Polycomb regulates circadian rhythms in Drosophila in clock neurons

This study demonstrated that Pc regulates the circadian rhythm. The targeted DamID analysis identified the Pc-binding profiles in different clock neuron clusters, indicating Pc regulates circadian rhythms by specific clock genes.

We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): Zhao X. and collegues have demonstrated a function for Polycomb protein Pc in the regulation of circadian rhythm within the Drosophila Clock neurons.The manuscript is well written, experiments are straightforward, the methodologies used are novel and cutting edge.However more details should be provided on the models chosen and some important and highly relevant controls are missing to warrant the publication of this work.Clarity should be implemented.
1.For this reviewer it was very difficult to approach all the genetic models presented in Figure1 and their relative meaning.It would be advisable to find a simple, short and effective way to explain what the various models are for, in order to make the understanding of the experiments possible also for a wider audience.Methods should be substantiated.Abstract is not fully understandable.
Example of what the reviewer is referring to (but not limited to that): Line 87-89: "In contrast to the w1118 and two control group flies including UAS-PcRNAi and tim-Gal4, both of which are 100% in percentage rhythmicity, 61.3% of nthe tim-Gal4/PcRNAi flies were rhythmic (Table 1)."It is very hard to discriminate between proper controls and knock down flies.Also in the methodological part these information are missing.This reviewer asks for a proper description of the models chosen, and why they have been chosen.
2. The subsequent analyzes are well conducted and followable.However, to derive information about the specificity of the binding and more importantly, about the function of Pc in the four clusters of neurons, one important control is missing: "general neurons (line 150)".
Are the Pc binding sites identified in the four clusters absent in other neural cells that do not belong to the four clusters of neurons?How the Pc binding is modulated between clock and non clock neurons?Ideally same experiments with TaDa should be performed in other neurons.However if this will be not possible, a validation on small scale with ChIP followed by qRT PCR may be of value.In this case would be interesting also to add ChIP for the histone modification.
3. How the regions bound by Pc have been identified?Line 168-169: To identify Pc binding genes enriched in the above four clock neurons, expression data were analyzed from these clock neurons.What does this mean?Why expression data have been included?4. Be sure to cite all the panels presented and in a proper chronological order.5. Be sure to perform statistics in all the panel reported (Ex.Figure 6 is missing) 6. Figure 6, the expression of Per and Tim in the validation with Pc KD are impossible to be evaluated by this reviewer...the genetic models used are imcomprehensible.

Minor:
1. Bar plot can be substituted by box plot or barplot that display the dots in order to understand how data are distributed.2. Figures will benefit by avoiding bold and italic font.3. Tables should be uniform for their esthetics.4. Line 84-85: "In the constant darkness (DD) conditions the tim-85 Gal4/PcRNAi flies showed a severely disrupted circadian rhythm (Table 1)."How it has been measured?1.A short summary of the paper, including description of the advance offered to the field.
In this paper, the authors investigate the role of Pc in circadian rhythms in Drosophila.Firstly, they show that knockdown of Pc in clock neurons causes a disruption to circadian rhythms.Then the paper centres on their TaDa-seq data -whereby they map the binding of Pc across the genome in different classes of neurons (l-LNvs, LNds, DN1s, s-LNvs), using DamID where the expression of Dam is driven in a targeted way.They analyse this data and show that Pc bind to promoters, and that target sites vary in the different classes of neuron.They identify the genes that are bound by Pc, the pathways/ontologies, and find significant overlap with known circadian rhythm genes.They show their TaDa profiles in the different neuron classes across known circadian genes, showing more prominent binding in l-LNvs and s-LNvs generally.They then validate their findings in the well characterised per and tim circadian genes, showing DamID qPCR binding in the different neuron classes, and the changes in expression that accompany Pc knockdown in the different neurons.Although the paper lacks mechanistic insight into how Pc is regulating the genes, the involvement of Pc in circadian rhythms is novel to my knowledge, and the TaDa data in the different neurons is of value to the field.
