F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.10428.1 Research Note Articles Marine & Freshwater Ecology Analysis of distribution of chromatin marks across "divergence islands" in three-spined stickleback ( Gasterosteus aculeatus)

[version 1; peer review: 2 approved with reservations]

 Sokolov Alexey https://orcid.org/0000-0002-3387-0649 a 1 Zhenilo Svetlana 1 Rastorguev Sergey 2 Mazur Alexander 1 Prokhortchouk Egor 1 Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russian Federation National Research Center 'Kurchatov Institute', Moscow, Russian Federation sokolovbiotech@gmail.com

SR designed the experiment. SZ and AM prepared ChIP-seq libraries. AS and EP carried out the research. AS wrote the manuscript.

Competing interests: The authors declare no conflict of interest.

 20 12 2016 2016 5 2880 19 12 2016 Copyright: © 2016 Sokolov A et al. 2016 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The three-spined stickleback ( Gasterosteus aculeatus) is a well-known 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.

 divergence islands chromatin marks Gasterosteus aculeatus Russian Science Foundation 14-24-00175 This work was supported by the Russian Science Foundation (RSF; grant #14-24-00175). 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.

 Methods 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 .

 Bioinformatics analysis

Reads were aligned to gasAcu1 reference genome from the UCSC Genome Browser Gateway ( https://genome-euro.ucsc.edu/) by Bowtie2 ( http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) with a "very-sensitive-local" parameter 4, 5 . For peaks calling we used MACS version 1.4.2 6 . For the intersection of peaks with divergence islands and genes we used bedtools version 2.26.0 7 .

 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).

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.

SamFle Name All reads Mapped reads Ratio Peaks
MMH3K27ac 16,319,162 13,242,763 81.14854795 11906
MMH3K4me1 15,805,735 12,244,401 77.46808991 13289
MMH3K4me3 7,354,166 6,872,442 93.44964473 12944
MFH3K27ac 16,917,583 9,700,338 57.33879361 13964
MFH3K4me1 12,773,849 10,303,455 80.66053544 20828
MFH3K4me3 14,099,289 7,836,369 55.57988775 18851
FFH3K27ac 14,567,425 11,367,244 78.0319377 9130
FFH3K4me1 12,243,305 10,737,653 87.70224216 8825
FFH3K4me3 15,176,697 9,476,326 62.43997623 14672
FMH3K27ac 14,850,800 11,887,597 80.04684596 12889
FMH3K4me1 13,213,512 10,426,786 78.91002786 7138
FMH3K4me3 14,512,630 11,658,225 80.33158015 15723
Results of intersection of chromatin peaks with divergence islands and differential chromatin peaks analysis between MM and MF, and between FF and FM.

“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.

Divergence islands coordinates MM MF FF FM
Chromosome Start End H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3
chrI 21,487,998 21,960,119 1 1 1 1 1 1 1 1 1 1 1 1
chrII 14,874,366 14,898,826 0 0 0 0 0 0 0 0 0 0 0 0
chrIV 12,803,780 12,881,296 1 1 1 1 1 1 1 1 1 1 1 1
chrIV 13,930,002 13,959,331 0 1 0 0 1 1 0 1 1 0 1 1
chrIV 19,811,922 19,914,666 1 1 1 1 1 1 1 1 1 1 1 1
chrIV 23,954,634 23,981,981 1 0 1 1 1 1 1 0 1 1 0 1
chrIV 26,016,955 26,166,536 1 1 1 1 1 1 1 1 1 1 1 1
chrV 2,482,209 2,501,295 1 1 1 1 1 1 1 0 1 1 0 1
chrVII 17,982,351 18,002,671 1 1 1 1 1 1 1 1 1 1 1 1
chrIX 8,521,935 8,537,559 1 1 1 1 1 1 1 1 1 1 0 1
chrIX 8,901,816 8,910,115 1 1 1 1 1 1 0 0 1 1 0 1
chrIX 9,208,158 9,227,809 0 0 0 0 0 0 0 0 0 0 0 0
chrIX 10334101 10353801 1 1 1 1 1 1 0 0 1 0 1 1
chrXI 5445757 5855124 1 1 1 1 1 1 1 1 1 1 1 1
chrXII 14338229 14358336 1 1 1 1 1 1 1 0 1 1 0 1
chrXII 16522028 16538810 0 0 0 0 0 0 0 0 0 0 0 0
chrXIX 2449903 2581858 1 1 1 1 1 1 1 1 1 1 1 1
chrXIX 14787904 14799088 0 0 0 0 0 0 0 0 0 0 0 0
chrXXI 5759879 7486635 1 1 1 1 1 1 1 1 1 1 1 1

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 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*.

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.

