Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells

Nature Structural & Molecular Biology volume 15, pages 259–267 (2008)Cite this article

Abstract

Loss of microRNA (miRNA) pathway components negatively affects differentiation of embryonic stem (ES) cells, but the underlying molecular mechanisms remain poorly defined. Here we characterize changes in mouse ES cells lacking Dicer (Dicer1). Transcriptome analysis of Dicer−/− cells indicates that the ES-specific miR-290 cluster has an important regulatory function in undifferentiated ES cells. Consistently, many of the defects in Dicer-deficient cells can be reversed by transfection with miR-290 family miRNAs. We demonstrate that Oct4 (also known as Pou5f1) silencing in differentiating Dicer−/− ES cells is accompanied by accumulation of repressive histone marks but not by DNA methylation, which prevents the stable repression of Oct4. The methylation defect correlates with downregulation of de novo DNA methyltransferases (Dnmts). The downregulation is mediated by Rbl2 and possibly other transcriptional repressors, potential direct targets of miR-290 cluster miRNAs. The defective DNA methylation can be rescued by ectopic expression of de novo Dnmts or by transfection of the miR-290 cluster miRNAs, indicating that de novo DNA methylation in ES cells is controlled by miRNAs.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Transcriptome analysis of Dicer−/− embryonic stem (ES) cells.
Figure 2: De novo DNA methyltransferases (Dnmts) are downregulated in Dicer−/− embryonic stem (ES) cells and their expression is rescued by miR-290 cluster miRNAs.
Figure 3: Retinoblastoma-like protein 2 (Rbl2) regulates the expression of Dnmt3a2 and Dnmt3b.
Figure 4: Oct4 expression during differentiation of Dicer+/− and Dicer−/− embryonic stem (ES) cells.
Figure 5: Deficient de novo DNA methylation of the Oct4 promoter in Dicer−/− embryonic stem (ES) cells can be rescued by expression of de novo DNA methyltransferases (Dnmts) or by transfection of miR-290 cluster miRNAs.
Figure 6: A model for a role of miRNAs in de novo DNA methylation in embryonic stem (ES) cells.

Similar content being viewed by others

References

  1. Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).

    Article  CAS  Google Scholar 

  2. He, L. & Hannon, G.J. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 5, 522–531 (2004).

    Article  CAS  Google Scholar 

  3. Meister, G. & Tuschl, T. Mechanisms of gene silencing by double-stranded RNA. Nature 431, 343–349 (2004).

    Article  CAS  Google Scholar 

  4. Zamore, P.D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005).

    Article  CAS  Google Scholar 

  5. Kim, V.N. MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6, 376–385 (2005).

    Article  CAS  Google Scholar 

  6. Bartel, D.P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297 (2004).

    Article  CAS  Google Scholar 

  7. Bushati, N. & Cohen, S.M. MicroRNA functions. Annu. Rev. Cell Dev. Biol. 23, 175–205 (2007).

    Article  CAS  Google Scholar 

  8. Pillai, R.S., Bhattacharyya, S.N. & Filipowicz, W. Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol. 17, 118–126 (2007).

    Article  CAS  Google Scholar 

  9. Valencia-Sanchez, M.A., Liu, J., Hannon, G.J. & Parker, R. Control of translation and mRNA degradation by miRNAs and siRNAs. Genes Dev. 20, 515–524 (2006).

    Article  CAS  Google Scholar 

  10. Brennecke, J., Stark, A., Russell, R.B. & Cohen, S.M. Principles of microRNA-target recognition. PLoS Biol. 3, e85 (2005).

    Article  Google Scholar 

  11. Lewis, B.P., Burge, C.B. & Bartel, D.P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005).

    Article  CAS  Google Scholar 

  12. Rajewsky, N. MicroRNA target predictions in animals. Nat. Genet. 38 Suppl, S8–S13 (2006).

    Article  CAS  Google Scholar 

  13. Smith, A.G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001).

    Article  CAS  Google Scholar 

  14. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  Google Scholar 

  15. Loh, Y.H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431–440 (2006).

    Article  CAS  Google Scholar 

  16. Suh, M.R. et al. Human embryonic stem cells express a unique set of microRNAs. Dev. Biol. 270, 488–498 (2004).

    Article  CAS  Google Scholar 

  17. Houbaviy, H.B., Murray, M.F. & Sharp, P.A. Embryonic stem cell-specific MicroRNAs. Dev. Cell 5, 351–358 (2003).

    Article  CAS  Google Scholar 

  18. Tang, F. et al. Maternal microRNAs are essential for mouse zygotic development. Genes Dev. 21, 644–648 (2007).

    Article  CAS  Google Scholar 

  19. Kanellopoulou, C. et al. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19, 489–501 (2005).

    Article  CAS  Google Scholar 

  20. Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S. & Hannon, G.J. Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci. USA 102, 12135–12140 (2005).