2. For each main point of the paper, please indicate if the data are strongly supportive.If not, explicitly state the additional experiments essential to support the claims made and the timeframe that these would require.
Pc gene expression is required for behavioral circadian rhythms (Figure 1).Data are strongly supportive.
Global features and functional classification of Pc bound genes identified by TaDa (Figure 2 and 3).Data are supportive.However, explanation or re-plotting is required for 2G, where the Dam signal is higher than the Dam-PC signal in all clusters.It would be interesting to analyse the published RNA-seq data that the authors cite and see where the Pc bound genes fall in terms of expression levels i.e. is Pc binding to active or inactive genes.This would support the discussion of Figure 6 data (see below).
The TaDa analysis shows that Pc binds to the genes involved in the circadian rhythm and rhythmic process pathways (Figure 4 and 5).Data are supportive.The group of 5045 circadian rhythm related genes is very large though -maybe there is a more refined published gene list that could be compared to the PC-bound gene lists.
There is a direct role for Pc in regulating circadian rhythms through specific clock genes (Figure 6).This requires some additional data to make the claim.
-the expression qRT-PCR analysis in Fig. 6 L-S should be carried out in samples from C929-Gal4-marked neurons since there is prominent binding of Pc to per and tim in this sample.
-To make the conclusion that "In addition, we checked the Pc binding on Per locus at four time points across 24 hours.The results showed that the Pc binding was not statistically oscillated (Figure S7)" the analysis should be conducted in C929-Gal marked neurons with primer set 1, where the strongest binding of Pc to per is observed (Fig. 6B).
-The authors should discuss more thoroughly the results in This study shows that the epigenetic regulator Polycomb (Pc) regulates the circadian rhythm by binding to the genes involved in the circadian rhythm in the Drosophila clock neurons.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-science-alliance.org/copyright-license-fee regulation of circadian rhythm within the Drosophila Clock neurons.The manuscript is well written, experiments are straightforward, the methodologies used are novel and cutting edge.However more details should be provided on the models chosen and some important and highly relevant controls are missing to warrant the publication of this work.Clarity should be implemented.
1.For this reviewer it was very difficult to approach all the genetic models presented in Figure1 and their relative meaning.It would be advisable to find a simple, short and effective way to explain what the various models are for, in order to make the understanding of the experiments possible also for a wider audience.Methods should be substantiated.Abstract is not fully understandable.
Example of what the reviewer is referring to (but not limited to that): Line 87-89: "In contrast to the w1118 and two control group flies including UAS-PcRNAi and tim-Gal4, both of which are 100% in percentage rhythmicity, 61.3% of nthe tim-Gal4/PcRNAi flies were rhythmic (Table 1)." It is very hard to discriminate between proper controls and knock down flies.Also in the methodological part these information are missing.This reviewer asks for a proper description of the models chosen, and why they have been chosen.
Answer: We apologize for the confusions.To distinguish the genotypes for the knockdown flies, we have labeled them in blue in Figure 1.In the revised table 1, three types of genotypes were Answer: We apologize for the inaccuracy of this statement.The statement "the general neuronal binding genes" in line 150 should be "the general background bindings".This has been revised in the updated version.The experimental design of TaDa assay in this experiment is to identify Pc binding in the four clusters of neurons, which was achieved by expressing Dam-Pc specifically in certain neuron clusters.The general background bindings were eliminated by including of Dam lines (eg.C929-Gal4/tub-Gal80ts;UAS-Dam/+, et al.), which were clarified in the last paragraph of the "TaDa sample preparation and data analysis" session of the material and methods.
Are the Pc binding sites identified in the four clusters absent in other neural cells that do not belong to the four clusters of neurons?How the Pc binding is modulated between clock and non clock neurons?
Ideally same experiments with TaDa should be performed in other neurons.However if this will be not possible, a validation on small scale with ChIP followed by qRT PCR may be of value.In this case would be interesting also to add ChIP for the histone modification.
