H4K20me1 plays a dual role in transcriptional regulation of regeneration and axis patterning in Hydra

H4K20me1 modification by SETD8 methyltransferase is typically associated with repression. In Hydra, it is associated with activation machinery locally at Wnt-responsive promoters indicating its dual role in transcriptional regulation.

1. SETD8 is known to activate the wnt target genes by interacting with TCF during development of bilaterians, and this paper shows that this system is conserved even in non-bilaterian Hydra and that H4K20me1, which is regulated by SETD8, plays an important role in head generation. This papers also demonstrates the genome wide methylation state during wnt signaling activation and regeneration. Hydra is well-studied for their signaling pathways in regeneration and development, and an important model organism in evolutionary developmental biology as a sister group of bilaterians for discussing the general biological mechanisms. On the other hand, the role of methylation in development and regeneration has not yet been well studied. Thus this study can be said unique and pioneering, and has potential for publication in Life science alliance.
2. While the first half of this paper clearly shows that methylation by SETD8 is involved in head generation via wnt signaling through the careful experiments, the second half of the paper regarding the comprehensive analysis of methylation states shown in Fig4-6 is critically lacking in explanation needed. It is unclear whether the method is consistent with the purpose of this paper, which is to elucidate the involvement of methylation in head generation. There appears to be a discrepancy between what is shown in Fig4-6 and the main text in Results. In addition, it is also unclear on which results some of the sentences in Discussion are based. Therefore, in order for this paper to be accepted, these issues (as described below in detail) would need to be resolved.
p6 "H4K20me1 occupancy is context-dependent and is linked to specific signaling pathways" , the genes whose methylation status is changed by ALP treatment compared to Cont, and for Fig. 5C, the genes whose methylation status is changed compared to 0hpa should be used in the analysis. Also, it is not clear what is meant by p7L19 "the Hippo signaling pathway had the least number of gene bodies with this modification".
L23~31 What the graphs in Figs 4D and 4E show and what is described in the text do not appear to match. It is also unclear which genes were used in this analysis. Given the purpose of this paper, genes with altered methylation status should be extracted.
p6L32-p7L8, Fig6: ATAC seq results It is unclear which genes were used in the analysis; Materials and Methods does not describe the method either. In addition, the description of the level of transcription and chromatin accessibility for the genes in each cluster in the main text does not seem to be consistent with Fig6. For example, the authors say "The cluster 1 genes have the highest level of H4K20me1, the least intensity of the ATAC seq reads, and the lowest levels of transcription" , but the peak height of transcription in Fig6 does not seem to be so different from other clusters. Also the authors say "the occupancy of H4K20me1 is also excluded from open chromatin regions displaying active transcription."(p10 L12), but it does not look that way in any of the clusters as seen in Fig6. The vertical axis in Fig6 (in particular, "Transcription") should be the same scale for comparison among CLSTRs.
The current presentation of DMSO and UNC at the same stage side by side makes it difficult to see the changes in DMSO and UNC over time. It would be easier to see both the difference between DMSO and UNC and their respective changes over time if DMSO and UNC were placed vertically.
-----p5 L15 "Brachyury has been established as a bonafide head specification marker. Brachyury in Hydra is a head organizer gene involved in head morphogenesis." Please cite appropriate references.
-----p5 L29 "The occurrence of both SETD8 and its target histone modification, H4K20me1, is relatively higher in the head and the foot of the polyps (Fig.2D)." Fig. 2D shows that the head (around the oral pore) does indeed appear to be stained, but not the foot.
-----p6 L21 "We used this experimental paradigm of ectopic activation of the Wnt signalling pathway and checked the occupancy of H4K20me1 on the promoter regions of Margin and setd8. We observed an enhanced occupancy of the modification at the promoter regions of both the genes (Fig. 3F).
