Analysis of distribution of chromatin marks across " divergence islands " in three-spined stickleback ( ) Gasterosteus aculeatus

The three-spined stickleback ( ) is a well-known Gasterosteus aculeatus model organism for studying adaptations to water salinity. In this work, we investigate the dynamics of an epigenetic landscape of water salinity adaptation using three chromatin marks: H3K27ac, H3K4me1 and H3K4me3. The choice of marks was determined by the fact that some adaptive genomic loci are situated in gene-free regions, suggesting their regulatory role as enhancers. Histone modifications seem to be a promising mechanism that could regulate such regions. Difference between histone modifications in sea and freshwater both in genes and intergenic enhancers may contribute to epigenetic plasticity of stickleback adaptation. As a result of this study, we found differential chromatin peaks in "divergence islands" at enhancer elements and promoters of genes, which are responsible for stress adaptation and homeostasis. However, a full genome study analysis is required to fully understand mechanism of adaptation to water salinity.


Introduction
The three-spined stickleback (Gasterosteus aculeatus) is a model organism that can be used to elucidate molecular mechanisms of adaption to various salinities.In this work, we analyze epigenetic signatures that can be specific for various salinities.We study three chromatin marks H3K27ac, H3K4me1 and H3K4me3, which were chosen as these marks label active enhancers and promoters 1 , in marine and fresh water sticklebacks in their natural habitats, as well as in foreign (change of salinity) environments.These marks were studied in 19 "divergence islands" 2 , short genomic regions, which are highly diverged between marine and fresh water species.In the present study, 17 divergence islands overlapped in protein-coding genes, which are relevant to fresh water adaptation.In addition, we report the changes of histone modifications between marine and freshwater fish in promoters of protein-coding genes and enhancers.

Sample preparation and ChIP experiment
In this study, we used six marine and six fresh water sticklebacks, which were collected in august of 2015 from the White Sea (near the Pertsov White Sea Biological Station of Lomonosov Moscow State University, Murmanskaya Oblast, Russia) and Mashinnoe Lake (located near the village of Chkalvosky, Repuyblic of Karelia, Russia), respectively.The fish were placed for four days in various water tanks: half of the sticklebacks from each group was kept in the water with their natural salinity (FF and MM for fresh and marine water, respectively) and the other half was kept in a modified environment (i.e. three fresh water fish was placed in salty water (FM) and three marine fish were placed in fresh water (MF)).The live fish were transferred to the laboratory to Moscow.
For the chromatin immunoprecipitation (ChIP)-seq experiments, gills were collected from all 12 sticklebacks.Chromatin was prepared from gills, as described by Cell Signalling (https://www.cellsignal.com/common/content/content.jsp?id=chip-agarose) and ChIP was performed as described by Filion et al. 3 .

Results
In our study, we determined between 7138 and 20828 histone modification peaks (Table 1).We selected histone modification peaks in "divergence islands" regions, which are highly divergent between marine and freshwater populations of sticklebacks 2 .We observed that the majority (17 out of 19) of the islands showed the same chromatin marks (H3K4me1, H3K4me3 and H3K27ac) in fresh water species in their natural salinity (FF) and fresh water species placed into a marine environment (FM).The same was true for marine water species in their natural salinity (MM) and marine water species placed into a fresh water environment (MF).In addition, the majority of the islands (14 out of 19) showed the same histone modifications between the whole set of marine and fresh water species (MM+MF vs. FF+FM; Table 2).
Nevertheless, we found 3 out of 19 "divergence islands" demonstrating differential chromatin marks in cases of short-term adaptation to water salinity for fish placed into foreign environments.Interestingly, one "divergence island" gained H3K4me1 in the promoter of the RPTOR gene in FM.The other two islands gained H3K27ac and lost H3K4me1 inf FM, suggesting their role as enhancers for genes outside the island.Also, two islands gained H3K4me3 and H3K4me1 at STC2 and PNPLA3 genes, respectively, in MF.
Finally, 5 out of 19 islands demonstrated differential histone modifications between fresh water species and marine species placed into fresh water (FF vs MF) (Table 3).In all these cases, MF gained a mark, which was absent in a FF.For example, H3K4me1 was gained in LRRC59 and BDH2 genes, which is involved in the "0" -no intersection; "1" -intersection.Regions with differential chromatin peaks are highlighted.MM -marine species in natural environment; MF -marine species in fresh water; FF -fresh water species in natural environment; FM -fresh water species in marine water.Differential peaks islands are highlighted in red.

