2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms

Summary Mouse embryonic stem cells (ESCs) are maintainedin serum with leukemia inhibitory factor(LIF) to maintainself-renewal and pluripotency. Recently, a 2i culture methodwas reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintainESCs in a naive ground state. How 2i maintainsa ground stateof ESCs remains elusive. Here we show that MEKi and GSK3i maintainthe ESC ground stateby downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine(5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintaina naive ground statethrough JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.