On the relations of phase separation and Hi-C maps to epigenetics

The relationship between compartmentalization of the genome and epigenetics is long and hoary. In 1928, Heitz defined heterochromatin as the largest differentiated chromatin compartment in eukaryotic nuclei. Müller's discovery of position-effect variegation in 1930 went on to show that heterochromatin is a cytologically visible state of heritable (epigenetic) gene repression. Current insights into compartmentalization have come from a high-throughput top-down approach where contact frequency (Hi-C) maps revealed the presence of compartmental domains that segregate the genome into heterochromatin and euchromatin. It has been argued that the compartmentalization seen in Hi-C maps is owing to the physiochemical process of phase separation. Oddly, the insights provided by these experimental and conceptual advances have remained largely silent on how Hi-C maps and phase separation relate to epigenetics. Addressing this issue directly in mammals, we have made use of a bottom-up approach starting with the hallmarks of constitutive heterochromatin, heterochromatin protein 1 (HP1) and its binding partner the H3K9me2/3 determinant of the histone code. They are key epigenetic regulators in eukaryotes. Both hallmarks are also found outside mammalian constitutive heterochromatin as constituents of larger (0.1–5 Mb) heterochromatin-like domains and smaller (less than 100 kb) complexes. The well-documented ability of HP1 proteins to function as bridges between H3K9me2/3-marked nucleosomes contributes to polymer–polymer phase separation that packages epigenetically heritable chromatin states during interphase. Contacts mediated by HP1 ‘bridging’ are likely to have been detected in Hi-C maps, as evidenced by the B4 heterochromatic subcompartment that emerges from contacts between large KRAB-ZNF heterochromatin-like domains. Further, mutational analyses have revealed a finer, innate, compartmentalization in Hi-C experiments that probably reflect contacts involving smaller domains/complexes. Proteins that bridge (modified) DNA and histones in nucleosomal fibres—where the HP1–H3K9me2/3 interaction represents the most evolutionarily conserved paradigm—could drive and generate the fundamental compartmentalization of the interphase nucleus. This has implications for the mechanism(s) that maintains cellular identity, be it a terminally differentiated fibroblast or a pluripotent embryonic stem cell.


Comments to the Author(s)
Singh and Newman provide a review of the state of the knowledge regarding the 3D conformation of chromosomes and epigenetics. I think this is a difficult task and they have done a commendable job. They do not provide new data/code/method/modelling and it appears that the only original contribution is to rename compartments in "Epigenetic compartmental domains". Because of this, I will referee this paper as if it was a review.
1. ".. could favour the merging of "clutches" to form ordered (fractal) "globules" that have been detected .. " is incorrect. Entropy works towards the equilibrium globule state. There is no direct connection between crowding/depletion attraction and fractal globule which is instead a non-equilibrium state of a polymer.
2. The authors do a very good job at connecting experiments with modelling work in many instances. They also correctly point out that LLPS is different from PPPS and that the latter is most likely the mechanism of phase separation in the nucleus.
I have a few more suggestions a. Regarding HP1 bridges: Barbieri et al PNAS 2013Brackley et al PNAS 2012 are to my knowledge the first using explicit protein bridges to study the folding of chromosomes.
b. An important work connecting co-polymer models with epigenetics is that of Daniel Jost et al NAR 2014. It was the first to my knowledge to make this connection clear.

5.
In several instances, it should be made clear that this is a review and that no new data is presented but simply reported from previous papers or obtained from online tools such as the genome browser.

