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Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm

  • Male Reproduction: Original Article
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Abstract

Gene expression during spermatogenesis undergoes significant changes due to a demanding sequence of mitosis, meiosis, and differentiation. We investigated the contribution of H3 histone modifications to gene regulation in the round spermatids. Round spermatids were purified from rat testes using centrifugal elutriation and Percoll density-gradient centrifugation. After enzymatic chromatin shearing, immuno-precipitation using antibodies against histone marks H3k4me3 and H3K9me3 was undertaken. The immunoprecipitated DNA fragments were subjected to massive parallel sequencing. Gene expression in round spermatids and sperm was analyzed by transcriptome sequencing using next-generation sequencing methods. ChIP-seq analysis showed significant peak enrichment in H3K4me3 marks in active chromatin regions and H3K9me3 peak enrichment in repressive regions. We found 53 genes which showed overlapping peak enrichment in both H3K4me3 and H3K9me3 marks. Some of the top H3K4me3-enriched genes were involved in sperm tail formation (Odf1, Odf3, Odf4, Oaz3, Ccdc42, Ccdc63, and Ccdc181), chromatin condensation (Dync1h1, Dynll1, and Kdm3a), and sperm functions such as acrosome reaction (Acrbp and Fabp9), energy generation (Gapdhs), and signaling for motility (Tssk1b, Tssk2, and Tssk4). Transcriptome sequencing in round spermatids found 64% transcripts of the H3K4me3-enriched genes at high levels and of about 25% of H3K9me3-enriched genes at very low levels. Transcriptome sequencing in sperm found that more than 99% of the ChIP-seq corresponding transcripts were also present in sperm. H3K4me3 enrichment in the round spermatids correlates significantly with gene expression and H3K9me3 correlates with gene silencing that contribute to sperm differentiation and setting the RNA payloads of sperm.

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Data Availability

All representative data are provided within the manuscript and also in the supporting information. Sequencing data can be accessed by GEO accession number GSE151608.

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References

  1. Pandey A, Yadav SK, Vishvkarma R, Singh B, Maikhuri JP, Rajender S, Gupta, G. The dynamics of gene expression during and post meiosis sets the sperm agenda. Mol Reprod Dev 2019;

  2. Kleene KCA. possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells. Mech Dev. 2001;106:3–23.

    Article  CAS  Google Scholar 

  3. Gatewood JM, Cook GR, Balhorn R, Schmid C, Bradbury EM. Isolation of four core histones from human sperm chromatin representing a minor subset of somatic histones. J Biol Chem. 1990;265:20662–6.

    Article  CAS  Google Scholar 

  4. Wang T, Gao H, Li W, Liu C. Essential role of histone replacement and modifications in male fertility. Front Genet. 2019;8(10):962.

    Article  Google Scholar 

  5. Bao J, Bedford MT. Epigenetic regulation of the histone-to-protamine transition during spermiogenesis. Reproduction (Cambridge, England). 2016;151(5):R55.

    Article  CAS  Google Scholar 

  6. Yuen BT, Bush KM, Barrilleaux BL, Cotterman R, Knoepfler PS. Histone H3. 3 regulates dynamic chromatin states during spermatogenesis. Development. 2014;141(18):3483–94.

  7. Shilatifard A. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu Rev Biochem. 2012;81:65–95.

    Article  CAS  Google Scholar 

  8. Lauberth SM, Nakayama T, Wu X, Ferris AL, Tang Z, Hughes SH, Roeder RG. H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation. Cell. 2013;152:1021–36.

    Article  CAS  Google Scholar 

  9. Flanagan JF, Mi LZ, Chruszcz M, Cymborowski M, Clines KL, Kim Y, Minor W, Rastinejad F, Khorasanizadeh S. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature. 2005;438:1181–5.

    Article  CAS  Google Scholar 

  10. Li H, Ilin S, Wang W, Duncan EM, Wysocka J, Allis CD, Patel DJ. Molecular basis for site-specific read-out of histone H3K4me3 by the BPTF PHD finger of NURF. Nature. 2006;442:91–5.