Divergence islands coordinates FM MM MF FF
Chromosome Start End H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3 H3K27ac H3K4me1 H3K4me3
chrI 21,487,998 21,960,119 1 1 1 1 1 1 1 1 1 1 1 1
chrII 14,874,366 14,898,826 0 0 0 0 0 0 0 0 0 0 0 0
chrIV 12,803,780 12,881,296 1 1 1 1 1 1 1 1 1 1 1 1
chrIV 13,930,002 13,959,331 0 1 1 0 1 0 0 1 1 0 1 1
chrIV 19,811,922 19,914,666 1 1 1 1 1 1 1 1 1 1 1 1
chrIV 23,954,634 23,981,981 1 0 1 1 0 1 1 1 1 1 0 1
chrIV 26,016,955 26,166,536 1 1 1 1 1 1 1 1 1 1 1 1
chrV 2,482,209 2,501,295 1 0 1 1 1 1 1 1 1 1 0 1
chrVII 17,982,351 18,002,671 1 1 1 1 1 1 1 1 1 1 1 1
chrIX 8,521,935 8,537,559 1 0 1 1 1 1 1 1 1 1 1 1
chrIX 8,901,816 8,910,115 1 0 1 1 1 1 1 1 1 0 0 1
chrIX 9,208,158 9,227,809 0 0 0 0 0 0 0 0 0 0 0 0
chrIX 10334101 10353801 0 1 1 1 1 1 1 1 1 0 0 1
chrXI 5445757 5855124 1 1 1 1 1 1 1 1 1 1 1 1
chrXII 14338229 14358336 1 0 1 1 1 1 1 1 1 1 0 1
chrXII 16522028 16538810 0 0 0 0 0 0 0 0 0 0 0 0
chrXIX 2449903 2581858 1 1 1 1 1 1 1 1 1 1 1 1
chrXIX 14787904 14799088 0 0 0 0 0 0 0 0 0 0 0 0
chrXXI 5759879 7486635 1 1 1 1 1 1 1 1 1 1 1 1
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.

Data availability

Fastq files can be found in the SRA archive under accession number SRX2403902 ( https://www.ncbi.nlm.nih.gov/sra/?term=SRX2403902).

 Acknowledgements

The authors are grateful to Konstantin G. Skryabin and Yulia Medvedeva (Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences) for ongoing support and valuable comments throughout the preparation of the manuscript.

 Shlyueva D Stampfel G Stark A : Transcriptional enhancers: from properties to genome-wide predictions. Nat Rev Genet. 2014;15(4):272–286. 24614317 10.1038/nrg3682 Terekhanova NV Logacheva MD Penin AA : Fast evolution from precast bricks: genomics of young freshwater populations of threespine stickleback Gasterosteus aculeatus. PLoS Genet. 2014;10(10):e1004696. 25299485 10.1371/journal.pgen.1004696 4191950 Filion GJ Zhenilo S Salozhin S : A family of human zinc finger proteins that bind methylated DNA and repress transcription. Mol Cell Biol. 2006;26(1):169–81. 16354688 10.1128/MCB.26.1.169-181.2006 1317629 Langmead B Salzberg SL : Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–359. 22388286 10.1038/nmeth.1923 3322381 Langmead B Trapnell C Pop M : Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10(3):R25. 19261174 10.1186/gb-2009-10-3-r25 2690996 Zhang Y Liu T Meyer CA : Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9(9):R137. 18798982 10.1186/gb-2008-9-9-r137 2592715 Quinlan AR Hall IM : BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010;26(6):841–842. 20110278 10.1093/bioinformatics/btq033 2832824 Devireddy LR Hart DO Goetz DH : A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell. 2010;141(6):1006–17. 20550936 10.1016/j.cell.2010.04.040 2910436 10.5256/f1000research.11237.r20811 Reviewer response for version 1 Kronholm Ilkka 1 Referee https://orcid.org/0000-0002-4126-0250 Centre of Excellence in Biological Interactions, Department of Biological and Environmental Sciences, University of Jyväskylä, Jyväskylä, Finland

Competing interests: No competing interests were disclosed.

 20 3 2017 Copyright: © 2017 Kronholm I 2017 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The study by Sokolov et al. investigates changes in chromatin marks among saline and freshwater populations of sticklebacks. While I like the idea of the study and I do appreciate that this manuscript is intended as a short note, the current state of this manuscript is not suitable for indexing at the moment. The authors need to rewrite most of the manuscript as major revision is necessary. I've given some suggestions below.

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.

Reviewer Expertise:

NA

I confirm that I have read this submission and 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.

 10.5256/f1000research.11237.r18627 Reviewer response for version 1 Ruzov Alexey 1 Referee https://orcid.org/0000-0002-1247-6634 Division of Cancer and Stem Cells, Centre for Biomolecular Sciences (CBS), School of Medicine, University of Nottingham, Nottingham, UK

Competing interests: No competing interests were disclosed.

 3 1 2017 Copyright: © 2017 Ruzov A 2017 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

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.

Reviewer Expertise:

NA

I confirm that I have read this submission and 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.