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. 2022 Jan 5;17(1):e0262277.
doi: 10.1371/journal.pone.0262277. eCollection 2022.

DNMT1 regulates the timing of DNA methylation by DNMT3 in an enzymatic activity-dependent manner in mouse embryonic stem cells

Takamasa Ito  1 , Musashi Kubiura-Ichimaru  1 , Yuri Murakami  1 , Aaron B Bogutz  2 , Louis Lefebvre  2 , Isao Suetake  3   4 , Shoji Tajima  4 , Masako Tada  1
Affiliations

DNMT1 regulates the timing of DNA methylation by DNMT3 in an enzymatic activity-dependent manner in mouse embryonic stem cells

Takamasa Ito et al. PLoS One. .

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. DNMT1 regulates de novo DNA methylation region and timing via DNMT3s.
(A) Immunofluorescence (IF) staining patterns for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) on metaphase chromosomes in DNMT1-deficient ESCs. WT, wild-type ESCs; 1KO, Dnmt1-/- ESCs.; DKO, Dnmt3a-/- and Dnmt3b-/- ESCs; TET TKO, Tet1-/-, Tet2-/-, and Tet3-/- ESCs. (B) Chromosomes show a typical IF staining pattern for 5mC and 5hmC in WT and 1KO ESCs. The dotted line indicates the space between two sister chromatids on a chromosome. (C) Schematic diagram showing the cell cycle information for the cell population that was used in this study. satDNA, pericentromeric heterochromatin rich in satellite repeats. (D) The late replicating region labeled with BrdU in S phase stains clearly with anti-BrdU antibody staining on both chromatids of each chromosome. (E) Mitotic cells were classified into the three categories: cells with most chromosomes that were equally positive for 5hmC on both sister chromatids (+/+); cells with the most chromosomes that were positive for 5hmC on one of the sister chromatids (+/−); cells that were mostly negative for 5hmC with some +/−chromosomes (+/−, −/−) (n = 66 WT and n = 51 1KO metaphases).
Fig 2
Fig 2. Restricted de novo DNA methylation after DNA replication in 1KO ESCs.
(A) Cells were subdivided and collected using fluorescence-activated cell sorting (FACS) into the following two populations: either in G1 to early S phase (G1) or in late S to M via G2 phase (G2). Using MeDIP-seq and hMeDIP-seq, the distribution patterns of 5mC and 5hmC in the G1 and G2 populations were compared. (B) Differences in DNA modification patterns between G1 to G2. Chromosome 1 is shown as an example. The vertical axis takes a positive value when G1 is higher and a negative value when the G2 is higher. (C) Line plots showing the difference in 5mC and 5hmC between G1 and G2 of 1KO ESCs around ATAC-seq and histone modification peaks (±2.5 kb). Sequencing data from WT ESCs (GSE90895) was used to identify peaks for ATAC-seq and each type of histone modification.
Fig 3
Fig 3. DNMT1CI, a catalytic null mutant of DNMT1, increases pericentric DNA methylation in 1KO ESCs.
(A) DNMT1 domains and their interacting proteins. In DNMT1CI, the C1229S mutation is introduced in the catalytic domain by a single base substitution. (B) Dot blot analysis showing increased levels of 5mC and 5hmC in 1KO+1CI ESCs compared to 1KO ESCs. (C) Quantification of the relative intensities of the 5mC and 5hmC signals obtained by dot blot analysis (n = 2; three blots). The average signal intensity obtained by WT ESCs was set to 1. (D) Chromosomes showing the typical IF staining patterns for 5mC and 5hmC in 1KO+1CI ESCs. A dotted line indicates a gap of two sister chromatids in the chromosome. (E) Increased 5mC levels around centromeres in 1KO+1CI ESCs detected by IF staining (***p<0.001).
Fig 4
Fig 4. IAPEz-int repression without apparent acquisition of DNA methylation in 1KO+1CI ESCs.
(A) DNAme levels of repeat sequences detected by targeted bisulfite sequencing in ESCs. Black and white circles mean methylated and unmethylated CpG, respectively. (B) The scatter plot shows the repressive effect of DNMT1CI on repetitive sequence expression in 1KO ESCs. Red dots, repeat sequence families, which are up-regulated in 1KO ESCs compared to WT ESCs (1KO / WT, log2 FC > 0.5), but down-regulated in 1KO+1CI ESCs compared to 1KO ESCs (1KO+1CI / 1KO, log2 FC < −0.5). (C) IAPEz-int expression located at Gm40354. A vertical axis, FPKM; a horizontal axis, corresponding genomic position. (D) IAPEz-int expression levels in WT, 1KO, and 1KO+1CI ESCs (***p<0.001).
Fig 5
Fig 5. No positive effect of DNMT1CI on the differentiation potency of 1KO ESCs.
(A) The ESC differentiation procedure used here. Embryoid body (EB) formation initiates the differentiation of ESCs into epiblast-like cells (EpiLCs). Adherent cell culture of 1-day-old EBs was continued for 3 days to induce functional epiblast maturation. (B) Cell differentiation potency was compared between WT, 1KO, and 1KO+1CI ESCs based on the gene expression profile detected by RNA-seq analysis. The heatmap shows Z-scores representing a relative difference in RNA expression levels in each gene between samples. 1KO+1CI cells are more likely to differentiate into primitive endoderm-like cells rather than EpiLCs. (C) Expression profiles of genes related to epigenetic regulation. (D) IF staining patterns for 5mC and 5hmC on metaphase chromosomes showing global DNAme changes during 4-day cell differentiation in WT, 1KO, and 1KO+1CI cells.
Fig 6
Fig 6. A model of DNA methylation regulation predicted from 5mC and 5hmC IF staining patterns in WT and 1KO ESCs.
CH, hemi-hydroxymethylated double-stranded DNA (dsDNA); HH, fully hydroxymethylated dsDNA; CC, unmodified dsDNA.
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Funds from the JSPS supported this work to MT (Grant 24613004) and MK (DC1, 2610904) https://www-shinsei.jsps.go.jp/kaken/english/index.html; and from the CIHR (PJT-165992) to LL, https://cihr-irsc.gc.ca/e/37788.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.