PPPS vs LLPS
In page 6, line 6-11 The authors said that "if HP1a does drive phase separation it does so in a manner different from multivalent intrinsically-disordered proteins [63], which are known to form endogenous liquidliquid phase separated condensates in the nucleus [70]. Based on these data we propose an alternative mechanism. Mammalian HP1 proteins drive phase separation by polymer-polymer phase separation (PPPS; [71]) rather than by LLPS." The reviewer thinks this interpretation is too biased. Reference [70] showed that only HP1a forms liquid droplets in vitro, but this does not deny the possibility of other HP1 isoforms as a critical component of phase-separated heterochromatin compartment induced by LLPS. Indeed, reference [69] showed such possibility. Also, LLPS-mediated but different compartments exist, suggesting that different intrinsically-disordered proteins which contribute to LLPS not always form same droplet/compartment. Data of ref [70] also does not deny that HP1-mediated heterochromatin may exist as LLPS or formation of LLPS may contribute to function or maintenance of heterochromatin. Therefore, the PPPS story is fine and interesting, but the authors should more carefully describe current situation and cite recent work of HP1 function in LLPS appropriately. Also, most recent Narlikar's nature paper (Sanulli et al 2019) should be integrated into this article.
2. Fig. 7 Page 14 first paragraph. The authors should cite more details of each panel for description of Fig.  7 to help understanding the authors points. Also, if any paper shows that HP1 level on H3K9me2/3+ high regions in compartment A is lower than that in compartment B, should be cited because this support the authors' Fig7 model.

conclusions and perspectives
It is nice to add one additional fig to show some of issues discussed in this section and how heterochromatin-like domains contribute to biological functions and evolution.
Decision letter (RSOS-191976.R0) 08-Jan-2020 Dear Dr Singh, The editors assigned to your paper ("On the relation of phase separation and Hi-C maps to epigenetics") have now received comments from reviewers.
Both reviewers are very positive about publication of your review, but each of them has some substantive suggestions for improving the paper before we can proceed to consider accepting the manuscript. We would like you to revise your paper in accordance with the referee suggestions which can be found below (not including confidential reports to the Editor). Please note this decision does not guarantee eventual acceptance.
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• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) Singh and Newman provide a review of the state of the knowledge regarding the 3D conformation of chromosomes and epigenetics. I think this is a difficult task and they have done a commendable job. They do not provide new data/code/method/modelling and it appears that the only original contribution is to rename compartments in "Epigenetic compartmental domains". Because of this, I will referee this paper as if it was a review.
1. ".. could favour the merging of "clutches" to form ordered (fractal) "globules" that have been detected .. " is incorrect. Entropy works towards the equilibrium globule state. There is no direct connection between crowding/depletion attraction and fractal globule which is instead a non-equilibrium state of a polymer.
2. The authors do a very good job at connecting experiments with modelling work in many instances. They also correctly point out that LLPS is different from PPPS and that the latter is most likely the mechanism of phase separation in the nucleus.

5.
In several instances, it should be made clear that this is a review and that no new data is presented but simply reported from previous papers or obtained from online tools such as the genome browser.
6. Do the authors have permission to reuse Figs.7A-E and other panels that are taken from published work?
Reviewer: 2 Comments to the Author(s) Singh and Newman (MS ID#RSOS-191976) In this review article, the authors describe how heterochromatin protein 1 (HP1) controls/orchestrates heterochromatin formation or heterochromatin compartmentalization. Especially, they propose that polymer-polymer phase separation (PPPS) is the major driving force of heterochromatin compartmentalization induced by HP1. Also, their interpretation of the Hi-C A/B compartment data in cohesion depleted cells is interesting and quite insightful. This article is definitely general interest to wide range of biologists and valuable to publish in ROYAL SOCIETY OPEN SCIENCE. The reviewer hopes the authors should address following comments before publication, 1. PPPS vs LLPS In page 6, line 6-11 The authors said that "if HP1a does drive phase separation it does so in a manner different from multivalent intrinsically-disordered proteins [63], which are known to form endogenous liquidliquid phase separated condensates in the nucleus [70]. Based on these data we propose an alternative mechanism. Mammalian HP1 proteins drive phase separation by polymer-polymer phase separation (PPPS; [71]) rather than by LLPS." The reviewer thinks this interpretation is too biased. Reference [70] showed that only HP1a forms liquid droplets in vitro, but this does not deny the possibility of other HP1 isoforms as a critical component of phase-separated heterochromatin compartment induced by LLPS. Indeed, reference [69] showed such possibility. Also, LLPS-mediated but different compartments exist, suggesting that different intrinsically-disordered proteins which contribute to LLPS not always form same droplet/compartment. Data of ref [70] also does not deny that HP1-mediated heterochromatin may exist as LLPS or formation of LLPS may contribute to function or maintenance of heterochromatin. Therefore, the PPPS story is fine and interesting, but the authors should more carefully describe current situation and cite recent work of HP1 function in LLPS appropriately. Also, most recent Narlikar's nature paper (Sanulli et al 2019) should be integrated into this article.
2. Fig. 7 Page 14 first paragraph. The authors should cite more details of each panel for description of Fig.  7 to help understanding the authors points. Also, if any paper shows that HP1 level on H3K9me2/3+ high regions in compartment A is lower than that in compartment B, should be cited because this support the authors' Fig7 model.

