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Identification of genetic elements that autonomously determine DNA methylation states

Abstract

Cytosine methylation is a repressive, epigenetically propagated DNA modification. Although patterns of DNA methylation seem tightly regulated in mammals, it is unclear how these are specified and to what extent this process entails genetic or epigenetic regulation. To dissect the role of the underlying DNA sequence, we sequentially inserted over 50 different DNA elements into the same genomic locus in mouse stem cells. Promoter sequences of approximately 1,000 bp autonomously recapitulated correct DNA methylation in pluripotent cells. Moreover, they supported proper de novo methylation during differentiation. Truncation analysis revealed that this regulatory potential is contained within small methylation-determining regions (MDRs). MDRs can mediate both hypomethylation and de novo methylation in cis, and their activity depends on developmental state, motifs for DNA-binding factors and a critical CpG density. These results demonstrate that proximal sequence elements are both necessary and sufficient for regulating DNA methylation and reveal basic constraints of this regulation.

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Figure 1: Ectopic Nanog promoter recapitulates the methylation state of the endogenous promoter.
Figure 2: One-kb elements autonomously set DNA methylation state.
Figure 3: Truncation experiments identify methylation-determining regions.
Figure 4: MDR function depends on CpG density and DNA-binding motifs.
Figure 5: MDRs control de novo methylation and function in cis on heterologous DNA.

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Acknowledgements

We are grateful to M. Pietrzak for sequencing. We thank M. Lorincz of the University of British Columbia–Vancouver for providing plasmids for RMCE and S. Fiering for advice. We would also like to thank members of the Schübeler group and S. Gasser for critical comments on the manuscript. F.L. is supported by a PhD fellowship of the Boehringer Ingelheim Fonds. Research in the laboratory of A.D. is supported by the Intramural Program of National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health. Research in the laboratory of D.S. is supported by the Novartis Research Foundation, by the European Union (NoE “EpiGeneSys” FP7-HEALTH-2010-257082, LSHG-CT-2006-037415), the European Research Council (ERC-204264) and by the RTD “Cellplasticity” of the Swiss initiative in Systems Biology (SystemsX.ch).

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F.L. and C.W. performed experiments. I.S. and A.D. generated the target ES cell line. F.L., F.M. and D.S. designed the study, analyzed data and wrote the manuscript.

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Correspondence to Dirk Schübeler.

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Lienert, F., Wirbelauer, C., Som, I. et al. Identification of genetic elements that autonomously determine DNA methylation states. Nat Genet 43, 1091–1097 (2011). https://doi.org/10.1038/ng.946

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