Abstract
DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT3 family enzymes, demethylation by the TET-mediated conversion of 5mC to 5-hydroxymethylation (5hmC), and maintenance of the remaining DNAme by DNMT1 are actively repeated throughout cell cycles, dynamically forming a constant 5mC profile. Nevertheless, the detailed mechanism and physiological significance of this active cyclic DNA modification in mouse ESCs remain unclear. Here by visualizing the localization of DNA modifications on metaphase chromosomes and comparing whole-genome methylation profiles before and after the mid-S phase in ESCs lacking Dnmt1 (1KO ESCs), we demonstrated that in 1KO ESCs, DNMT3-mediated remethylation was interrupted during and after DNA replication. This results in a marked asymmetry in the distribution of 5hmC between sister chromatids at mitosis, with one chromatid being almost no 5hmC. When introduced in 1KO ESCs, the catalytically inactive form of DNMT1 (DNMT1CI) induced an increase in DNAme in pericentric heterochromatin and the DNAme-independent repression of IAPEz, a retrotransposon family, in 1KO ESCs. However, DNMT1CI could not restore the ability of DNMT3 to methylate unmodified dsDNA de novo in S phase in 1KO ESCs. Furthermore, during in vitro differentiation into epiblasts, 1KO ESCs expressing DNMT1CI showed an even stronger tendency to differentiate into the primitive endoderm than 1KO ESCs and were readily reprogrammed into the primitive streak via an epiblast-like cell state, reconfirming the importance of DNMT1 enzymatic activity at the onset of epiblast differentiation. These results indicate a novel function of DNMT1, in which DNMT1 actively regulates the timing and genomic targets of de novo methylation by DNMT3 in an enzymatic activity-dependent and independent manner, respectively.
Highlights
DNA methylation (DNAme) is an essential epigenetic mark in mammalian development, and it regulates chromatin structure and gene expression through interaction with other proteins [1,2,3]
We performed IF staining of mitotic chromosomes that were prepared from mouse embryonic stem cells (ESCs) completely lacking the DNMT1 protein (1KO ESCs), and we found that DNMT1 may regulate the timing of DNAme by DNMT3s during the cell cycle
The results demonstrate that DNMT3 family enzymes are basically unable to methylate hemi-hydroxymethylated double-stranded DNA (dsDNA) throughout the cell cycle, and DNMT3s require DNMT1 enzymatic activity to remethylate unmodified newly synthesized dsDNA in euchromatin during the S phase (Fig 6)
Summary
DNA methylation (DNAme) is an essential epigenetic mark in mammalian development, and it regulates chromatin structure and gene expression through interaction with other proteins [1,2,3]. An accessory function that enhances de novo DNMT activity is found in DNMT3B Both the catalytically inactive (CI) forms DNMT3B3, an isoform lacking key exons, and DNMT3BCI, an artificial amino acid substitution, replace DNMT3B function almost completely in mouse embryonic stem cells (ESCs) and mice, respectively [9, 10]. The ubiquitin E3 ligase UHRF1/Np95 (ubiquitin-like with PHD and RING finger domains) that ubiquitinates both PAF15 and histone H3 in the region that is rich in lysine 9-methylated histone H3 (H3K9me2/3) is an essential protein that directs the recruitment of DNMT1 to replicating regions and heterochromatin [14,15,16,17,18]. The cooperative relationship of DNMT-interacting proteins in DNAme and transcriptional regulation is an active field of study, especially with regards to DNMT1 activity
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