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De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes

Nature Structural & Molecular Biology volume 15, pages 1176–1183 (2008)Cite this article

Abstract

The pluripotency-determining gene Oct3/4 (also called Pou5f1) undergoes postimplantation silencing in a process mediated by the histone methyltransferase G9a. Microarray analysis now shows that this enzyme may operate as a master regulator that inactivates numerous early-embryonic genes by bringing about heterochromatinization of methylated histone H3K9 and de novo DNA methylation. Genetic studies in differentiating embryonic stem cells demonstrate that a point mutation in the G9a SET domain prevents heterochromatinization but still allows de novo methylation, whereas biochemical and functional studies indicate that G9a itself is capable of bringing about de novo methylation through its ankyrin domain, by recruiting Dnmt3a and Dnmt3b independently of its histone methyltransferase activity. These modifications seem to be programmed for carrying out two separate biological functions: histone methylation blocks target-gene reactivation in the absence of transcriptional repressors, whereas DNA methylation prevents reprogramming to the undifferentiated state.

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Figure 1: Genome-wide G9a-dependent de novo methylation.
Figure 2: Heterochromatinization is prevented by point mutation in the G9a SET domain.
Figure 3: G9a recruits Dnmt3a and Dnmt3b.
Figure 4: Mapping the G9a- and Dnmt-interacting domains.
Figure 5: ANK-dependent Oct3/4 methylation.
Figure 6: Reactivation and reprogramming of Oct3/4, Nanog, Dnmt3L and Tnfsrf8 genes are affected by their epigenetic state.

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Acknowledgements

We are grateful to S. Tajima (Osaka University) for Dnmt3a and 3b expression vectors, K. Wright (University of South Florida) for the hemagglutinin-G9a expression vector and S. Pradhan (New England Biolabs) for pGEX-G9a. This work was supported by grants from the Israel Academy of Science (Y.B. and H.C.), Philip Morris USA Inc. and Philip Morris International (S.E.-L., Y.B. and H.C.), the National Institutes of Health (Y.B. and H.C.), the Israel Cancer Research Fund (Y.B. and H.C.) and the Prostate Cancer Foundation (H.C.).

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  1. Silvina Epsztejn-Litman, Nirit Feldman and Monther Abu-Remaileh: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Experimental Medicine and Cellular Biochemistry, Hebrew University Medical School, Ein Kerem, 91120, Jerusalem, Israel

    Silvina Epsztejn-Litman, Nirit Feldman, Monther Abu-Remaileh, Ariela Gerson, Howard Cedar & Yehudit Bergman

  2. The Department of OB/GYN, IVF Unit, The Hadassah Human Embryonic Stem Cell Research Center, Hadassah Hebrew University Medical Center, 91120, Jerusalem, Israel

    Yoel Shufaro

  3. Experimental Research Center for Infectious Disease, Institute for Virus Research, Kyoto University, Sakyo-Ku, 606-8507, Kyoto, Japan

    Jun Ueda & Yoichi Shinkai

  4. Laboratory of Cancer Epigenetics, Faculty of Medicine, Free University of Brussels, 1070, Brussels, Belgium

    Rachel Deplus & François Fuks

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Contributions

S.E.-L., N.F., M.A.-R. and A.G. performed the ChIP, mDIP microarray, bisulfite sequencing, RNA analyses, some of the immunoprecipitation experiments and western blots, and reversal experiments; Y. Shufaro performed the nuclear transfer experiments; J.U. and Y. Shinkai generated the knockout and stable cell lines; R.D. and F.F. carried out some of the immunoprecipitation and western blot analyses. H.C. and Y.B. planned and supervised the research and wrote the manuscript. All authors contributed to the preparation of the manuscript.

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Correspondence to Howard Cedar.

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Epsztejn-Litman, S., Feldman, N., Abu-Remaileh, M. et al. De novo DNA methylation promoted by G9a prevents reprogramming of embryonically silenced genes. Nat Struct Mol Biol 15, 1176–1183 (2008). https://doi.org/10.1038/nsmb.1476

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