conclusions and perspectives
It is nice to add one additional fig to show some of issues discussed in this section and how heterochromatin-like domains contribute to biological functions and evolution.

Author's Response to Decision Letter for (RSOS-191976.R0)
See Appendix A.

Recommendation?
Accept with minor revision (please list in comments)

Comments to the Author(s)
The authors have made an effort to address some of my comments. Still, I feel that this is a strange review in that it is not limited to provide an overview of current work in the field but also appears to propose new models. Some of them are not new although they are presented as such using misleading wording, e.g. ".. in our scheme .." pg 12 line 4, "we propose an alternative mechanism .." pg 6 ln 9, "we suggest that tethering .." pg 17 ln 35, "we suggest that the B-type compartmental ..." pg 19 ln 50, etc). I feel that I would not do my job as a reviewer if I didn't point out that the authors should be clear and careful in attributing merit. I'd warmly encourage the authors to reword ambiguous sentences, such as, -we suggest that tethering -> It was recently suggested that tethering ... -we propose an alternative mechanism -> A an alternative mechanism was proposed -in our scheme -> remove -we suggest that the B-type compartmental .. -> Existing models suggest that B-compartments may be associated to epigenetic marks such as HP1 and Pc (and add references to, e.g., Rao Cell 2014, di Pierro PNAS 2017, Michieletto PRL 2019, ..., where the idea of the relationship between compartments and epigenetics is actually being tested experimentally and with computational models).

Are the interpretations and conclusions justified by the results? No
Is the language acceptable? Yes

Do you have any ethical concerns with this paper? No
Have you any concerns about statistical analyses in this paper? No

Recommendation?
Accept as is

Comments to the Author(s)
Unfortunately the author basically ignore the comment 1 which is only the critical issue from the reviewer. " the authors should more carefully describe current situation and cite recent work of HP1 function in LLPS appropriately." No further comment.
Decision letter (RSOS-191976.R1) 27-Jan-2020 Dear Dr Singh: On behalf of the Editors, I am pleased to inform you that your Manuscript RSOS-191976.R1 entitled "On the relations of phase separation and Hi-C maps to epigenetics" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email.
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• Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list.
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• Authors' contributions All submissions, other than those with a single author, must include an Authors' Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published.
All contributors who do not meet all of these criteria should be included in the acknowledgements.
We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
• Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria.
• Funding statement Please list the source of funding for each author.
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Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch. Comments to the Author(s) Unfortunately the author basically ignore the comment 1 which is only the critical issue from the reviewer. " the authors should more carefully describe current situation and cite recent work of HP1 function in LLPS appropriately." No further comment.