    Article  CAS  Google Scholar 

  11. Nishioka K, Chuikov S, Sarma K, Erdjument-Bromage H, Allis CD, Tempst P, Reinberg D. Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 2002;16:479–89.

    Article  CAS  Google Scholar 

  12. Schneider R, Bannister AJ, Weise C, Kouzarides T. Direct binding of INHAT to H3 tails disrupted by modifications. J Biol Chem. 2004;279:23859–62.

    Article  CAS  Google Scholar 

  13. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K. High-resolution profiling of histone methylations in the human genome. Cell. 2007;129:823–37.

    Article  CAS  Google Scholar 

  14. Lehnertz B, Ueda Y, Derijck AA, Braunschweig U, Perez-Burgos L, Kubicek S, Chen T, Li E, Jenuwein T, Peters AH. Suv39h-mediated histone H3 lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr Biol. 2003;13:1192–200.

    Article  CAS  Google Scholar 

  15. Otani J, Nankumo T, Arita K, Inamoto S, Ariyoshi M, Shirakawa M. Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX–DNMT3–DNMT3L domain. EMBO Rep. 2009;10(11):1235–41.

    Article  CAS  Google Scholar 

  16. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature. 2009;460:473–8.

    Article  CAS  Google Scholar 

  17. Yadav SK, Pandey A, Kumar L, Devi A, Kushwaha B, Vishvkarma R, Maikhuri JP, Rajender S, Gupta G. The thermo-sensitive gene expression signatures of spermatogenesis. Reprod Biol Endocrinol. 2018;16:56.

    Article  Google Scholar 

  18. Mercier E, Droit A, Li L, Robertson G, Zhang X, Gottardo R. An integrated pipeline for the genome-wide analysis of transcription factor binding sites from ChIP-Seq. PLoS One 2011;6:e16432

  19. Yu G, Wang LG, He QY. ChIPseeker: an R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics. 2015;31:2382–3.

    Article  CAS  Google Scholar 

  20. Yu G, He QY. ReactomePA: an R/Bioconductor package for reactome pathway analysis and visualization. Mol Biosyst. 2016;12:477–9.

    Article  CAS  Google Scholar 

  21. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–7.

    Article  CAS  Google Scholar 

  22. Hou CC, Yang WX. New insights to the ubiquitin-proteasome pathway (UPP) mechanism during spermatogenesis. Mol Biol Rep. 2013;40:3213–30.

    Article  CAS  Google Scholar 

  23. Escalier D, Bai XY, Silvius D, Xu PX, Xu X. Spermatid nuclear and sperm periaxonemal anomalies in the mouse Ube2b null mutant. Mol Reprod Dev. 2003;65:298–308.

    Article  CAS  Google Scholar 

  24. Suryavathi V, Khattri A, Gopal K, Rani DS, Panneerdoss S, Gupta NJ, Chakravarty B, Deenadayal M, Singh L, Thangaraj K. Novel variants in UBE2B gene and idiopathic male infertility. J Androl. 2008;29:564–71.

    Article  CAS  Google Scholar 

  25. Yatsenko AN, Georgiadis AP, Murthy LJ, Lamb DJ, Matzuk MM. UBE2B mRNA alterations are associated with severe oligozoospermia in infertile men. Mol Hum Reprod. 2013;19:388–94.

    Article  CAS  Google Scholar 

  26. Nakamura N. Ubiquitination regulates the morphogenesis and function of sperm organelles. Cells. 2013;2:732–50.

    Article  Google Scholar 

  27. Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A, Opravil S, Doyle M, Sibilia M, Jenuwein T. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell. 2001;107:323–37.

    Article  CAS  Google Scholar 

  28. O’Carroll D, Scherthan H, Peters AH, Opravil S, Haynes AR, Laible G, Rea S, Schmid M, Lebersorger A, Jerratsch M, Sattler L, Mattei MG, Denny P, Brown SD, Schweizer D, Jenuwein T. Isolation and characterization of Suv39h2, a second histone H3 methyltransferase gene that displays testis-specific expression. Mol Cell Biol. 2000;20:9423–33.

