additionally, the literature suggests a dual role for H4K20me1 in gene regulation, from facilitating transcriptional elongation in certain contexts to acting as a repressor in others.

Unlike in group A genes which had low enrichment of H4K20me1 before heat shock (Fig 4B and D), the high enrichment of H4K20me1 in group B genes (Fig 4C and E) could imply a repressive role for this mark prior to heat stress. Thus, in the context of group B genes, it's conceivable that the removal of H4K20me1 might be necessary for their activation during heat stress. Thus, PR-SET7 may possess functions beyond its role as a histone methylase, which are crucial for activating group B genes under heat stress conditions. These functions could include methylation of non-histone substrates and non-catalytic activities.

Furthermore, our analysis of gene expression in pr-set720 and parp-1C03256 mutants indicates that while PARP-1 and H4K20me1 interaction may have overlapping roles in gene regulation, they also possess distinct functions in the modulation of gene expression (Fig 3E). Thus, we propose that the relationship between PR-SET7 and PARP-1 in transcriptional regulation involves a complex regulatory mechanism that extends beyond the presence of H4K20me1.

We modified the discussion section to address this point: Another plausible explanation could be that the recruitment of PARP-1 to group B genes loci promotes H4 dissociation and then leads to a reduction of H4K20me1. However, our findings suggest an alternative interpretation: the decrease in H4K20me1 at group B genes during heat shock does not seem to impede PARP-1's role in transcriptional activation, (Fig.4A, C and E). Rather than disrupting PARP-1 function, we propose that this reduction in H4K20me1 may signify a regulatory shift in chromatin structure, priming these genes for transcriptional activation during heat shock, with PARP-1 playing an independent facilitating role. Moreover, existing studies have highlighted the dual role of H4K20me1, acting as a promoter of transcription elongation in certain contexts and as a repressor in others (13, 25, 38, 39, 41-45). The elevated enrichment of H4K20me1 in group B genes under normal conditions may indicate a repressive state that requires alleviation for transcriptional activation. Additionally, we cannot discount the possibility of unique regulatory functions associated with PR-SET7, extending beyond its recognized role as a histone methylase. Non-catalytic activities and potential interactions with non-histone substrates might contribute to the nuanced control exerted by PR-SET7 on group B genes during heat stress (46, 47). Furthermore, our exploration of pr-set720 and ParpC03256 mutants reveals distinct roles for PARP-1 and H4K20me1 in modulating gene expression (Fig 3E). This reinforces the notion that the interplay between PR-SET7 and PARP-1 involves a multifaceted regulatory mechanism. Understanding the intricate relationship between these molecular players is crucial for elucidating the complexities of gene expression modulation under heat stress conditions.

We hope that this modification will adequately address Reviewer 2 concerns and enhance the clarity of our conclusions.

Reviewer #1 (Recommendations For The Authors):

(1) Please check the entire manuscript for grammatical errors and typos. PR-set7 has been wrongly written as PR-ste7 in quite a few places in the manuscript. Poly (ADP)-ribosylation has been written as poly(ADP-ribosyl)ation in the last result heading. There are more such errors. Please rectify them.

We express our sincere appreciation to Reviewer 1 for their meticulous review of our manuscript, and we acknowledge the importance of ensuring grammatical accuracy and clarity. We have taken your feedback seriously and conducted a comprehensive revision of the entire manuscript to rectify the identified typos and grammatical errors. We hope that these revisions contribute to an improved overall presentation of our research, and we appreciate the reviewer's diligence in ensuring the accuracy of the manuscript.

(2) The authors can also look up publicly available mammalian ChIP-seq data for H4K20me1 and PARP1, in order to further ossify their findings and increase the breadth of their work.

We appreciate the suggestion from Reviewer 1 and have taken steps to further validate and broaden the scope of our findings. Specifically, we compared the distribution of PARP1 and H4K20me1 in Human K562 cells. The results of this analysis revealed a correlation in their distribution, supporting the idea that the observed correlation between PARP-1 and H4K20me1 is not limited to fruit flies. We have incorporated these findings into the Results section and added a new Supplemental Fig. S6 to visually highlight this correlation: Finally, to extend the generalizability of our observations beyond Drosophila, we compared the distribution of PARP1 and H4K20me1 in Human K562 cells. Strikingly, we observed a correlation in their distribution, suggesting that the interplay between PARP-1 and H4K20me1 is not limited to fruit flies (Supplemental Fig. S6).

We believe that this modification addresses Reviewer 1's suggestion by providing additional evidence that supports the broader relevance of our findings beyond the Drosophila model system.

(3) Please discuss in greater detail how the PARP1-H4K20me1 axis orchestrates the repression program (metabolic pathways in this case) with proper references.

We appreciate Reviewer 1's continued engagement with our manuscript and have adjusted the discussion section to provide a more detailed insight into how the PARP1-H4K20me1 axis orchestrates the repression program, particularly focusing on metabolic pathways. The modified discussion section now reads: In our previous study, we discovered that PARP-1 plays a crucial role in repressing highly active metabolic genes during the development of Drosophila by binding directly to their loci (34). Also, PARP-1 is required for maintaining optimum glucose and ATP levels at the third-instar larval stage (34). During Drosophila development, repression of metabolic genes is crucial for larval to pupal transition (35, 36). This repression is linked to the reduced energy requirements as the organism prepares for its sedentary pupal stage (35, 37). Notably, we observed that PARP-1 shows a high affinity for binding to the gene bodies of these metabolic genes (34). Our data indicates that in both parp-1 and pr-set7 mutant animals, there was a preferential repression of metabolic genes at sites where PARP-1 and H4K20me1 are co-bound (Fig.3E), while these metabolic genes are highly active during third-instar larval stage (Supplemental Fig.S6). Thus, we propose that the presence of H4K20me1 may be essential for the binding of PARP-1 at these gene bodies, contributing to their repression. Importantly, this mechanism of gene repression has broader developmental implications. As earlier stated, mutant animals lacking functional PARP-1 and PR-SET7 undergo developmental arrest during larval to pupal transition. This arrest could be directly linked to the disruption of the normal metabolic gene repression during development. Without the repressive action of PARP-1 and PR-SET7, key metabolic processes might remain unchecked, leading to metabolic imbalances that are incompatible with the normal progression to the pupal stage.

We hope these modifications provide a more comprehensive discussion on how the PARP1-H4K20me1 axis influences the repression program, particularly within metabolic pathways, and how this mechanism contributes to the broader context of Drosophila development.