Answer: I agree.Exploring the Pc binding profile in neurons other than clock neurons is indeed an intriguing question.To gain insight into Pc binding differences between clock neurons and non-clock neurons, we conducted TaDa assays followed by PCR to assess Pc bindings at specific gene loci.Experiments were done using the following genotypes: To verify the histone modification on these sites, we also performed tissue specific ChIP for the histone modification by using the UAS-unc84GFP labeling and isolation of nuclei of specific neuron clusters.Experiments were done using the following genotypes: The result has been put into Figure 7. 4. Be sure to cite all the panels presented and in a proper chronological order.
Answer: In the updated version, we have thoroughly revised the manuscript and figures.We have also ensured that all the panels are properly cited in a chronological order.(eg. Figure S1).
5. Be sure to perform statistics in all the panel reported (Ex.Figure 6 is missing) Answer: Thank you for the suggestion.We have performed the statistical analysis as recommended by the reviewer, and the results have been incorporated into the updated Figure 6.
6. Figure 6, the expression of Per and Tim in the validation with Pc KD are impossible to be evaluated by this reviewer...the genetic models used are imcomprehensible.
Answer: The inclusion of the tub-Gal80 ts was to activate the Gal4 in a temporally specific manner and preventing the early expression-associated lethality (McGuire et al., 2003).Gal80's presence has an inhibitory effect on Gal4 activity.The inclusion of Gal80 ts enables Gal80 be deactivated upon the high temperature treatment, which activates the Gal4 in a temporally specific manner.In

Figures will benefit by avoiding bold and italic font.
Answer: Thank you for the suggestion.As suggested by the reviewer, we have eliminated the use of bold fonts entirely.Italic font was also avoided except when it is necessary, as italic fonts are typically employed for gene names and genotypes in the Drosophila research field.
3. Tables should be uniform for their esthetics.
Answer: As suggested by the reviewer, all main Tables and supplementary Tables are uniform.4. Line 84-85: "In the constant darkness (DD) conditions the tim-85 Gal4/PcRNAi flies showed a severely disrupted circadian rhythm (Table 1)."How it has been measured?Answer: According to the standard method for measuring circadian rhythm of Drosophila, Drosophila Activity Monitoring (DAM) system (Trikinetics, MA, U.S.) were used in this study.
Briefly, individual fly is placed in tube where their movement was continuously monitored using a simple and robust array of infrared beams, recording movements per second..The resulting data collected over periods of days is uploaded periodically to a computer for subsequent analysis.A diagram has been included below to better illustrate this mechanism (As show at https://trikinetics.com/).A detailed description of the method can be found in the material and method session under the title "Drosophila Activity Monitor-based method for circadian rhythm measurement".Pc gene expression is required for behavioral circadian rhythms (Figure 1).Data are strongly supportive.
Global features and functional classification of Pc bound genes identified by TaDa (Figure 2 and 3).Data are supportive.However, explanation or re-plotting is required for 2G, where the Dam signal is higher than the Dam-PC signal in all clusters.It would be interesting to analyse the published RNA-seq data that the authors cite and see where the Pc bound genes fall in terms of expression levels i.e. is Pc binding to active or inactive genes.This would support the discussion of Figure 6 data (see below).
Answer: Thanks for pointing this out.Yes, all analysis conducted was compared with the Dam controls to eliminate non-specific effects (See material and method session for details).Figure 2F showed values for each sample, while Figure 2G showed the final results compared with the Dam controls.All the other analysis were done by eliminating the background effects of Dam.
The Dam signal was higher than the Dam-Pc signal in Figure 2F troubled us for a while.