Reviewer: 1
Comments to the Author(s) The authors have made an effort to address some of my comments. Still, I feel that this is a strange review in that it is not limited to provide an overview of current work in the field but also appears to propose new models. Some of them are not new although they are presented as such using misleading wording, e.g. ".. in our scheme .." pg 12 line 4, "we propose an alternative mechanism .." pg 6 ln 9, "we suggest that tethering .." pg 17 ln 35, "we suggest that the B-type compartmental ..." pg 19 ln 50, etc). I feel that I would not do my job as a reviewer if I didn't point out that the authors should be clear and careful in attributing merit. I'd warmly encourage the authors to reword ambiguous sentences, such as, -we suggest that tethering -> It was recently suggested that tethering ... -we propose an alternative mechanism -> A an alternative mechanism was proposed -in our scheme -> remove -we suggest that the B-type compartmental .. -> Existing models suggest that B-compartments may be associated to epigenetic marks such as HP1 and Pc (and add references to, e.g., Rao Cell 2014, di Pierro PNAS 2017, Michieletto PRL 2019, ..., where the idea of the relationship between compartments and epigenetics is actually being tested experimentally and with computational models).

03-Feb-2020
Dear Dr Singh, It is a pleasure to accept your manuscript entitled "On the relations of phase separation and Hi-C maps to epigenetics" in its current form for publication in Royal Society Open Science. There were no further comments from reviewers or editors on your manuscript.
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Thank you for your fine contribution. On behalf of the Editors of Royal Society Open Science, we look forward to your continued contributions to the Journal. b. An important work connecting co-polymer models with epigenetics is that of Daniel Jost et al NAR 2014. It was the first to my knowledge to make this connection clear. We are grateful to the reviewer for suggesting these models and are respectful of his/her scholarship. For what it's worth, some models regarding HP1 and Pc proteins were proposed by ourselves during an earlier epoch (Singh and Huskisson, Developmental Genetics, 1998). Given that we already have ~250 references we focussed on those references suggested by reviewer 1 that are key. Accordingly, we have added Barbieri et al PNAS 2012 andDaniel Jost et al NAR 2014 to the references at references 87 and 165 respectively. 4. Cross-linking typically has an irreversible connotation so I would not use it to describe the reversible bridging of HP1 or other proteins on the chromatin. Accepted. We have used "bridging" instead of "cross-linking" where appropriate in the text.

5.
In several instances, it should be made clear that this is a review and that no new data is presented but simply reported from previous papers or obtained from online tools such as the genome browser.
With all due respects to the reviewer, this is an odd request. Yes, the paper is submitted as a review; the cover to the manuscript, generated by the journal, states under "Article type" that it is.
That said, in keeping with the guidelines to authors, it is more than a simple review: it is a synthesis that has explanatory force and makes specific testable predictions.
The synthesis presented is founded upon mammalian HP1 proteins and H3K9me2/3-marked heterochromatin-like domains/complexes they assemble (see Table 1), which places current knowledge on phase separation and Hi-C maps in mammalian cells within a single paradigm that can be tested experimentally. It has explanatory force and posits testable predictions for a variety of chromatin-templated phenomena ranging from cytologically-visible compartmentalisation, through epigenetic compartmental domains and the mechanisms that maintain cellular identity to the evolution of phylotypic restriction during mammalian development.
Our synthesis enabled us, for the first time, to make a prediction as to the contacts that likely result in macro-and micro-phase separation by PPPS. We have also confirmed that there are likely to be enough nucleation sites within the B4 sub-compartment for it to be inherited as an epigenetic domain.
Experiment will determine the veracity of this paradigm.
6. Do the authors have permission to reuse Figs.7A-E and other panels that are taken from published work?
Yes. Panel A of Figure 1 is taken and modified from Hiragami-Hamada, et al., (2016) and is used according to the Creative Commons License CC BY that allows for maximum dissemination and reuse of open access materials. The source is properly acknowledged. Panels B through to G of Figure 1 are from Billur et al., (2010). PBS is corresponding author on that paper and re-use was granted by Elsevier through Copyright Clearance Center's RightsLink service.