    Article  CAS  Google Scholar 

  29. Soufi A, Donahue G, Zaret KS. Facilitators and impediments of the pluripotency reprogramming factors’ initial engagement with the genome. Cell. 2012;151:994–1004.

    Article  CAS  Google Scholar 

  30. Lesch BJ, Silber SJ, McCarrey JR, Page DC. Parallel evolution of male germline epigenetic poising and somatic development in animals. Nat Genet. 2016;48(8):888–94. Erratum in: Nat Genet. 2016;28;48(10):1296.

  31. Wang X, Kang JY, Wei L, Yang X, Sun H, Yang S, Lu L, Yan M, Bai M, Chen Y, Long J, Li N, Li D, Huang J, Lei M, Shao Z, Yuan W, Zuo E, Lu K, Liu MF, Li J. PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis. Development. 2019 10;146(13):dev175547. Erratum in: Development. 2020;147(8): PMID: 31189663.

  32. Maezawa S, Hasegawa K, Yukawa M, Kubo N, Sakashita A, Alavattam KG, Sin HS, Kartashov AV, Sasaki H, Barski A, Namekawa SH. Polycomb protein SCML2 facilitates H3K27me3 to establish bivalent domains in the male germline. Proc Natl Acad Sci U S A. 2018;115(19):4957–62.

    Article  CAS  Google Scholar 

  33. Erkek S, Hisano M, Liang CY, Gill M, Murr R, Dieker J, Schübeler D, van der Vlag J, Stadler MB, Peters AH. Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa. Nat Struct Mol Biol. 2013;20(7):868–75. Erratum in: Nat Struct Mol Biol. 2013 ;20(10):1236.

  34. Liu Y, Zhang Y, Yin J, Gao Y, Li Y, Bai D, He W, Li X, Zhang P, Li R, Zhang L, Jia Y, Zhang Y, Lin J, Zheng Y, Wang H, Gao S, Zeng W, Liu W. Distinct H3K9me3 and DNA methylation modifications during mouse spermatogenesis. J Biol Chem. 2019;294(49):18714–25.

    Article  Google Scholar 

  35. Bryant JM, Donahue G, Wang X, Meyer-Ficca M, Luense LJ, Weller AH, Bartolomei MS, Blobel GA, Meyer RG, Garcia BA, Berger SL. Characterization of BRD4 during mammalian postmeiotic sperm development. Mol Cell Biol. 2015;35(8):1433–48.

    Article  CAS  Google Scholar 

  36. Teperek M, Simeone A, Gaggioli V, Miyamoto K, Allen GE, Erkek S, Kwon T, Marcotte EM, Zegerman P, Bradshaw CR, Peters AH, Gurdon JB, Jullien J. Sperm is epigenetically programmed to regulate gene transcription in embryos. Genome Res. 2016;26:1034–46.

    Article  CAS  Google Scholar 

  37. Oikawa M, Simeone A, Hormanseder E, Teperek M, Gaggioli V, O’Doherty A, Falk E, Sporniak M, D’Santos C, Franklin VN, Kishore K. Epigenetic homogeneity in histone methylation underlies sperm programming for embryonic transcription. Nat Commun. 2020;11(1):1–6.

    Article  Google Scholar 

  38. Harbuz R, Zouari R, Pierre V, Ben Khelifa M, Kharouf M, Coutton C, Merdassi G, Abada F. Escoffier J, Nikas Y, Vialard F, Koscinski I, Triki C, et al. A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation. Am J Hum Genet 2011;88:351–361

  39. Doran J, Walters C, Kyle V, Wooding P, Hammett-Burke R, Colledge WH. Mfsd14a (Hiat1) gene disruption causes globozoospermia and infertility in male mice. Reproduction. 2016;152:91–9.

    Article  CAS  Google Scholar 

  40. Takasaki N, Tachibana K, Ogasawara S, Matsuzaki H, Hagiuda J, Ishikawa H, Mochida K, Inoue K, Ogonuki N, Ogura A, Noce T, Ito C, Toshimori K, Narimatsu H. A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility. Proc Natl Acad Sci U S A. 2014;111:1120–5.

