The distinct effects of MEK and GSK3 inhibition upon the methylome and transcriptome of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) were first cultured in vitro in serum-containing medium with leukaemia inhibitory factor, in which they exhibit heterogeneous expression of both pluripotency and some early differentiation markers. Over the last decade, however, it has become commonplace to grow mESCs with inhibitors of MEK and GSK3 signalling, which together elicit a more homogeneously 'naive' state of pluripotency. Whilst 2i/L-cultured mESCs have been shown to be globally hypomethylated, a comprehensive understanding of the distinct effects of these signalling inhibitors upon the DNA methylome is still lacking. Here we carried out whole genome bisulphite and RNA sequencing of mESCs grown with MEK or GSK3 inhibition alone, including different time points and concentrations of MEK inhibitor treatment. This demonstrated that MEK inhibition causes a dose-dependent impairment of maintenance methylation via loss of UHRF1 protein, as well as rapid impairment of de novo methylation. In contrast, GSK3 inhibition triggers impairment of de novo methylation alone, and consequent hypomethylation is enriched at enhancers with a 2i/L-specific chromatin signature and coincides with upregulation of nearby genes. Our study provides detailed insights into the epigenetic and transcriptional impacts of inhibiting MEK or GSK3 signalling in mouse pluripotent cells.