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:

Trapped in action: direct visualization of DNA methyltransferase activity in living cells

Nature Methods volume 2, pages 751–756 (2005)Cite this article

Abstract

DNA methyltransferases have a central role in the complex regulatory network of epigenetic modifications controlling gene expression in mammalian cells. To study the regulation of DNA methylation in living cells, we developed a trapping assay using transiently expressed fluorescent DNA methyltransferase 1 (Dnmt1) fusions and mechanism-based inhibitors 5-azacytidine (5-aza-C) or 5-aza-2′-deoxycytidine (5-aza-dC). These nucleotide analogs are incorporated into the newly synthesized DNA at nuclear replication sites and cause irreversible immobilization, that is, trapping of Dnmt1 fusions at these sites. We measured trapping by either fluorescence bleaching assays or photoactivation of photoactivatable green fluorescent protein fused to Dnmt1 (paGFP-Dnmt1) in mouse and human cells; mutations affecting the catalytic center of Dnmt1 prevented trapping. This trapping assay monitors kinetic properties and activity-dependent immobilization of DNA methyltransferases in their native environment, and makes it possible to directly compare mutations and inhibitors that affect regulation and catalytic activity of DNA methyltransferases in single living cells.

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: Schematic representation of the DNA methylation reaction.
Figure 2: Trapping of active Dnmt1 after 5-aza-C treatment.
Figure 3: Time and dose dependence of Dnmt1 immobilization after 5-aza-C and 5-aza-dC treatment.
Figure 4: Direct visualization of trapping by photoactivation of paGFP-Dnmt1.
Figure 5: Postreplicative action of Dnmt1 and rationale of the trapping assay.

Similar content being viewed by others

References

  1. Li, E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662–673 (2002).

    Article  CAS  Google Scholar 

  2. Jaenisch, R. & Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 33 (Suppl.), 245–254 (2003).

    Article  CAS  Google Scholar 

  3. Bestor, T.H. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395–2402 (2000).

    Article  CAS  Google Scholar 

  4. Robertson, K.D. DNA methylation and chromatin—unraveling the tangled web. Oncogene 21, 5361–5379 (2002).

    Article  CAS  Google Scholar 

  5. Tang, L.Y. et al. The eukaryotic DNMT2 genes encode a new class of cytosine-5 DNA methyltransferases. J. Biol. Chem. 278, 33613–33616 (2003).

    Article  CAS  Google Scholar 

  6. Hermann, A., Schmitt, S. & Jeltsch, A. The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity. J. Biol. Chem. 278, 31717–31721 (2003).

    Article  CAS  Google Scholar 

  7. Kunert, N., Marhold, J., Stanke, J., Stach, D. & Lyko, F.A. Dnmt2-like protein mediates DNA methylation in Drosophila. Development 130, 5083–5090 (2003).

    Article  CAS  Google Scholar 

  8. Leonhardt, H., Page, A.W., Weier, H.U. & Bestor, T.H. A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865–873 (1992).

    Article  CAS  Google Scholar 

  9. Chuang, L.S. et al. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 277, 1996–2000 (1997).

    Article  CAS  Google Scholar 

  10. Rountree, M.R., Bachman, K.E. & Baylin, S.B. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet. 25, 269–277 (2000).

    Article  CAS  Google Scholar 

  11. Robertson, K.D. et al. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet. 25, 338–342 (2000).

    Article  CAS  Google Scholar 

  12. Fuks, F., Hurd, P.J., Deplus, R. & Kouzarides, T. The DNA methyltransferases associate with HP1 and the SUV39H1 histone methyltransferase. Nucleic Acids Res. 31, 2305–2312 (2003).

    Article  CAS  Google Scholar 

  13. Kimura, H. & Shiota, K. Methyl-CpG-binding protein, MeCP2, is a target molecule for maintenance DNA methyltransferase, Dnmt1. J. Biol. Chem. 278, 4806–4812 (2003).

    Article  CAS  Google Scholar 

  14. Esteve, P.O., Chin, H.G. & Pradhan, S. Human maintenance DNA (cytosine-5)-methyltransferase and p53 modulate expression of p53-repressed promoters. Proc. Natl. Acad. Sci. USA 102, 1000–1005 (2005).

    Article  CAS  Google Scholar 

  15. Jones, P.A. & Baylin, S.B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3, 415–428 (2002).

    Article  CAS  Google Scholar 

  16. Gaudet, F. et al. Induction of tumors in mice by genomic hypomethylation. Science 300, 489–492 (2003).

    Article  CAS  Google Scholar 

  17. Rhee, I. et al. CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 404, 1003–1007 (2000).

    Article  CAS  Google Scholar 

  18. Cheng, X. & Roberts, R.J. AdoMet-dependent methylation, DNA methyltransferases and base flipping. Nucleic Acids Res. 29, 3784–3795 (2001).