How it was verified should be described (ChIP-qPCR?). Also, more detail information such as the realtime PCR system, reagents, primers need to be described in the corresponding section of materials and methods (p. 16). For examples; p11 L6 "in the context of Hydra head regeneration, where there is a necessity for various signalling pathways to interact, the trend indicates that H4K20me1 is negatively correlated with the transcription of genes. " p11 L16 "The specificity is at TF binding sites, and a deeper investigation of the TCF binding sites displayed distinct regulation of H4K20me1 at these motifs in response to Wnt signalling activation." Which results support these discussions? -----Materials and Methods p15 L10 Please provide information of α-SETD8 antibody.
p15 "RNA sequencing" Please provide information of sequencing, such as the sequencer, the number of cycles (read length) and the number of reads obtained.

p17-18
The authors listed all commands to run the analysis software, but need not be listed unless special options were set.
Reviewer #2 (Comments to the Authors (Required)): Gungi et al. treat Hydra with a compound that is known to inhibit the H4K20me1 methyltransferase SETD8, and find that this impairs the regeneration process. They show that this correlates with the regulation of Wnt pathway genes, that SETD8 interacts with beta-catenin, and SETD8 is upregulated upon an increase in Wnt signaling. They then go on to profile H4K20me1, and show that it anti-correlates with chromatin accessibility at gene promoters.
If all points below are addressed, this manuscript describes a role of SETD8 in Hydra regeneration, as well as the genomic distribution of H4K20me1 across the Hydra genome, and its relationship with transcriptional regulation during regeneration.
Major points: 1. The authors use a compound which has been described as a chemical inhibitor of SETD8. It is not clear if this inhibitor is specific to SETD8 at the concentration used (200uM) in Hydra. They should perform experiments showing that they inhibit SETB8 specifically, or at least state clearly any off-target effects they observe. For example, in Figure EV2 they show a small reduction of H4K20me1when treated with UNC0379 for 8h. Why is this specific to 8h? What concentration was used in this Figure? Did they test different concentrations? And what were the effects on other histone modifications? What about the effects on non-histone substrates, eg PCNA (https://pubmed.ncbi.nlm.nih.gov/25032507/)? Maybe they can try to inhibit SETD8 genetically, and compare the effects to the compound? 2. Figure 3F: The authors should show a control region where H4K20me1 does not increase. Also, since ChIP-seq was eventually performed, screen shots of these regions in the ChIP-seq data would be informative to show that the ChIP-qPCR and ChIP-seq data are comparable, as well as a more detailed analysis of H4K20me1 on Wnt (target) genes, and how this correlates with gene expression (changes) on these genes. Especially since the authors claim that H4K20me1 can activate or repress genes, and overall H4K20me1 seems to correlate with reduced chromatin accessibility. 3. It seems to me that Figure 4 is really Figure 5, and Figure 4 is obsolete (except 4a). Indicate the y axis units on the metaplots (is it cpm?). How was the clustering performed? Are there any genes that are not in a cluster? 4. What is the exact analysis underlying Figure 4B? As I understand it simply shows the number of genes associated with a particular pathway. The authors state that the Hippo pathway is underrepresented in H4K20me1 covered genes, but it also seems to have less genes in cluster 4, which as far as I understand is all genes that are not H4K20me1 covered? The authors should perform a statistical analysis to check for enrichments of genes with certain GO terns in their clusters. 5. Figure 6: While I can see an increase in ATAC seq signal, I cannot see increased transcription in cluster 3 and 4 vs cluster 1 and 2 genes. 6. It would be good to perform ChIP for total H3 or H4 and compare the profiles (heatmaps) to the h4K20me1 profiles, to exclude that the H4K20me1 profiles are specific for the modification and do not simply reflect histone binding.
Minor points: 1. The authors should provide more details on the analysis of the RNAseq experiments: PCA plots of all samples, how was the differential expression calculated, volcano plots, a list of all genes with their logFCs and adjusted p -values,...If regeneration is delayed by inhibitor treatment, then how can the authors be sure they are not just comparing different points in the regeneration process? 2. Figure 2D: The staining at the foot regions are very difficult to see. 3. In general, the y-axis scales of metaplots should be kept the same across the plots of a particular data type to make it easier to compare levels. The y-axis units should be indicated. 4. The authors could further explore the H4K20me1 distribution in the genome beyond gene bodies, for example at enhancers, or repetitive elements.
