Skip to main content
SpringerLink
Log in

G9a co-localized with histone H3 lysine 9 monomethylation but not dimethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development

  • Epigenetics
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

Histone H3 lysine 9 (H3K9) methylation plays an important role in the regulation of preimplantation embryo development. G9a has been reported to be a major H3K9mono (m1)/dimethylation(m2) methyltransferase and to contain nuclear localization signals. This study was performed to investigate the correlation between H3K9 methylation level and G9a localization when the nuclear membrane undergoes periodic reconstruction in the cell cycle during preimplantation embryo development.

Methods

The fluorescence intensity was examined via immunofluorescence. The mRNA expression of G9awas determined using real-time reverse transcriptase (RT)-PCR. Eight-cell embryos were cultured in KSOM supplemented with nocodazole (0.5 μM) for 12 h.

Results

In this study, it was observed that the fluorescence intensity of H3K9m2 and G9a began to increase significantly from the 4-cell stage and reached the peak at the morula stage (p < 0.001), but the fluorescence intensity declined to 4-cell-stage levels when it reached the blastula stage. We observed a similar pattern when we examined G9a mRNA expression. Once the nuclear membrane disintegrated, G9a and H3K9m1 were not detectable by immunofluorescence; when it was reconstructed, G9a and H3K9m1 had relocated to the cell nucleus. However, no significant change was observed in the H3K9m2 localization or in the G9a mRNA level (p > 0.05) during the whole process. JHDM2A was consistently localized in the cytoplasm irrespective of the presence or absence of a nuclear membrane.

Conclusion

These results indicate dynamic changes in the expression level of H3K9m2 and G9a as preimplantation embryogenesis progresses. G9a co-localized with H3K9 m1 in a nuclear membrane-dependent manner during mouse preimplantation embryo development.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Strahl B, Allis C. The language of covalent histone modifications. Nature. 2000;403:41–5.

    Article  PubMed  CAS  Google Scholar 

  2. Margueron R, Trojer P, Reinberg D. The key to development: interpreting the histone code? Curr Opin Genes Dev. 2005;15:163–76.

    Article  CAS  Google Scholar 

  3. Loh YH, Zhang WW, Chen X, George J, Ng HH. Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes Dev. 2007;21:2545–57.

    Article  PubMed  CAS  Google Scholar 

  4. Cabot R. Chromatin remodeling and embryo development. Biol Reprod. 2010;83:98.

    Google Scholar 

  5. Pickard B, Dean W, Engemann S, Bergmann K, Fuermann M, Jung M, Reis A, Allen N, Piotrowska K, Modliński JA, Korwin-Kossakowski M, Karasiewicz J. Effects of preactivation of ooplasts or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos. Biol Reprod. 2000;63:677–82.

    Article  Google Scholar 

  6. Wu SC, Zhang Y. Active DNA demethylation: many roads lead to rome. Nat Rev Mol Cell Biol. 2010;9:607–20.

    Article  Google Scholar 

  7. Tachibana M, Sugimoto K, Fukushima T, Shinkai Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem. 2001;276:25309–17.

    Article  PubMed  CAS  Google Scholar 

  8. Estève PO, Chin HG, Smallwood A, Feehery GR, Gangisetty O, Karpf AR, Carey MF, Pradhan S. Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Gene Dev. 2006;20:3089–103.

    Article  PubMed  Google Scholar 

  9. Myant K, Termanis A, Sundaram AY, Boe T, Li C, Merusi C, Burrage J, de Las Heras JI, Stancheva I. LSH and G9a/GLP complex are required for developmentally programmed DNA methylation. Genome Res. 2011;21:83–94.

    Article  PubMed  CAS  Google Scholar 

  10. Kubicek S, O’Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Leung DC, Dong KB, Maksakova IA, Goyal P, Appanah R, Lee S, Tachibana M, Shinkai Y, Lehnertz B, Mager DL, Rossi F, Lorincz MC. Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing. PNAS. 2011;108:5718–23.

    Article  Google Scholar 

  11. Collins RE, Tachibana M, Tamaru H, Smith KM, Jia D, Zhang X, Selker EU, Shinkai Y, Cheng X. In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases. J Biol Chem. 2005;280:5563–70.

    Article  PubMed  CAS  Google Scholar 

  12. Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 2002;16:1779–91.

