MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells

Sinkkonen L, Hugenschmidt T, Berninger P, Gaidatzis D, Mohn F, Artus-Revel CG, Zavolan M, Svoboda P, Filipowicz W
Source: Nat Struct Mol Biol
Publication Date: (2008)
Issue: 15(3): 259-67
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
Embryonic stem cell (ES), mouse
Species: mouse
Tissue Origin: embryo
Nucleofector™ I/II/2b
Approximately 3x10^6 Dicer –/– cells were used per transfection and the cells were plated immediately after nucleofection. Transfections of siRNAs were performed using 300 pmol of siRNA against RL mRNA (siRL) (Eurogentec), 50 pmol of siGENOME smartPOOL siRNAs against Rbl2 (Dharmacon), 50 pmol of each of the mmu-mir-290, mmu-mir 291a-3p, mmu-mir-292-3p, mmu-mir-293, mmu-mir-294 and mmu-mir-295 miRNA mimics (Dharmacon), or 300 pmol of mmu-mir-291a-3p, together with 2 mg of pCX-EGFP50, which served as control for transfection efficiency. For rescue of de novo DNA methylation by a mixture of pCag-EGFP-Dnmt3a2, pCag-EGFP-Dnmt3b and pCag-EGFP-Dnmt3L plasmids, the Dicer –/– cells were co-transfected with 7 mg of each of these plasmids. The EGFP-expressing cells were collected using a MoFlow cell sorter after 3 d of culture in the presence of 100 nM RA and the absence of LIF.
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.