MEK inhibitor PD0325901 and vitamin C synergistically induce hypomethylation of mouse embryonic stem cells.

A rationally selected combination of small-molecule chemicals can affect cell plasticity and fate, suggesting an open chemistry way to manipulate cells to achieve a specific goal. Here we for the first time demonstrate that a combination of vitamin C (Vc) and PD0325901 can achieve about 90% erasure of 5-methylcytosine (5mC) within 5 days (decreasing from 3.2 to ~ 0.3 5mC per 100 C) in mouse embryonic stem cells (ESCs). The hypomethylated level is comparable to that of gonadal primordial germ cells (PGCs), whose pluripotency is closely associated with the global DNA hypomethylation. In contrast, Vc or PD0325901 alone only induces a moderately reduced level of global DNA methylation. Our mechanistic study suggested that PD0325901 elevated expression of Prdm14, which repressed de novo methyltransferase Dnmt3b and its cofactor Dnmt3l at levels of protein, via the mode to eliminate 5mC from de novo synthesis. By further addition of Vc, the oxidation of 5mC as catalyzed by Tet1/Tet2 dioxygenases was significantly increased as manifested by the elevated level of 5-hydroxymethylcytosine. However, by the depletion of Tet1/Tet2, Vc failed to enhance PD0325901-stimulated hypomethylation of ESCs' genomic DNA. Furthermore, mouse ESCs in Vc/PD0325901-supplemented medium show great morphology and pluripotency. Therefore, we demonstrate a novel and synergistic chemical approach for promoting hypomethylation and sustaining pluripotency of ESCs.