Table 3. Results of intersection of chromatin peaks with divergence islands and differential chromatin peaks analysis between FM and MM, and between MF and FF.
"0" -no intersection; "1" -intersection.Regions with differential chromatin peaks are highlighted.MM -marine species in natural environment; MF -marine species in fresh water; FF -fresh water species in natural environment; FM -fresh water species in marine water.Differential peaks islands are highlighted in red.adaption to stress and homeostasis 8 , suggesting that these genes might be activated after the placement of marine fish into a fresh water environment.In addition, 6 islands out of 19 demonstrated differential histone modifications between marine and fresh water species placed in marine water, with both gains and losses of marks (Table 3).Among these, we found that H3K4me3 was gained at STC2 in FM* compared to MM*, H3K4me1 at LRRC59 and BDH2 in MM* compared with FM*, and H3K27ac at RPTR in MM* compared with FM*.

Conclusions
In this study, we analyzed the epigenetic profile of "divergence islands" with three chromatin marks, H3K4me1, H3K4me3 and H3K27ac.We report differential histone modifications that might be involved in the regulation of promoters and enhancers located in "divergent islands", and therefore contribute to adaptation to water salinity.Furthermore, we found differential chromatin peaks at promoters of genes that are responsible for stress adaptation and homeostasis.The results of this study contributes to our understanding of molecular mechanisms of adaptation to water salinity.However, a full genome histone modification analysis is required in order to further understand these mechanisms of adaptation.

Introduction
The introduction needs to be expanded so that the authors give some background on the system as well, there is plenty of previous research on the stickleback system that has been done and more of it needs to be cited here.Please also explain the ideas behind the divergence islands, give some information about these chromatin marks etc.

Methods
More information needs to be given about the chromatin immunoprecipitation experiments and the bioinformatic analysis.Currently the methods do not stand on their own.For example, at the moment the manuscript only states that MACS software was used for peak calling with no information how the method works or what parameters were used.

Results
I was wondering would it be helpful if the some of the results were shown as figures.Perhaps at least those peaks where differences were found.

Discussion
Currently the results are not really discussed at all.The authors should properly discuss their results.The authors found changes in certain genes, but the biological functions of those genes are barely mentioned or whether these are candidates for explaining adaptation to marine and freshwater environments.
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.Referee Report https://doi.org/10.5256/f1000research.11237.r18627

Alexey Ruzov
Division of Cancer and Stem Cells, Centre for Biomolecular Sciences (CBS), School of Medicine, University of Nottingham, Nottingham, UK In my opinion, the results on differential distribution of histone modifications in sea and freshwater three-spined sticklebacks may potentially be interesting.Despite this, I strongly believe that text of the manuscript is currently beyond the minimal standards required for paper indexing.
Specifically: the abstract does not describe main findings of the paper in sufficient detail; the introduction does not provide an adequate background information for the study but, instead, mainly repeats the abstract; any Discussion comparing the results with already available literature is missing; some crucial details of the methodologies used (e.g. which antibodies have been used for CHIP) are missing from Methods; some sentences in the text are difficult to understand.I recommend the authors to rewrite the text of manuscript addressing these points.
No competing interests were disclosed.

Competing Interests:
I have read this submission.I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative Your article is indexed in PubMed after passing peer review Dedicated customer support at every stage For pre-submission enquiries, contact research@f1000.com Sokolov A, Zhenilo S, Rastorguev S How to cite this article: et al.Analysis of distribution of chromatin marks across "divergence islands"

Table 1 . Results of reads mapping and peaks calling
. MM -marine species in natural environment; MF -marine species in fresh water; FF -fresh water species in natural environment; FM -fresh water species in marine water.