However, when we look back into the literature, we found a paper from Nature communication has data got from Dam-Pc (Marshall et al., 2017).We analyzed their data using the same procedure and got the similar result (Figure showed below, comparing with figure 2F).We believe that the reason behind this phenomenon may be attributed the weak toxicity of Dam-Pc expression to the cells.This potential toxicity could have influenced the cell cycle or cell survival and resulted in a much less cell number in the Dam-Pc samples than the Dam only samples.polycomb gene regulation disrupt lineage fidelity in intestinal stem cells.Elife 2021 Mar 16;10.PMID: 33724181 The TaDa analysis shows that Pc binds to the genes involved in the circadian rhythm and rhythmic process pathways (Figure 4 and 5).Data are supportive.The group of 5045 circadian rhythm related genes is very large though -maybe there is a more refined published gene list that could be compared to the PC-bound gene lists.
Answer: The results of the separate comparisons for the 5045 circadian rhythm-related genes are shown in the figure below.This additional analysis allows for a more detailed examination of the binding patterns of Pc protein to these genes and provides further support for our findings.
There is a direct role for Pc in regulating circadian rhythms through specific clock genes (Figure 6).This requires some additional data to make the claim.
the expression qRT-PCR analysis in Fig. 6 L-S should be carried out in samples from C929-Gal4-marked neurons since there is prominent binding of Pc to per and tim in this sample.
Answer: As suggested by the reviewer, RT-qPCR carried out in samples from C929-Gal4 marked neurons revealed that the Per and Tim expression were down regulated by Pc knocking down in the C929-Gal4 marked neurons (Figure 6P, U).This result aligns with the trends observed in the down-regulation of Pc in the pdf-Gal4 marked neurons.These results have been added in the updated figure 6P, U.
-To make the conclusion that "In addition, we checked the Pc binding on Per locus at four time points across 24 hours.The results showed that the Pc binding was not statistically oscillated (Figure S7)" the analysis should be conducted in C929-Gal marked neurons with primer set 1, where the strongest binding of Pc to per is observed (Fig. 6B).
Answer: As suggested by the reviewer, we conducted TaDa-qPCR with Per primer set 1 at four time points across 24 hours in C929-Gal4 marked neurons.We found that Pc binding Per primer set 1 was statistically oscillated (Figure S7G).These results have been included in the revised manuscript and updated Figure S7G.
-The authors should discuss more thoroughly the results in "The relationship between expression of the Pc downstream targets and Pc bound targets were complex.Pc has direct downstream targets (bound by Pc) and indirect downstream targets (not bound by Pc).As shown in figure 6L-U, we checked the expression level of clock genes.Pc can function as both repressor or activator (Morey L et al., 2010;Lv X et al., 2017).Moreover, as discussed above, in some cases, non-clock neurons were targeted in this study.So, when Pc is knockdown, its direct downstream target genes can be either activated or repressed under different conditions in this study.That's why we see either up regulation or down regulation of Pc targets.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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Sincerely, Novella Guidi, PhD Scientific Editor Life Science Alliance
Unfortunately, this reviewer finds the current form of the work still very difficult to be read.In the previous review, this reviewer asks to improve the clarity: numerous suggestions were made and the authors were strongly urged to improve the way the data were presented, to more easily clarify for example the genetic models used for a broad audience.This reviewer does not feel that the work has been much improved in this regard.Even genomic traces are difficult to interpret and of poor graphical quality, the comparisons to be made are not immediate.This reviewer believes that, to be published, this work must benefit from a global reorganization that considers the good of the reader, the readability of the article, the clarity in the presentation of the data as the first priority.
Reviewer #2 (Comments to the Authors (Required)): The manuscript is improved from the initial submission, with some extra supportive data added, and improved clarity and figures (particularly the graph presentation).
From my comments in the first round of revision, most have been discussed and/or additional data added to the paper.However, the graphs provided in the rebuttal in response to my query: "The group of 5045 circadian rhythm related genes is very large though -maybe there is a more refined published gene list that could be compared to the PC-bound gene lists" needs a little more information.How many genes are in each of the groups they examine, and where do those lists come from?Is the percentage circadian-related Pc bound (TaDa data) targets similar in each cell type for the different groups of circadian gene lists?Could this be provided in Supplementary data for all readers?