    Article  CAS  Google Scholar 

  19. Bestor, T.H. & Verdine, G.L. DNA methyltransferases. Curr. Opin. Cell Biol. 6, 380–389 (1994).

    Article  CAS  Google Scholar 

  20. Santi, D.V., Garrett, C.E. & Barr, P.J. On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell 33, 9–10 (1983).

    Article  CAS  Google Scholar 

  21. Chen, L. et al. Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. Biochemistry 30, 11018–11025 (1991).

    Article  CAS  Google Scholar 

  22. Christman, J.K. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene 21, 5483–5495 (2002).

    Article  CAS  Google Scholar 

  23. Easwaran, H.P., Schermelleh, L., Leonhardt, H. & Cardoso, M.C. Replication-independent chromatin loading of Dnmt1 during G2 and M phases. EMBO Rep. 5, 1181–1186 (2004).

    Article  CAS  Google Scholar 

  24. Leonhardt, H. et al. Dynamics of DNA replication factories in living cells. J. Cell Biol. 149, 271–280 (2000).

    Article  CAS  Google Scholar 

  25. Sporbert, A., Gahl, A., Ankerhold, R., Leonhardt, H. & Cardoso, M.C. DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol. Cell 10, 1355–1365 (2002).

    Article  CAS  Google Scholar 

  26. Wyszynski, M.W., Gabbara, S. & Bhagwat, A.S. Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes. Nucleic Acids Res. 20, 319–326 (1992).

    Article  CAS  Google Scholar 

  27. Jones, P.A. & Taylor, S.M. Cellular differentiation, cytidine analogs and DNA methylation. Cell 20, 85–93 (1980).

    Article  CAS  Google Scholar 

  28. Cihak, A. Biological effects of 5-azacytidine in eukaryotes. Oncology 30, 405–422 (1974).

    Article  CAS  Google Scholar 

  29. Patterson, G.H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    Article  CAS  Google Scholar 

  30. Robert, M.F. et al. DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat. Genet. 33, 61–65 (2003).

    Article  CAS  Google Scholar 

  31. Weisenberger, D.J. et al. Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation. Mol. Cancer Res. 2, 62–72 (2004).

    CAS  PubMed  Google Scholar 

  32. Liu, K., Wang, Y.F., Cantemir, C. & Muller, M.T. Endogenous assays of DNA methyltransferases: Evidence for differential activities of DNMT1, DNMT2, and DNMT3 in mammalian cells in vivo. Mol. Cell. Biol. 23, 2709–2719 (2003).

    Article  CAS  Google Scholar 

  33. Juttermann, R., Li, E. & Jaenisch, R. Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc. Natl. Acad. Sci. USA 91, 11797–11801 (1994).

    Article  CAS  Google Scholar 

  34. Easwaran, H.P., Leonhardt, H. & Cardoso, M.C. Cell cycle markers for live cell analyses. Cell Cycle 4, 453–455 (2005).

    Article  CAS  Google Scholar 

  35. Cardoso, M.C. et al. Mapping and use of a sequence that targets DNA ligase I to sites of DNA replication in vivo. J. Cell Biol. 139, 579–587 (1997).

    Article  CAS  Google Scholar 

  36. Sporbert, A., Domaing, P., Leonhardt, H. & Cardoso, M.C. PCNA acts as a stationary loading platform for transiently interacting Okazaki fragment maturation proteins. Nucleic Acids Res. 33, 3521–3528 (2005).

    Article  CAS  Google Scholar 

  37. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R.Y. Tsien for providing mRFP1 cDNA, J. Lippincott-Schwartz for providing paGFP cDNA, E. Li for mutant Dnmt1 ES cells and P. Vertino for the human DNMT1 cDNA. We thank M. Grohmann for sharing expression constructs. We are grateful to I. Grunewald and A. Gahl for technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Max Delbrück Center to H.L. and M.C.C.

Author information

Authors and Affiliations

  1. Department of Biology II, Ludwig Maximilians University Munich, Großhaderner Str. 2, Planegg-Martinsried, 82152, Germany

    Lothar Schermelleh, Fabio Spada, Kourosh Zolghadr & Heinrich Leonhardt

  2. Max Delbrueck Center for Molecular Medicine, FVK, Wiltbergstr. 50, Berlin, 13125, Germany

    Hariharan P Easwaran, Jean B Margot, M Cristina Cardoso & Heinrich Leonhardt

Authors
  1. Lothar Schermelleh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fabio Spada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hariharan P Easwaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kourosh Zolghadr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jean B Margot
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Cristina Cardoso
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Heinrich Leonhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Heinrich Leonhardt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

GFP-Dnmt1 fusion protein restores methylation at chromocenters in dnmt1−/− ES cells. (PDF 399 kb)

Supplementary Fig. 2

Trapping of DNMT1 after 5–aza–dC treatment in human SH–EP N14 neuroblastoma cells. (PDF 204 kb)

Supplementary Video 1

Dnmt1 mobility before and during 5–aza–dC treatment in a single living cell. (MOV 2476 kb)

Supplementary Methods (PDF 75 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schermelleh, L., Spada, F., Easwaran, H. et al. Trapped in action: direct visualization of DNA methyltransferase activity in living cells. Nat Methods 2, 751–756 (2005). https://doi.org/10.1038/nmeth794

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth794

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