    Article  PubMed  CAS  Google Scholar 

  13. Peters AH, Kubicek S, Mechtler K, O’Sullivan RJ, Derijck AA, Perez-Burgos L, Kohlmaier A, Opravil S, Tachibana M, Shinkai Y, Martens JH, Jenuwein T. Partitioning and plasticity of repressive histone methylation states in mammalian chromatin. Mol Cell. 2003;12:1577–89.

    Article  PubMed  CAS  Google Scholar 

  14. Yokochi T, Poduch K, Ryba T, Lu J, Hiratani I, Tachibana M, Shinkai Y, Gilbert DM. G9a selectively represses a class of late-replicating genes at the nuclear periphery. PNAS. 2009;106:19363–8.

    Article  PubMed  CAS  Google Scholar 

  15. Estève PO, Patnaik D, Chin HG, Benner J, Teitell MA, Pradhan S. Functional analysis of the N-and C-terminus of mammalian G9a histone H3 methyltransferase. Nucleic Acids Res. 2005;33:3211–23.

    Article  PubMed  Google Scholar 

  16. Yamane K, Toumazou C, Tsukada Y, Erdjument-Bromage H, Tempst P, Wong J, Zhang Y. JHDM2A, a JmjC-containing H3K9demethylase, facilitates transcription activation by androgen receptor. Cell. 2006;125:483–95.

    Article  PubMed  CAS  Google Scholar 

  17. Doherty AS, Mann MRW, Tremblay KD, Bartolomei MS, Schultz RM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod. 2000;62:1526–35.

    Article  PubMed  CAS  Google Scholar 

  18. Otaegui PJ, O’Neill GT, Campbell KH, Wilmut I. Transfer of nuclei from 8-cell stage mouse embryos following use of nocodazole to control the cell cycle. Mol Reprod Dev. 1994;39:147–52.

    Article  PubMed  CAS  Google Scholar 

  19. Santos F, Peters AH, Otte AP, Reik W, Dean W. Dynamic chromatin modifications characterize the first cell cycle in mouse embryos. Dev Biol. 2005;280:225–36.

    Article  PubMed  CAS  Google Scholar 

  20. Samake S, Smith LC. Effects of cell-cycle-arrest agents on cleavage and development of mouse embryos. J Exp Zool. 1996;274:111–20.

    Article  PubMed  CAS  Google Scholar 

  21. Matsui Y, Nakayama Y, Okamoto M, Fukumoto Y, Yamaguchi N. Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: a novel method suitable for examining dynamic changes in proteins during mitotic progression. Eur J Cell Biol. 2012;91:413–9.

    Article  PubMed  CAS  Google Scholar 

  22. Sun QY, Wu GM, Lai L, Park KW, Cabot R, Cheong HT, Day BN, Prather RS, Schatten H. Translocation of active mitochondria during pig oocyte maturation, fertilization and early embryo development in vitro. Reproduction. 2001;122:155–63.

    Article  PubMed  CAS  Google Scholar 

  23. Korfiatis N, Trounson A, Lacham-Kaplan O. Cell synchronization for the purposes of nuclear transfer in the bovine. Cloning Stem Cells. 2001;3:125–38.

    Article  PubMed  CAS  Google Scholar 

  24. Zhang LS, Jiang MX, Lei ZL, Li RC, Sang D, Sun QY, Chen DY. Development of goat embryos reconstituted with somatic cells: the effect of cell-cycle coordination between transferred nucleus and recipient oocytes. J Reprod Dev. 2004;50:661–6.

    Article  PubMed  Google Scholar 

  25. Danilchik MV, Bedrick SD, Brown EE, Ray K. Furrow microtubules and localized exocytosis in cleaving Xenopuslaevis embryos. J Cell Sci. 2003;116:273–83.

    Article  PubMed  CAS  Google Scholar 

  26. Hoebeke JC, Van Nijen G, De Brabander M. Interaction of nocodazole(R17934), a new anti-tumoral drug, with rat brain tubulin. Biochem Biophys Res Commun. 1976;69:319–24.

    Article  PubMed  CAS  Google Scholar 

  27. Maro B, Bornens M. The centriole-nucleus association: effects of cytochalasin B and nocodazole. Biol Cells. 1980;39:287–90.