    Article  CAS  Google Scholar 

  41. Escoffier J, Lee HC, Yassine S, Zouari R, Martinez G, Karaouzene T, Coutton C, Kherraf ZE, Halouani L, Triki C, Nef S, Thierry-Mieg N, Savinov SN, Fissore R, Ray PF, Arnoult C. Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP. Hum Mol Genet. 2016;25:878–91.

    Article  CAS  Google Scholar 

  42. Zhu F, Liu C, Wang F, Yang X, Zhang J, Wu H, Zhang Z, He X, Zhang Z, Zhou P, Wei Z, Shang Y, Wang L, Zhang R, Ouyang YC, Sun QY, Cao Y, Li W. Mutations in PMFBP1 cause acephalic spermatozoa syndrome. Am J Hum Genet. 2018;103:188–99.

    Article  CAS  Google Scholar 

  43. Okutman O, Muller J, Skory V, Garnier JM, Gaucherot A, Baert Y, Lamour V, Serdarogullari M, Gultomruk M, Ropke A, Kliesch S, Herbepin V, et al. A no-stop mutation in MAGEB4 is a possible cause of rare X-linked azoospermia and oligozoospermia in a consanguineous Turkish family. J Assist Reprod Genet. 2017;34:683–94.

    Article  Google Scholar 

  44. Jimenez A, Zu W, Rawe VY, Pelto-Huikko M, Flickinger CJ, Sutovsky P, Gustafsson JA, Oko R, Miranda-Vizuete A. 2Spermatocyte/spermatid-specific thioredoxin-3, a novel Golgi apparatus-associated thioredoxin, is a specific marker of aberrant spermatogenesis. J Biol Chem. 2004;279:34971–82.

    Article  CAS  Google Scholar 

  45. Esfahani MH, Abbasi H, Mirhosseini Z, Ghasemi N, Razavi S, Tavalaee M, Tanhaei S, Deemeh MR, Ghaedi K, Zamansoltani F, Rajaei F. Can altered expression of hspa2 in varicocele patients lead to abnormal spermatogenesis? International Journal of Fertility and Sterility. 2010;4(3):104–13.

    Google Scholar 

  46. Motiei M, Tavalaee M, Rabiei F, Hajihosseini R, Nasr-Esfahani MH. Evaluation of HSPA 2 in fertile and infertile individuals. Andrologia. 2013;45(1):66–72.

    Article  CAS  Google Scholar 

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Funding

The authors received funding from the Council of Scientific and Industrial Research (CSIR) under the network scheme of projects (BSC0101). Poonam Mehta received a graduate fellowship from the University Grants Commission (460/CSIR-UGC NET DEC.2017).

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Authors and Affiliations

  1. Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, India

    Saumya Sarkar, Santosh Yadav, Poonam Mehta, Gopal Gupta & Singh Rajender

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

    Poonam Mehta, Gopal Gupta & Singh Rajender

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  1. Saumya Sarkar
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  2. Santosh Yadav
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  3. Poonam Mehta
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  4. Gopal Gupta
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  5. Singh Rajender
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Contributions

SS, RS and GG have conceived the idea. SS, SY and RS have performed the experimental procedures. SS, PM and RS have analyzed the data. SS, PM and RS wrote the manuscript.

Corresponding author

Correspondence to Singh Rajender.

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The study was approved by the institutional ethics committee of CSIR-CDRI (IAEC/2014/49/Renew03(135/16)) and the study was performed in accordance with the ethical standards as mentioned in the 1964 Declaration of Helsinki and its later amendments.

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The authors declare no competing interests.

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Council of Scientific and Industrial Research,India,BSC0101,Singh Rajender

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Sarkar, S., Yadav, S., Mehta, P. et al. Histone Methylation Regulates Gene Expression in the Round Spermatids to Set the RNA Payloads of Sperm. Reprod. Sci. 29, 857–882 (2022). https://doi.org/10.1007/s43032-021-00837-3

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  • DOI: https://doi.org/10.1007/s43032-021-00837-3

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