My other remaining concern is that no negative control region is provided for the TaDa-qPCR (Fig 6) or for the H3K27me3 ChIP-qPCR (Fig 7), and so it is impossible to say whether or not there is binding e.g. are levels that are below those at iab-7 still sig above background?

Minor comments:
-Define acronyms at first use e.g.PDF, s-LNvs, l-LNvs, and LNd -Figure citations incorrect for statement line 304 "While, in DVpdf-Gal4, pdf-Gal80 marked neurons, Pc had binding comparable with iab-7 on Tim but not on Per gene locus (Figure 6E, J).In R18H11-Gal4 marked neurons, Pc had much less binding on both Per and Tim gene locus (Figure 6F, K)." -these are the wrong way round Unfortunately, this reviewer finds the current form of the work still very difficult to be read.
In the previous review, this reviewer asks to improve the clarity: numerous suggestions were made and the authors were strongly urged to improve the way the data were presented, to more easily clarify for example the genetic models used for a broad audience.This reviewer does not feel that the work has been much improved in this regard.Even genomic traces are difficult to interpret and of poor graphical quality, the comparisons to be made are not immediate.
This reviewer believes that, to be published, this work must benefit from a global reorganization that considers the good of the reader, the readability of the article, the clarity in the presentation of the data as the first priority.
Answer: We apologize for any confusion caused.To provide clearer clarification of the genetic models used, we have made further improvements to the data.In Figure 1 and Figure S1, we have now labeled the meaning of the genetic models.Additionally, in the method session (Last paragraph under the title: Drosophila Activity Monitor-based method for circadian rhythm measurement), we have included descriptions of the parameters, namely Activity, Power, periods, and rhythmicity.
"For the circadian analysis, activity counts for each fly were binned every 30 minutes.Power represents the relative strength of the rhythm during DD.Flies with a power value greater than or equal to 10, along with a "width" value of 1.5 or more (indicating the number of peaks above the periodogram 95% confidence line in 30-minute increments), were considered rhythmic.Similarly, for DD rhythmicity, rhythmic flies were defined as those with a minimum period peak power of 10 or above.Period calculations took into account all flies with power-significance values greater than or equal to 10. Periods were calculated for each individual fly using X2 periodogram analysis and then pooled to obtain a group average for each genotype." Regarding graphical quality, we have provided figures in a higher quality TIFF format.These Thank you for submitting your revised manuscript entitled "Polycomb regulates circadian rhythms in Drosophila in clock neurons".We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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You can contact the journal office with any questions, contact@life-science-alliance.orgAgain, congratulations on a very nice paper.I hope you found the review process to be constructive and are pleased with how the manuscript was handled editorially.We look forward to future exciting submissions from your lab.Sincerely, Novella Guidi, PhD Scientific Editor Life Science Alliance Fig 6 L-S a) why they think they see the phenotype in Fig 6L-S, in which tim-Gal neurons show upregulation of per and tim in Pc knockdown, whereas other classes of clock neurons show downregulation in Pc knockdown.b) Pc is commonly a repressor, so why do they think they see downregulation?c) Why do they see downregulation in Pc knockdown of genes where there is no prominent Pc binding e.g.tim and per in R18H11-Gal4 marked neurons 3. Lastly, indicate any additional issues you feel should be addressed (text changes, data presentation, statistics etc.blurb (enter in submission system): A short text summarizing in a single sentence the study (max.200 characters including spaces).This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title.It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person.Author names should not be mentioned.