1st Authors' Response to Reviewers December 11, 2022 Reviewer #1: 1. SETD8 is known to activate the wnt target genes by interacting with TCF during development of bilaterians, and this paper shows that this system is conserved even in non-bilaterian Hydra and that H4K20me1, which is regulated by SETD8, plays an important role in head generation. This papers also demonstrates the genome wide methylation state during wnt signaling activation and regeneration. Hydra is wellstudied for their signaling pathways in regeneration and development, and an important model organism in evolutionary developmental biology as a sister group of bilaterians for discussing the general biological mechanisms. On the other hand, the role of methylation in development and regeneration has not yet been well studied. Thus this study can be said unique and pioneering, and has potential for publication in Life science alliance.
2. While the first half of this paper clearly shows that methylation by SETD8 is involved in head generation via wnt signaling through the careful experiments, the second half of the paper regarding the comprehensive analysis of methylation states shown in Fig4-6 is critically lacking in explanation needed. It is unclear whether the method is consistent with the purpose of this paper, which is to elucidate the involvement of methylation in head generation. There appears to be a discrepancy between what is shown in Fig4-6 and the main text in Results. In addition, it is also unclear on which results some of the sentences in Discussion are based. Therefore, in order for this paper to be accepted, these issues (as described below in detail) would need to be resolved.
Response: We thank the reviewer for encouraging comments and suggestions on our manuscript. The further analysis suggested has helped in refining the manuscript and shaping the discussion around the role of H4K20me1 in a better manner.
p6 "H4K20me1 occupancy is context-dependent and is linked to specific signaling pathways" , the genes whose methylation status is changed by ALP treatment compared to Cont, and for Fig. 5C, the genes whose methylation status is changed compared to 0hpa should be used in the analysis. Also, it is not clear what is meant by p7L19 "the Hippo signaling pathway had the least number of gene bodies with this modification".
Response: Thank you for this suggestion. We have performed the analysis of the ChIP seq data sets, retrieving the regions with differential occupancy of H4K20me1 in both ALP treatment and regeneration time points (Fig. 4-6). We have also done different analyses to understand the significance of the changing histone mark occupancy on the genomic regions in relation to the genes associated with it and this is included in the revised version of the manuscript (Fig. 4-6).
L23~31 What the graphs in Figs 4D and 4E show and what is described in the text do not appear to match. It is also unclear which genes were used in this analysis. Given the purpose of this paper, genes with altered methylation status should be extracted.
Response: As suggested by this and the second reviewer, we have retrieved the genes with altered methylation status and interpreted the results in this condition. Following this, we retrieved the genes and performed further annotation of genes, GO term enrichment, and STRING interaction analysis (Lines 207-216). There was no significant GO term enrichment in the ALP condition and we have therefore depicted the STRING results in the revised manuscript ( Fig. 4C-F; Fig. S9-S24).
p6L32-p7L8, Fig6: ATAC seq results It is unclear which genes were used in the analysis; Materials and Methods does not describe the method either. In addition, the description of the level of transcription and chromatin accessibility for the genes in each cluster in the main text does not seem to be consistent with Fig6. For example, the authors say "The cluster 1 genes have the highest level of H4K20me1, the least intensity of the ATAC seq reads, and the lowest levels of transcription" , but the peak height of transcription in Fig6 does not seem to be so different from other clusters. Also the authors say "the occupancy of H4K20me1 is also excluded from open chromatin regions displaying active transcription."(p10 L12), but it does not look that way in any of the clusters as seen in Fig6. The vertical axis in Fig6 (in particular, "Transcription") should be the same scale for comparison among CLSTRs.
Response: We thank the reviewer for this comment and suggestion. We have performed this analysis too on the differentially methylated regions of the Hydra genome and associated genes. We have maintained the scale of the vertical axis constant for each type of data we have analysed to enable comparison between the clusters ( Does any statistical analysis support this result? Response: The enrichment is based on the proportion of the genes with H4K20me1 occupancy which is statistically supported. However, in the revised version of the manuscript, we have retrieved the differentially occupied regions and performed an interaction analysis on the associated genes ( Fig. 4C-F; Fig S9-S24).
The current presentation of DMSO and UNC at the same stage side by side makes it difficult to see the changes in DMSO and UNC over time. It would be easier to see both the difference between DMSO and UNC and their respective changes over time if DMSO and UNC were placed vertically.