    CAS  Google Scholar 

  28. Johnson MH, Pickering SJ, Dhiman A, Radcliffe GS, Maro B. Cytocortical organization during natural and prolonged mitosis of mouse 8-cell blastomeres. Development. 1988;102:143–55.

    PubMed  CAS  Google Scholar 

  29. Sun F, Betzendahl I, Pacchierotti F, Ranaldi R, Smitz J, Cortvrindt R, Eichenlaub-Ritter U. Aneuploidy in mouse metaphase II oocytes exposed in vivo and in vitro in preantral follicle culture to nocodazole. Mutagenesis. 2005;20:65–75.

    Article  PubMed  CAS  Google Scholar 

  30. Yu Y, Xia P, Li S, Yan Y, Tan J. Determination and synchronisation of G1-phase of the cell cycle in 2-and 4-cell mouse embryos. Zygote. 2002;10:245–51.

    PubMed  Google Scholar 

  31. Zeng F, Baldwin DA, Schultz RM. Transcript profiling during preimplantation mouse development. Dev Biol. 2004;272:483–96.

    Article  PubMed  CAS  Google Scholar 

  32. Zeng F, Schultz RM. RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo. Dev Biol. 2005;283:40–57.

    Article  PubMed  CAS  Google Scholar 

  33. Morgan HD, Santos F, Green K, Dean W, Reik W. Epigenetic reprogramming in mammals. Hum Mol Genet. 2005;14:47–58.

    Article  Google Scholar 

  34. Kato Y, Ushijima Y, Yamaguchi M. Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase. Cell Struct Funct. 2011;36:121–9.

    Article  PubMed  CAS  Google Scholar 

  35. Weiss SL, Lee EA, Diamond J. Evolutionary matches of enzyme and transporter capacities to dietary substrate loads in the intestinal brush border. PNAS. 1998;95:2117–21.

    Article  PubMed  CAS  Google Scholar 

  36. Diamond JM. In: Noble D, Boyd CAR, editors. Logic of life: the challenge of integrative physiology. Oxford: Oxford Univ. Press; 1993. p. 89–111.

    Google Scholar 

  37. Diamond J, Hammond K. The matches, achieved by natural selection, between biological capacities and their natural loads. Experientia. 1992;48:551–7.

    Article  PubMed  CAS  Google Scholar 

  38. Tachibana M, Nozaki M, Takeda N, Shinkai Y. Functional dynamics of H3K9 methylation during meiotic prophase progression. EMBO J. 2007;26:3346–59.

    Article  PubMed  CAS  Google Scholar 

  39. Rydberg B, Lindahl T. Nonenzymatic methylation of DNA by the intracellular methyl group donor S-adenosyl-L-methionine is a potentially mutagenic reaction. EMBO J. 1982;1:211–6.

    PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work is supported by the National Key Basic Research (973) program of China (Grant No. 2007CB948101) and Scientific and Technological Developing Scheme of Shaanxi Province (Grant No. 2012K17-02-03). We thank Dr. Lei Pan from Institute of Genetics and Developmental Biology, Chinese Academy of Sciences for skillful technical assistance. We also appreciate the valuable comments from other members of our laboratory.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Obstetrics and Gynecology, Tang-du Hospital, the Fourth Military Medical University, Xi’an, 710038, People’s Republic of China

    Bo Li & Xiaohong Wang

  2. Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100080, People’s Republic of China

    Bo Li, Shuqiang Chen, Xue Li, Xiuying Huang & Fangzhen Sun

  3. Shaanxi Institute for Food and Drug Control, Xi’an, 710038, People’s Republic of China

    Na Tang

Authors
  1. Bo Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Na Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shuqiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xue Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiuying Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaohong Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fangzhen Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xiaohong Wang or Fangzhen Sun.

Additional information

Capsule

G9a specifically regulates histone H3 lysine 9 monomethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development.

Bo Li and Na Tang contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Li, B., Tang, N., Chen, S. et al. G9a co-localized with histone H3 lysine 9 monomethylation but not dimethylation in a nuclear membrane-dependent manner during mouse preimplantation embryo development. J Assist Reprod Genet 30, 441–448 (2013). https://doi.org/10.1007/s10815-012-9911-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-012-9911-y

Keywords

Search

Navigation