Reviewer: 2
Comments to the Author(s) Singh and Newman (MS ID#RSOS-191976) In this review article, the authors describe how heterochromatin protein 1 (HP1) controls/orchestrates heterochromatin formation or heterochromatin compartmentalization. Especially, they propose that polymer-polymer phase separation (PPPS) is the major driving force of heterochromatin compartmentalization induced by HP1. Also, their interpretation of the Hi-C A/B compartment data in cohesion depleted cells is interesting and quite insightful. This article is definitely general interest to wide range of biologists and valuable to publish in ROYAL SOCIETY OPEN SCIENCE. The reviewer hopes the authors should address following comments before publication, Accepted. We would add that it is only because we propose that HP1 proteins regulate PPPS that it is possible to gain insight into the changes observed in "the Hi-C A/B compartments data in cohesion (sic) depleted cells".

PPPS vs LLPS
In page 6, line 6-11 The authors said that "if HP1a does drive phase separation it does so in a manner different from multivalent intrinsically-disordered proteins [63], which are known to form endogenous liquidliquid phase separated condensates in the nucleus [70]. Based on these data we propose an alternative mechanism. Mammalian HP1 proteins drive phase separation by polymer-polymer phase separation (PPPS; [71]) rather than by LLPS." The reviewer thinks this interpretation is too biased. Reference [70] showed that only HP1a forms liquid droplets in vitro, but this does not deny the possibility of other HP1 isoforms as a critical component of phase-separated heterochromatin compartment induced by LLPS. Indeed, reference [69] showed such possibility. Also, LLPS-mediated but different compartments exist, suggesting that different intrinsically-disordered proteins which contribute to LLPS not always form same droplet/compartment. Data of ref [70] also does not deny that HP1-mediated heterochromatin may exist as LLPS or formation of LLPS may contribute to function or maintenance of heterochromatin. Therefore, the PPPS story is fine and interesting, but the authors should more carefully describe current situation and cite recent work of HP1 function in LLPS appropriately. Also, most recent Narlikar's nature paper (Sanulli et al 2019) should be integrated into this article.
With all due respects to reviewer 2, reviewer 1 agrees with us (see point 2 of reviewer 1 above). Also, as mentioned above, it is only because we propose that HP1 proteins mediate PPPS that it is possible to gain insight into the changes observed in "the Hi-C A/B compartments data in cohesion (sic) depleted cells".
Nevertheless, there is some merit to reviewer 2's comments and in order to assuage his/her concerns we have added the following sentences: "These data indicate that phase separation of mammalian constitutive heterochromatin is unlikely to be mediated by HP1-driven LLPS, albeit work in Drosophila (Strom et al., 2017;[70]) and fission yeast (Sanulli et al., 2019;[71]) shows that HP1 proteins can drive LLPS. Rather, we suggest that the major mechanism by which mammalian HP1 proteins drive phase separation is polymer-polymer phase separation (PPPS; [72]) as opposed to LLPS." 2. Fig. 7 Page 14 first paragraph. The authors should cite more details of each panel for description of Fig. 7 to help understanding the authors points. Also, if any paper shows that HP1 level on H3K9me2/3+ high regions in compartment A is lower than that in compartment B, should be cited because this support the authors' Fig7 model.
We are grateful for the reviewer's comment. We have provided very detailed legends to Figure 7 (over a page long). Also, the paragraph that goes over the page, from page 14 to 15, describes the panels in detail; we have tried to make this accessible by limiting the mathematical treatment and giving clear and simple explanations of the panels. At the risk of repeating ourselves and taking away from the clarity we not added more to the first paragraph.
We agree that one test of our model would be to measure HP1 proteins on H3K9me2/3-marked nucleosomes in A and B compartments in wt cells and to compare this to HP1 at the same H3K9me2/3-marked nucleosomes after cohesin depletion. This experiment is beyond the scope of the current review.

conclusions and perspectives
It is nice to add one additional fig to show some of issues discussed in this section and how heterochromatin-like domains contribute to biological functions and evolution.
With all due respects to this reviewer, we appreciate the opportunity to add another figure but feel that the paper is already too long.