Response: We have modified the representation of the figure so that the time course of regeneration is displayed in the two different conditions -DMSO and UNC treated. We agree with the reviewer that this makes the visualisation of the molecular dynamics easy and thank the reviewer for this suggestion (Fig. 2B).
-----p5 L15 "Brachyury has been established as a bonafide head specification marker. Brachyury in Hydra is a head organizer gene involved in head morphogenesis." Please cite appropriate references.
Response: Thank you for pointing this out; the appropriate references have been added (Lines 114-115).
-----p5 L29 "The occurrence of both SETD8 and its target histone modification, H4K20me1, is relatively higher in the head and the foot of the polyps (Fig.2D)." Fig. 2D shows that the head (around the oral pore) does indeed appear to be stained, but not the foot.
Response: We have now used images of polyps with a clearer representation of the increased occurrence of the KMT and its histone mark at both the poles (Fig. 2D). We have also provided more images in the supplementary data to support this (Fig.  S5).
-----p6 L21 "We used this experimental paradigm of ectopic activation of the Wnt signalling pathway and checked the occupancy of H4K20me1 on the promoter regions of Margin and setd8. We observed an enhanced occupancy of the modification at the promoter regions of both the genes (Fig. 3F).
How it was verified should be described (ChIP-qPCR?). Also, more detail information such as the realtime PCR system, reagents, primers need to be described in the corresponding section of materials and methods (p. 16). For examples; p11 L6 "in the context of Hydra head regeneration, where there is a necessity for various signalling pathways to interact, the trend indicates that H4K20me1 is negatively correlated with the transcription of genes. " p11 L16 "The specificity is at TF binding sites, and a deeper investigation of the TCF binding sites displayed distinct regulation of H4K20me1 at these motifs in response to Wnt signalling activation." Which results support these discussions?

Response
Response: This suggestion has indeed helped increase clarity the manuscript. As asked above: 1. "in the context of Hydra head regeneration, where there is a necessity for various signalling pathways to interact, the trend indicates that H4K20me1 is negatively correlated with the transcription of genes. " (in the revised manuscript Fig. 6A, 6B indicating an increase of transcription upon inhibition of SETD8 by UNC0379, Fig. S9-S24 indicating the large network of genes with differential occupancy of H4K20me1 vs the STRING interaction networks in Fig.  4C-4E showing very few genes with differential methylation). 2. "The specificity is at TF binding sites, and a deeper investigation of the TCF binding sites displayed distinct regulation of H4K20me1 at these motifs in response to Wnt signalling activation." (in the revised manuscript Fig. 3F shows occupancy at localised TCF motifs studied using ChIP-qPCRs) 3. The interpretations of Fig 7B,  p15 "RNA sequencing" Please provide information of sequencing, such as the sequencer, the number of cycles (read length) and the number of reads obtained.

p17-18
The authors listed all commands to run the analysis software, but need not be listed unless special options were set.
Response: We have provided all the relevant information about the antibody, sequencer, and sequencing reads in the methods section of the revised manuscript (Lines 424-425, 441-445). The codes used did not require custom options and, as suggested, have been removed to avoid redundant information.

Reviewer #2:
Gungi et al. treat Hydra with a compound that is known to inhibit the H4K20me1 methyltransferase SETD8, and find that this impairs the regeneration process. They show that this correlates with the regulation of Wnt pathway genes, that SETD8 interacts with beta-catenin, and SETD8 is upregulated upon an increase in Wnt signaling. They then go on to profile H4K20me1, and show that it anti-correlates with chromatin accessibility at gene promoters.
If all points below are addressed, this manuscript describes a role of SETD8 in Hydra regeneration, as well as the genomic distribution of H4K20me1 across the Hydra genome, and its relationship with transcriptional regulation during regeneration.