Reviewer # 1 (
Comments to the Authors (Required)): Zhao X. and collegues have demonstrated a function for Polycomb protein Pc in the indicated, including Control phenotypes, Pc down regulation phenotypes, Pc up regulation phenotypes.In the method session, the corresponding descriptions were added to the last paragraph of the "fly stocks" session, including description of the models chosen, and why they have been chosen."In this study, downregulation of Polycomb (Pc) levels by Pc RNAi in Drosophila clock neurons (tim-Gal4 marked most of the clock neurons) resulted in abnormal circadian rhythms.To further elucidate which clock neurons are required for the Pc gene to regulate the circadian rhythm, we selected several Gal4s specifically expresses in key clusters of clock neurons to drive the Pc RNAi.These drivers include C929-Gal4 (marked clusters including l-LNvs), R6-Gal4 (marked clusters including s-LNvs), R18H11-Gal4 (marked clusters including DN1) and DVpdf-Gal4, pdf-Gal80 (marked clusters including LNds).Based on the purpose of our experiments, we checked the phenotypes of flies with altered Pc levels in LD/DD conditions." 2. The subsequent analyzes are well conducted and followable.However, to derive information about the specificity of the binding and more importantly, about the function of Pc in the four clusters of neurons, one important control is missing: "general neurons (line 150)".
3. How the regions bound by Pc have been identified?Line 168-169: To identify Pc binding genes enriched in the above four clock neurons, expression data were analyzed from these clock neurons.What does this mean?Why expression data have been included?Answer: We apologize for the inaccuracy of this statement.Thank you for bringing this to our attention.This sentence should be "To identify Pc binding genes enriched in the above four clock neurons, TaDa data was analyzed from these clock neurons."This has been revised in the updated version.

Figure 6 L
Figure 6 L-U, the red line represents genotypes involving temporal and spatial RNAi of Pc, while the black line corresponds to the control group.Ref: McGuire SE, Le PT, Osborn AJ, Matsumoto K, Davis RL.Spatiotemporal rescue of memory dysfunction in Drosophila.Science.2003 Dec 5;302(5651):1765-8.doi: 10.1126/science.1089035.

Reviewer # 2 (
Comments to the Authors (Required)):1.A short summary of the paper, including description of the advance offered to the field.In this paper, the authors investigate the role of Pc in circadian rhythms in Drosophila.Firstly, they show that knockdown of Pc in clock neurons causes a disruption to circadian rhythms.Then the paper centres on their TaDa-seq data -whereby they map the binding of Pc across the genome in different classes of neurons (l-LNvs, LNds, DN1s, s-LNvs), using DamID where the expression of Dam is driven in a targeted way.They analyse this data and show that Pc bind to promoters, and that target sites vary in the different classes of neuron.They identify the genes that are bound by Pc, the pathways/ontologies, and find significant overlap with known circadian rhythm genes.They show their TaDa profiles in the different neuron classes across known circadian genes, showing more prominent binding in l-LNvs and s-LNvs generally.They then validate their findings in the well characterised per and tim circadian genes, showing DamID qPCR binding in the different neuron classes, and the changes in expression that accompany Pc knockdown in the different neurons.Although the paper lacks mechanistic insight into how Pc is regulating the genes, the involvement of Pc in circadian rhythms is novel to my knowledge, and the TaDa data in the different neurons is of value to the field.2.For each main point of the paper, please indicate if the data are strongly supportive.If not, explicitly state the additional experiments essential to support the claims made and the timeframe that these would require.
Fig 6 L-S a) why they think they see the phenotype in Fig 6L-S, in which tim-Gal neurons show upregulation of per and tim in Pc knockdown, whereas other classes of clock neurons show downregulation in Pc knockdown.b)Pc is commonly a repressor, so why do they think they see downregulation?c) Why do they see downregulation in Pc knockdown of genes where there is no prominent Pc binding e.g.tim and per in R18H11-Gal4 marked neurons Answer: Thank you for pointing out this.I agree that we should have been discussed more thoroughly about it.One paragraph was added to the discussion session.
Comments to the Authors (Required)): figures have been revised in the updated version.
Pc could also have indirect target, whose expression could be regulated by direct downstream targets of Pc.This could explain what we see in tim and per in R18H11-Gal4 marked neurons."3.Lastly, indicate any additional issues you feel should be addressed (text changes, data presentation, statistics etc.).Thank you for submitting your revised manuscript entitled "Polycomb regulates circadian rhythms in Drosophila in clock neurons" -Thank you for submitting your Research Article entitled "Polycomb regulates circadian rhythms in Drosophila in clock neurons".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.