FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency. Mus musculus

Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos but the signalling mechanisms that induce reprogramming are unknown. Here we show that inhibition of Erk1/2 and Gsk3b signalling in mouse embryonic stem cells (ESCs) by small molecule inhibitors (PD0325901 and CHIR99021, hereafter called 2i) induces genome-wide demethylation on a scale similar to that in PGCs and early embryos, with only major satellites, intracisternal A particles (IAPs) and imprinted genes relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) in part by Tet1 together with repression of the de novo methyltransferases (Dnmt3a, Dnmt3b) and their regulator Dnmt3L and we identify a cis-acting regulatory region in Dnmt3b that is highly responsive to signalling. Notably, this epigenetic and transcriptional ground state of pluripotency resembles closely that of inner cell mass (ICM) cells of the blastocyst. These insights provide a novel framework for understanding how signalling pathways regulate epigenetic reprogramming. Overall design: mRNA and methylation profiles of ES cells passaged with or without 2i inhibitors were generated by deep sequencing, using Illumina GAIIx and HiSeq.