Major points: 1. The authors use a compound which has been described as a chemical inhibitor of SETD8. It is not clear if this inhibitor is specific to SETD8 at the concentration used (200uM) in Hydra. They should perform experiments showing that they inhibit SETB8 specifically, or at least state clearly any off-target effects they observe. For example, in Figure EV2 they show a small reduction of H4K20me1when treated with UNC0379 for 8h. Why is this specific to 8h? What concentration was used in this Figure Response: Thank you for this insightful question. We have performed time-course experiments and observed the effects of various concentrations of the compound UNC0379. At a higher concentration of 250 µM, the polyps start to disintegrate within 4 h and cannot be used for performing regeneration assays. At the concentration used for regeneration assays (150 µM), the polyps were harvested at different time points to establish the duration of treatment for regeneration assays. We have mentioned this information in the revised version of the manuscript. Further, we are aware of the compounding effects on the function of SETD8 on its non-histone substrates, but we still attribute this to effects on catalytic function. Such effects will also be present upon genetic inhibition and more extensive assays will be required to delineate these that are out of the scope of this manuscript due to time constraints. We have tried genetically inhibiting the function using siRNA-mediated knockdowns. However, unlike transcription factor knockdowns that are quite stable (Reddy et. al. 2019), knockdown of epigenetic regulators, and specifically histone methyltransferases, is very transient, and the feedback regulation does not allow enough loss of the enzyme expression to affect morphogenetic events. Regeneration assays performed after knockdown suggest the same, where the effect on regeneration is less pronounced than with chemical inhibition. The data is shown below.
2. Figure 3F: The authors should show a control region where H4K20me1 does not increase. Also, since ChIP-seq was eventually performed, screen shots of these regions in the ChIP-seq data would be informative to show that the ChIP-qPCR and ChIP-seq data are comparable, as well as a more detailed analysis of H4K20me1 on Wnt (target) genes, and how this correlates with gene expression (changes) on these genes. Especially since the authors claim that H4K20me1 can activate or repress genes, and overall H4K20me1 seems to correlate with reduced chromatin accessibility.
Response: We thank the reviewer for this comment. We have shown a genomic desert region (used in Reddy et. al. 2020) as a control that does not display altered H4K20me1 status. Also, in the analysis we have performed for the revised version of the manuscript, we have retrieved and plotted the IGV screenshots for the genomic regions in different clusters ( Fig. 4C-F, Fig. 6L-O). Isolating Wnt-related genes with high accuracy is difficult due to the lack of complete annotation of the genome, and we have tried to do this using the new version of the Hydra genome available (v 3). However, in the ALP-treated polyps, we do find a positive correlation of H4K20me1 occupancy with few genomic regions around Wnt target transcription factors (Fig. 4). The negative correlation with transcription is more prominent in the regeneration phenotype, where upon inhibition of SETD8, we observed increased transcription at multiple time points (Fig. 5 and Fig. 6).
3. It seems to me that Figure 4 is really Figure 5, and Figure 4 is obsolete (except 4a ). Indicate the y axis units on the metaplots (is it cpm?). How was the clustering performed? Are there any genes that are not in a cluster?
Response: Figure 4 depicts the dynamics of H4K20me1 in ALP-treated polyps, while Figure 5 shows this in regenerating tips of the polyps. In the revised version of the manuscript, we have only shown the differentially occupied regions in the main figures and the whole genome occupancy in the extended information ( Fig. S7 and Fig. S8). The clustering was performed using Deeptools and it was a k-means based clustering showing the occupancy across treatment conditions or timepoints. Since the previous analysis did not look at differentially occupied regions, almost all the genes were displayed in the heatmap. The detailed analysis we have performed to identify the differentially occupied genomic regions has allowed us to differentiate between the behaviour of H4K20me1 in both physiological conditions and make better interpretations. This has now been incorporated in the revised manuscript.
4. What is the exact analysis underlying Figure 4B? As I understand it simply shows the number of genes associated with a particular pathway. The authors state that the Hippo pathway is underrepresented in H4K20me1 covered genes, but it also seems to have less genes in cluster 4, which as far as I understand is all genes that are not H4K20me1 covered? The authors should perform a statistical analysis to check for enrichments of genes with certain GO terns in their clusters.
