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Cell Cycle-Dependent Turnover of 5-Hydroxymethyl Cytosine in Mouse Embryonic Stem Cells

<div><p>Hydroxymethylcytosine in the genome is reported to be an intermediate of demethylation. In the present study, we demonstrated that maintenance methyltransferase Dnmt1 scarcely catalyzed hemi-hydroxymethylated DNA and that the hemi-hydroxymethylated DNA was not selectively recognized by the SRA domain of Uhrf1, indicating that hydroxymethylcytosine is diluted in a replication-dependent manner. A high level of 5-hydroxymethylcytosine in mouse embryonic stem cells was produced from the methylcytosine supplied mainly by <i>de novo</i>-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the <i>HoxA</i> gene cluster showed a high hydroxymethylation level whilst the methylcytosine level was quite low, suggesting that methylated CpG is actively hydroxylated during proliferation. All the results indicate that removal and production of hydroxymethylcytosine are regulated in replication-dependent manners in mouse embryonic stem cells.</p> </div

Cell cycle-dependent change in the 5hmC content.

<p><b>A</b>. The 5hmC content in mESCs treated with aphidicolin, hydroxyurea, serum depletion, or nocodazole was determined by β-GT assaying. The values represent the fold change normalized as to that without treatment. The values for each treatment are averages ± SD (n=3). <b>B</b>. The 5hmC content in mESCs sorted by FACS (left panel) was determined (right panel). The values are averages ± SE (n=3). <b>C</b>. The non-synchronized (w/o S) and synchronized mESCs were collected after the indicated times and the 5hmC contents were determined. The left panels show the results of FACS analyses and the right panel the 5hmC content.</p

Enrichment of 5mC and 5hmC in specific promoters.

<p>5hmC (blue bars) and 5mC (red bars) were determined by DNA microarray analysis in the promoters of the <i>Pcdha</i> gene cluster (<b>A</b>), maternal imprinting genes (<b>B</b>), and <i>HoxA</i> gene cluster (<b>C</b>). The abscissas indicate enrichment of 5hmC or 5mC on a log₂ scale.</p

Dnmt3a and Dnmt3b-dependent 5mC are responsible for the production of 5hmC.

<p>The 5hmC (<b>A</b>) and 5mC (<b>B</b>) contents of J1 (blue bars), <i>Dnmt1</i> (1-KO, red bars), and <i>Dnmt3a</i> and <i>Dnmt3b</i> (3-DKO, light green bars) knockout mESCs were determined by q-PCR in the promoters of five representative 5hmC-enriched genes. The values are the averages + SD determined for three independent genomic DNA samples. </p