Passive DNA demethylation preferentially up-regulates pluripotency-related genes and facilitates the generation of induced pluripotent stem cells

Abstract A high proliferation rate has been observed to facilitate somatic cell reprogramming, but the pathways that connect proliferation and reprogramminghave not been reported. DNA methyltransferase1 ( DNMT1) methylates hemimethylated CpG sitesproduced during S phase and maintains stable inheritance of DNA methylation. Impairing this process results in passive DNA demethylation. In this study, we show that the cell proliferation rate positively correlated with the expression of Dnmt1in G1 phase. In addition, as determined by whole-genome bisulfate sequencing and high-performance liquid chromatography, global DNA methylationof mouse embryonic fibroblastswas significantly higher in G1 phasethan in G2/M phase. Thus, we suspected that high cellular proliferation requires more Dnmt1expression in G1 phaseto prevent passive DNA demethylation. The methylation differences of individual CpG sitesbetween G1 and G2/M phase were related to the methylation status and the positions of their surrounding CpG sites. In addition, larger methylation differences were observed on the promoters of pluripotency-related genes; for example, Oct4, Nanog, Sox2, Esrrb, Cdh1, and Epcam. When such methylation differences or passive DNA demethylationaccumulated with Dnmt1suppression and proliferation acceleration, DNA methylationon pluripotency-related genes was decreased, and their expression was up-regulated, which subsequently promoted pluripotency and mesenchymal–epithelial transition, a necessary step for reprogramming. We infer that high cellular proliferation rates promote generation of induced pluripotent stem cellsat least partially by inducing passive DNA demethylationand up-regulating pluripotency-related genes. Therefore, these results uncover a connection between cell reprogrammingand DNA methylation.