Response: In the original version, we retrieved the number of genes associated with specific GO terms with occupancy of H4K20me1. However, for more physiological relevance to the process being investigated, we have performed a differential occupancy analysis and retrieved the genomic regions and genes associated with them. Since we do not find significant GO term enrichment due to the lack of good annotation of the genes in Hydra, we performed a STRING interaction analysis to identify significantly interacting networks with differentially H4K20 mono-methylated regions ( Fig. 4C-F; Fig. S9-S24).
5. Figure 6: While I can see an increase in ATAC seq signal, I cannot see increased transcription in cluster 3 and 4 vs cluster 1 and 2 genes.
Response: We have now retrieved the differentially occupied regions with respect to the 0 h time point during regeneration and performed further analysis on these regions ( Fig. 5A-C; Fig. 6A-D). To understand the effect of H4K20me1 on transcription, we have also plotted the UNC-treated RNA seq data (Fig. 6E-H). This has allowed us to better understand the role of H4K20me1 in transcriptional regulation. The discussion has been added in the revised version of the manuscript.
6. It would be good to perform ChIP for total H3 or H4 and compare the profiles (heatmaps) to the H4K20me1 profiles, to exclude that the H4K20me1 profiles are specific for the modification and do not simply reflect histone binding.
Response: The reviewer has raised an important point here. Our previously published report on understanding enhancer-mediated regulation of Wnt signalling (Reddy & Gungi et. al., Epigenetics & Chromatin 2020) includes the data for H3 ChIPseq and demonstrates a conserved loss of H3 occupancy at the +1 nucleosome in Hydra. Comparing both the profile and the level of H4K20me1 occupancy to that, we are able to ensure that we are not observing general histone binding. H4K20me1 is a very low occurrence histone mark and does not display the +1 nucleosome loss as seen with H3. Further, we see a clear spreading of H4K20me1 occupancy along the gene bodies with differential occupancy at exons and introns as well. This needs deeper analysis to decipher the role of H4K20me1 in splicing, gene expression regulation, or isoform regulation, and will be a worthwhile new line of investigation. We therefore believe this is currently out of scope for this manuscript.
Minor points: 1. The authors should provide more details on the analysis of the RNAseq experiments: PCA plots of all samples, how was the differential expression calculated, volcano plots, a list of all genes with their logFCs and adjusted p -values,...If regeneration is delayed by inhibitor treatment, then how can the authors be sure they are not just comparing different points in the regeneration process?
Response: Thank you for pointing this out. We have now provided the relevant information in the extended information of the revised manuscript. From the volcano plots obtained comparing the same time points in control and treated conditions, we can clearly see changes in gene expression that are due to the inhibition of SETD8 (Fig. S25). The plots in the main figure panel represent the logCPM+1 values of genes that show differential expression to depict the dynamics.
2. Figure 2D: The staining at the foot regions are very difficult to see.
Response: We thank the reviewer for pointing this out. We have changed the representative images to reflect better the occurrence of both SETD8 and the histone marks in the head and foot of the polyps (Fig. 2D, Fig. S5). We have also provided additional images in the extended information as evidence for the same.
3. In general, the y-axis scales of metaplots should be kept the same across the plots of a particular data type to make it easier to compare levels. The y-axis units should be indicated.
Response: We thank the reviewer for this comment. As suggested by you and the first reviewer, we have kept the y-axis of all the plots the same for a single type of data to enable comparison across clusters obtained in the new analysis. These revised figures are provided in the revised version of the manuscript (Fig. 4,5,6).
4. The authors could further explore the H4K20me1 distribution in the genome beyond gene bodies, for example at enhancers, or repetitive elements.
Response: We thank the reviewer for this insightful suggestion. We have retrieved the intergenic, intronic, and promoter sequences for the whole genome of Hydra using the available version of the genome and Bedtools. However, we did not find significantly differential trends of H4K20me1 occupancy in these regions and have therefore not pursued this further. However, further analysis could provide us with information to understand the role of H4K20me1 in regulating enhancer regions, and we plan to explore this in the future. Thank you for submitting your revised manuscript entitled "H4K20me1 plays a dual role in transcriptional regulation of regeneration & axis patterning in Hydra" to Life Science Alliance. The manuscript has been seen by the original reviewers whose comments are appended below. While the reviewers continue to be overall positive about the work in terms of its suitability for Life Science Alliance, some important issues remain.
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