Abstract
Loss of microRNA (miRNA) pathway components negatively affects differentiation of embryonic stem (ES) cells, but the underlying molecular mechanisms remain poorly defined. Here we characterize changes in mouse ES cells lacking Dicer (Dicer1). Transcriptome analysis of Dicer−/− cells indicates that the ES-specific miR-290 cluster has an important regulatory function in undifferentiated ES cells. Consistently, many of the defects in Dicer-deficient cells can be reversed by transfection with miR-290 family miRNAs. We demonstrate that Oct4 (also known as Pou5f1) silencing in differentiating Dicer−/− ES cells is accompanied by accumulation of repressive histone marks but not by DNA methylation, which prevents the stable repression of Oct4. The methylation defect correlates with downregulation of de novo DNA methyltransferases (Dnmts). The downregulation is mediated by Rbl2 and possibly other transcriptional repressors, potential direct targets of miR-290 cluster miRNAs. The defective DNA methylation can be rescued by ectopic expression of de novo Dnmts or by transfection of the miR-290 cluster miRNAs, indicating that de novo DNA methylation in ES cells is controlled by miRNAs.
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Acknowledgements
We thank G. Hannon and E. Murchison (Cold Spring Harbor Laboratory, New York, USA), for providing Dicer−/− ES cells, K. Ura (Osaka University, Japan), for providing DNMT3 plasmids, A. Peters for providing antibodies, D. Schubeler for helpful suggestions and comments (both Friedrich Miescher Institute, Basel, Switzerland). We also thank E. Oakeley, H. Angliker and M. Pietrzak for their contributions to array analysis and sequencing (Friedrich Miescher Institute). P.S. is supported by the European Molecular Biology Organization (EMBO) SDIG program #1488, GAAV IAA501110701 and the Purkynje Fellowship. P.B. is supported by the Swiss National Science Foundation (SNF) grant #3100A0-114001 to M.Z., and D.G. is supported by the Swiss Institute of Bioinformatics. L.S. is partially supported by the EC FP6 STREP program LSHG-CT-2004. The Friedrich Miescher Institute is supported by the Novartis Research Foundation.
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L.S., P.S. and W.F. designed the study; L.S., P.S. and M.Z. designed the computational part; L.S. carried out most of the experiments; T.H. contributed to some experiments with Dicer−/− ES cells and most of the western analyses; C.G.A.-R. contributed to Rbl2 knockdown and western analyses; D.G. and P.B. performed computational analyses; P.S. carried out some of the bisulfite sequencing and initial analysis of microarray data; F.M. helped with Sox30 and Tsp50 methylation analysis; L.S., P.S., M.Z. and W.F. wrote the manuscript.
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Sinkkonen, L., Hugenschmidt, T., Berninger, P. et al. MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nat Struct Mol Biol 15, 259–267 (2008). https://doi.org/10.1038/nsmb.1391
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DOI: https://doi.org/10.1038/nsmb.1391
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