    Article  CAS  Google Scholar 

  21. Wang, Y., Medvid, R., Melton, C., Jaenisch, R. & Blelloch, R. DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39, 380–385 (2007).

    Article  CAS  Google Scholar 

  22. Krutzfeldt, J. et al. Silencing of microRNAs in vivo with 'antagomirs'. Nature 438, 685–689 (2005).

    Article  Google Scholar 

  23. Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005).

    Article  CAS  Google Scholar 

  24. Schmitter, D. et al. Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic Acids Res. 34, 4801–4815 (2006).

    Article  CAS  Google Scholar 

  25. Griffiths-Jones, S., Grocock, R.J., van Dongen, S., Bateman, A. & Enright, A.J. miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34, D140–D144 (2006).

    Article  CAS  Google Scholar 

  26. Zeng, F., Baldwin, D.A. & Schultz, R.M. Transcript profiling during preimplantation mouse development. Dev. Biol. 272, 483–496 (2004).

    Article  CAS  Google Scholar 

  27. Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007).

    Article  CAS  Google Scholar 

  28. Chen, T., Ueda, Y., Xie, S. & Li, E. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J. Biol. Chem. 277, 38746–38754 (2002).

    Article  CAS  Google Scholar 

  29. Aapola, U., Maenpaa, K., Kaipia, A. & Peterson, P. Epigenetic modifications affect Dnmt3L expression. Biochem. J. 380, 705–713 (2004).

    Article  CAS  Google Scholar 

  30. Jinawath, A., Miyake, S., Yanagisawa, Y., Akiyama, Y. & Yuasa, Y. Transcriptional regulation of the human DNA methyltransferase 3A and 3B genes by Sp3 and Sp1 zinc finger proteins. Biochem. J. 385, 557–564 (2005).

    Article  CAS  Google Scholar 

  31. Su, A.I. et al. Large-scale analysis of the human and mouse transcriptomes. Proc. Natl. Acad. Sci. USA 99, 4465–4470 (2002).

    Article  CAS  Google Scholar 

  32. Harris, M.A. et al. The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 32, D411–D417 (2004).

    Article  CAS  Google Scholar 

  33. Litovchick, L. et al. Evolutionarily conserved multisubunit RBL2/p130 and E2F4 protein complex represses human cell cycle-dependent genes in quiescence. Mol. Cell 26, 539–551 (2007).

    Article  CAS  Google Scholar 

  34. Gu, P., Le Menuet, D., Chung, A.C. & Cooney, A.J. Differential recruitment of methylated CpG binding domains by the orphan receptor GCNF initiates the repression and silencing of Oct4 expression. Mol. Cell. Biol. 26, 9471–9483 (2006).

    Article  CAS  Google Scholar 

  35. Feldman, N. et al. G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Nat. Cell Biol. 8, 188–194 (2006).

    Article  CAS  Google Scholar 

  36. Li, J.Y. et al. Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog. Mol. Cell Biol. 27, 8748–8759 (2007).

    Article  CAS  Google Scholar 

  37. Gartel, A.L. & Radhakrishnan, S.K. Lost in transcription: p21 repression, mechanisms, and consequences. Cancer Res. 65, 3980–3985 (2005).

    Article  CAS  Google Scholar 

  38. Murakami, M. et al. mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol. Cell. Biol. 24, 6710–6718 (2004).

    Article  CAS  Google Scholar 

  39. Giraldez, A.J. et al. Zebrafish miR-430 promotes deadenylation and clearance of maternal mRNAs. Science 312, 75–79 (2006).

    Article  CAS  Google Scholar 

  40. Schultz, R.M. The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. Hum. Reprod. Update 8, 323–331 (2002).

    Article  CAS  Google Scholar 

  41. Choi, W.Y., Giraldez, A.J. & Schier, A.F. Target protectors reveal dampening and balancing of nodal agonist and antagonist by miR-430. Science 318, 271–274 (2007).

    Article  CAS  Google Scholar 

  42. Dotto, G.P. p21(WAF1/Cip1): more than a break to the cell cycle? Biochim. Biophys. Acta 1471, M43–M56 (2000).

    CAS  PubMed  Google Scholar 

  43. He, L. et al. A microRNA polycistron as a potential human oncogene. Nature 435, 828–833 (2005).

    Article  CAS  Google Scholar 

  44. Zvetkova, I. et al. Global hypomethylation of the genome in XX embryonic stem cells. Nat. Genet. 37, 1274–1279 (2005).

