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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.

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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.

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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.

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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

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  1. Lothar Schermelleh
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Correspondence to Heinrich Leonhardt.

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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)

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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

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