Overall reduction in 5hmC levels during the S phase of DNMT1-deficient mouse embryonic stem cells

DNA methylation is an essential for genome stability and mammalian embryonic development. DNA methyltransferase 1 (DNMT1) maintains cellular DNA methylation profile and its loss causes embryonic lethality. In this study, immunofluorescence staining, which can compare the difference between sister chromatids, were performed in mouse embryonic stem cells (ESCs) and suggest that the timing and region of de novo methylation by DNMT3 are restricted in DNMT1-deficient ESCs. To reanalyze the timing of de novo methylation by DNMT3, we collected cells from G1 to early S phases (called G1), cells in the mid-S phase, and cells in late S, G2, and M phases (called G2) by FACS, respectively. Among them, two cell populations, G1 and G2, were used for Me/hMeDIP-seq. As a result, the 5mC and 5hmC were globally decreased in the G2 cells. MeDIP-seq and hmeDIP-seq were performed on DNMT1 KO mESCs in G1 and G2, respectively.