Cyclical DNA methylation of a transcriptionally active promoter

A Corrigendum to this article was published on 21 January 2010

Abstract

Processes that regulate gene transcription are directly under the influence of the genome organization. The epigenome contains additional information that is not brought by DNA sequence, and generates spatial and functional constraints that complement genetic instructions. DNA methylation on CpGs constitutes an epigenetic mark generally correlated with transcriptionally silent condensed chromatin. Replication of methylation patterns by DNA methyltransferases maintains genome stability through cell division. Here we present evidence of an unanticipated dynamic role for DNA methylation in gene regulation in human cells. Periodic, strand-specific methylation/demethylation occurs during transcriptional cycling of the pS2/TFF1 gene promoter on activation by oestrogens. DNA methyltransferases exhibit dual actions during these cycles, being involved in CpG methylation and active demethylation of 5mCpGs through deamination. Inhibition of this process precludes demethylation of the pS2 gene promoter and its subsequent transcriptional activation. Cyclical changes in the methylation status of promoter CpGs may thus represent a critical event in transcriptional achievement.

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Figure 1: Cyclical methylation of the pS2 promoter.
Figure 2: Kinetic association of Dnmts with transcriptionally active pS2 promoter in MDA::ERα cells.
Figure 3: Inhibition of Dnmt activity modifies pS2 transcriptional cycling.
Figure 4: Mobilization of TDG, p68, Dnmt3a/b and BER proteins during pS2 promoter demethylation.
Figure 5: Dnmt3a/b deaminate CpGs and 5m CpGs.

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Acknowledgements

We thank B. Brueckner for the gift of the RG108 compound, P. Chambon for the pSG5-TDGwt plasmid, F. Fuller-Pace for the anti-p68 RNA helicase antibody, and V. Legagneux and U. K. Laemmli for the anti-CAPD2 and anti-CAPH antibodies, respectively. This work was supported by funds from the Ministère de l’Education Nationale de l’Enseignement Supérieur et de la Recherche (MENESR), the Centre National de la Recherche Scientifique (CNRS), the University of Rennes I, the Association pour la Recherche contre le Cancer (ARC), the Ligue contre le Cancer, and by funding from EMBO and EMBL. R.G. was supported by a grant from the MENESR and a fellowship from the Ligue Nationale Contre le Cancer. We also thank C. Ralliere for DNA sequencing, C. Martin, F. Percevault and C. Tascon for their technical assistance, and B.Stride and S. Kangaspeska for their comments during the writing of the manuscript.

Authors Contributions ChIP, sequential-ChIP experiments, bisulphite and run-on assays were performed by R.M. Methylation/deamination assays were set up by R.G. and performed by R.G. and R.M. C.T. ran all RT-PCR experiments on siRNA-treated cells, and set up the analysis on the Wisp-2 gene. Co-immunoprecipitations and proteomic controls were performed by C.L.P., R.G. and R.M. Synthesis and purification of TDG protein were performed by P.B. and F.D., as the preparation of the anti-TDG antibody. R.Z.J. purified all the Dnmt3 catalytic domains. R.P.C. and D.I. were involved, under the supervision of V.B., in the mass sequencing of the clones generated during the bisulphite-mediated analysis of CpG methylation and through the in vitro deamination assays. G.R. introduced R.M. to FACS analysis and bisulphite-modification of DNA. R.M., G.R., A.J., F.G. and G.S. were responsible for the overall project management, strategy and data interpretation. R.M., G.R. and G.S. prepared the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Raphaël Métivier.

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Supplementary Information

The file contains Supplementary Discussion with additional references, Supplementary Figures S1 to S10 with Legends and Supplementary Tables 1 to 4. Supplementary Figures and Supplementary Tables present additional controls and data that support both the main text and supplementary discussion. Supplementary Figure S10 depicts a scheme summarizing the proposed mechanism. Supplementary Figures 1a, 3d, 7c, e and 8a-d were replaced on 21 Jan 2010. (PDF 15174 kb)

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Métivier, R., Gallais, R., Tiffoche, C. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008). https://doi.org/10.1038/nature06544

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