    Article  CAS  Google Scholar 

  45. Robertson, K.D., Keyomarsi, K., Gonzales, F.A., Velicescu, M. & Jones, P.A. Differential mRNA expression of the human DNA methyltransferases (DNMTs) 1, 3a and 3b during the G(0)/G(1) to S phase transition in normal and tumor cells. Nucleic Acids Res. 28, 2108–2113 (2000).

    Article  CAS  Google Scholar 

  46. Fabbri, M. et al. MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc. Natl. Acad. Sci. USA 110, 15805–15810 (2007).

    Article  Google Scholar 

  47. Chen, T., Ueda, Y., Dodge, J.E., Wang, Z. & Li, E. Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b. Mol. Cell. Biol. 23, 5594–5605 (2003).

    Article  CAS  Google Scholar 

  48. Ishida, C. et al. Genomic organization and promoter analysis of the Dnmt3b gene. Gene 310, 151–159 (2003).

    Article  CAS  Google Scholar 

  49. Nimura, K. et al. Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation. Genes Cells 11, 1225–1237 (2006).

    Article  CAS  Google Scholar 

  50. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997).

    Article  CAS  Google Scholar 

  51. Sinkkonen, L., Malinen, M., Saavalainen, K., Vaisanen, S. & Carlberg, C. Regulation of the human cyclin C gene via multiple vitamin D3-responsive regions in its promoter. Nucleic Acids Res. 33, 2440–2451 (2005).

    Article  CAS  Google Scholar 

  52. Svoboda, P., Stein, P., Filipowicz, W. & Schultz, R.M. Lack of homologous sequence-specific DNA methylation in response to stable dsRNA expression in mouse oocytes. Nucleic Acids Res. 32, 3601–3606 (2004).

    Article  CAS  Google Scholar 

  53. Kotaja, N. et al. The chromatoid body of male germ cells: similarity with processing bodies and presence of Dicer and microRNA pathway components. Proc. Natl. Acad. Sci. USA 103, 2647–2652 (2006).

    Article  CAS  Google Scholar 

  54. Nordhoff, V. et al. Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences. Mamm. Genome 12, 309–317 (2001).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank G. Hannon and E. Murchison (Cold Spring Harbor Laboratory, New York, USA), for providing Dicer−/− ES cells, K. Ura (Osaka University, Japan), for providing DNMT3 plasmids, A. Peters for providing antibodies, D. Schubeler for helpful suggestions and comments (both Friedrich Miescher Institute, Basel, Switzerland). We also thank E. Oakeley, H. Angliker and M. Pietrzak for their contributions to array analysis and sequencing (Friedrich Miescher Institute). P.S. is supported by the European Molecular Biology Organization (EMBO) SDIG program #1488, GAAV IAA501110701 and the Purkynje Fellowship. P.B. is supported by the Swiss National Science Foundation (SNF) grant #3100A0-114001 to M.Z., and D.G. is supported by the Swiss Institute of Bioinformatics. L.S. is partially supported by the EC FP6 STREP program LSHG-CT-2004. The Friedrich Miescher Institute is supported by the Novartis Research Foundation.

Author information

Authors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel, 4058, Switzerland

    Lasse Sinkkonen, Tabea Hugenschmidt, Fabio Mohn, Caroline G Artus-Revel & Witold Filipowicz

  2. Division of Bioinformatics, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland, and the Swiss Institute of Bioinformatics.,

    Philipp Berninger, Dimos Gaidatzis & Mihaela Zavolan

  3. Institute of Molecular Genetics, Videnska, Prague, 1083, Czech Republic

    Petr Svoboda

Authors
  1. Lasse Sinkkonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tabea Hugenschmidt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Philipp Berninger
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dimos Gaidatzis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fabio Mohn
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Caroline G Artus-Revel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mihaela Zavolan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Petr Svoboda
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Witold Filipowicz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L.S., P.S. and W.F. designed the study; L.S., P.S. and M.Z. designed the computational part; L.S. carried out most of the experiments; T.H. contributed to some experiments with Dicer−/− ES cells and most of the western analyses; C.G.A.-R. contributed to Rbl2 knockdown and western analyses; D.G. and P.B. performed computational analyses; P.S. carried out some of the bisulfite sequencing and initial analysis of microarray data; F.M. helped with Sox30 and Tsp50 methylation analysis; L.S., P.S., M.Z. and W.F. wrote the manuscript.

Corresponding authors

Correspondence to Petr Svoboda or Witold Filipowicz.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–4 and Supplementary Methods (PDF 1543 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sinkkonen, L., Hugenschmidt, T., Berninger, P. et al. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15, 259–267 (2008). https://doi.org/10.1038/nsmb.1391

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1391

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing