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DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei

Nature Cell Biology volume 6, pages 984–990 (2004)Cite this article

Abstract

Nuclear transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation1,2,3; however, very little is known about the molecular mechanism of nuclear reprogramming. Here we have used nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.

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Figure 1: Deproteinization of nuclei and unmethylated DNA accelerate the time when oct4 transcription starts.
Figure 2: The mouse oct4 promoter is demethylated by Xenopus oocytes.
Figure 3: The regulatory region of mouse oct4 in plasmid DNA can be used to analyse the demethylating activity of Xenopus oocytes.
Figure 4: Mutational analysis of oct4 promoter demethylation.

References

  1. Gurdon, J.B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. J. Embryol. Exp. Morphol. 10, 622–640 (1962).

    CAS  PubMed  Google Scholar 

  2. Wilmut, I. et al. Somatic cell nuclear transfer. Nature 419, 583–586 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Gurdon, J.B., Byrne, J.A. & Simonsson, S. Nuclear reprogramming and stem cell creation. Proc. Natl Acad. Sci. USA 100, 11819–11822 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. De Robertis, E.M. & Gurdon, J.B. Gene activation in somatic nuclei after injection into amphibian oocytes. Proc. Natl Acad. Sci. USA 74, 2470–2474 (1977).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Byrne, J.A., Simonsson, S., Western, P.S. & Gurdon, J.B. Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4 stem cell gene expression by amphibian oocytes. Curr. Biol. 13, 1206–1213 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Surani, M.A. Reprogramming of genome function through epigenetic inheritance. Nature 414, 122–128 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Stancheva, I., El-Maarri, O., Walter, J., Niveleau, A. & Meehan, R.R. DNA methylation at promoter regions regulates the timing of gene activation in Xenopus laevis embryos. Dev. Biol. 243, 155–165 (2002).

    Article  CAS  PubMed  Google Scholar 

  8. Boiani, M., Eckardt, S., Scholer, H.R. & McLaughlin, K.J. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev. 16, 1209–1219 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Bortvin, A. et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673–1680 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Dean, W., Santos, F. & Reik, W. Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin. Cell Dev. Biol. 14, 93–100 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Matsuo, K. et al. An embryonic demethylation mechanism involving binding of transcription factors to replicating DNA. EMBO J. 17, 1446–1453 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Kimura, H., Suetake, I. & Tajima, S. Xenopus maintenance-type DNA methyltransferase is accumulated and translocated into germinal vesicles of oocytes. J. Biochem. (Tokyo) 125, 1175–1182 (1999).

    Article  CAS  Google Scholar 

  13. Curradi, M., Izzo, A., Badaracco, G. & Landsberger, N. Molecular mechanisms of gene silencing mediated by DNA methylation. Mol. Cell. Biol. 22, 3157–3173 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pesce, M. & Scholer, H.R. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19, 271–278 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genet. 24, 372–376 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Gidekel, S. & Bergman, Y. A unique developmental pattern of Oct-3/4 DNA methylation is controlled by a cis-demodification element. J. Biol. Chem. 277, 34521–34530 (2002).

    Article  CAS  PubMed  Google Scholar 

  18. Gurdon, J.B. & Melton, D.A. Gene transfer in amphibian eggs and oocytes. Annu. Rev. Genet. 15, 189–218 (1981).

    Article  CAS  PubMed  Google Scholar 

  19. Belikov, S., Gelius, B., Almouzni, G. & Wrange, O. Hormone activation induces nucleosome positioning in vivo. EMBO J. 19, 1023–1033 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Modak, S.P., Principaud, E. & Spohr, G. Regulation of Xenopus c-myc promoter activity in oocytes and embryos. Oncogene 8, 645–654 (1993).

    CAS  PubMed  Google Scholar 

  21. Luckow, B. & Schutz, G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 15, 5490 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mohun, T.J., Garrett, N. & Gurdon, J.B. Upstream sequences required for tissue-specific activation of the cardiac actin gene in Xenopus laevis embryos. EMBO J. 5, 3185–3193 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Steinbeisser, H., Hofmann, A., Stutz, F. & Trendelenburg, M.F. Different regulatory elements are required for cell-type and stage specific expression of the Xenopus laevis skeletal muscle actin gene upon injection in X. laevis oocytes and embryos. Nucleic Acids Res. 16, 3223–3238 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Siegfried, Z. et al. DNA methylation represses transcription in vivo. Nature Genet. 22, 203–206 (1999).

    Article  CAS  PubMed  Google Scholar 

  25. Hattori, N. et al. Epigenetic control of mouse Oct-4 gene expression in ES cells and TS cells. J. Biol. Chem. 279, 17063–17069 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Hinkley, C.S., Martin, J.F., Leibham, D. & Perry, M. Sequential expression of multiple POU proteins during amphibian early development. Mol. Cell. Biol. 12, 638–649 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kikyo, N., Wade, P.A., Guschin, D., Ge, H. & Wolffe, A.P. Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI. Science 289, 2360–2362 (2000).

    Article  CAS  PubMed  Google Scholar 

  28. Wade, P.A. & Kikyo, N. Chromatin remodeling in nuclear cloning. Eur. J. Biochem. 269, 2284–2287 (2002).

    CAS  Google Scholar 

  29. Gurdon, J.B. Nuclear transplantation in eggs and oocytes. J. Cell Sci. Suppl. 4, 287–318 (1986).

    Article  CAS  PubMed  Google Scholar 

  30. Gonda, K. et al. Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b. Nature Cell Biol. 5, 205–210 (2003).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank T.J. Mohun, G. Spohr and A. Smith for gifts of DNA constructs; N. Garrett for help, especially with the construction of DNA mutations; C. Lee for a supply of mouse thymocytes; and P. Hurd, A. Bannister, P. Hajkova and J. Byrne for discussion. Our work is supported by the Biotechnology and Biological Sciences Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education, the Wellcome Trust and the European Commission.

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  1. Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Zoology, University of Cambridge, Cambridge, CB2 1QR, UK

    Stina Simonsson & John Gurdon

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  1. Stina Simonsson
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  2. John Gurdon
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Correspondence to John Gurdon.

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Simonsson, S., Gurdon, J. DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei. Nat Cell Biol 6, 984–990 (2004). https://doi.org/10.1038/ncb1176

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