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Transient cyclical methylation of promoter DNA

Abstract

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division1,2,3. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated4. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor α (ERα) promoters could be partially methylated by treatment with deacetylase inhibitors5, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERα and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERα and of pS2 transcripts.

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Figure 1: The methylation status of the pS2 promoter cycles on release from synchronization.
Figure 2: Cyclical changes in methylation status occur on promoters of several genes.
Figure 4: ERα and pS2 are re-expressed at low levels after transitory treatment with doxorubicin.
Figure 3: Doxorubicin induces cyclical changes in the methylation status of transcriptionally inactive promoters.

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Primary accessions

Gene Expression Omnibus

Data deposits

The gene expression data set reported has been deposited in the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE10145.

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Acknowledgements

The authors thank L. Cortesi, S. Denger and J. Lewis for their critical reading of this manuscript, and J. Blake for his assistance in expression array analysis. This work was supported by the EC 6th framework programme grant CRESCENDO and by the European Molecular Biology Organisation (EMBO).

Author Contributions G.R. and F.G. conceived the study, designed the experimental strategy and analysed data; S.K., B.S. and R.M. contributed to the experimental design. S.K. and B.S. prepared the GST–MBD fusion protein, validated the pull-down method, performed the MBD pull-downs and RT–PCR, and prepared DNA and chromatin for methylation analysis and for ChIP. R.M. performed restriction-sensitive methylation analysis and ChIP. M.P.-S. generated RNA for expression analysis and conducted the western blot studies. D.I., R.P.C. and V.B. produced the bisulphite sequencing results and expression array data from material provided by S.K., B.S. and M.P.-S. G.R., R.M. and F.G. wrote the paper, S.K. prepared the Supplementary Information, and all authors discussed the results and commented on the manuscript. F.G. and G.R. are joint senior authors.

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Correspondence to George Reid.

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The file contains Supplementary Notes, Supplementary Methods and additional references, followed by Supplementary Table 1 and Supplementary Figures S1-S5 with Legends. (PDF 5170 kb)

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Kangaspeska, S., Stride, B., Métivier, R. et al. Transient cyclical methylation of promoter DNA. Nature 452, 112–115 (2008). https://doi.org/10.1038/nature06640

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