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

Cuiping Li,Hailin Wang,Shangwei Zhong,Baodong Liu

doi:10.18632/oncotarget.9452

Abstract

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.

Highlights

Mammalian cells demonstrate an amazing plasticity that allows them to develop from one type to the other functionally distinct cells
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 in mouse embryonic stem cells (ESCs)
By the use of highly sensitive approach of ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry coupled with multiple-reaction monitoring (UHPLC-multiple reaction monitoring (MRM)-MS/MS) [13], we observed that the co-treatment of Vc and 2i can dramatically reduce 5mC content of mouse ESCs

Summary

INTRODUCTION

Mammalian cells demonstrate an amazing plasticity that allows them to develop from one type to the other functionally distinct cells. Establishment of ESCs can provide the chance to investigate the developmental processes in vitro [3] Due to their pluripotency, various cell types can be generated for regenerative medicine [3]. As manifested by recent discovery of full chemically induced pluripotent stem cells, neural progenitor cells, and cardiomyocytes [10,11,12], small-molecule compounds show promising applications in reprogramming and trans-differentiation and the potentials in clinical developments. We present a chemical approach to rapidly and effectively promote hypomethylation in mouse embryonic stem cells (ESCs) using two small-molecule compounds. It is known that both vitamin C [13, 14] and 2i (two smallmolecule kinase inhibitors, PD0325901 and CHIR99021) [9, 15] can induce a decrease in genomic 5mC, respectively They exert 5mC erasure through two distinct mechanisms. We will investigate which factor in 2i contributes to the combined DNA demethylation and unveil the mechanisms of action

AND DISCUSSION

MATERIALS AND METHODS

CONFLICTS OF INTEREST