Global DNA methylation of <i>Dnmt1</i> and <i>Uhrf1</i> double-knockout, or TKO ESC expressing ectopic Dnmt1.

<p>(<b>A</b>) Genome DNA prepared from <i>Dnmt1</i> and <i>Uhrf1</i> conditional double-knockout ESC expressing either no ectopic Dnmt1 (Double F/F), or full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), or Dnmt1(602–1620) clone #1 (602#1) treated without (-) or with OHT (+) for ten days was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel) or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>B</b>) The genome DNA in panel A and Dnmt1(602–1620) clone #5 (602#5) were fragmented by sonication, and then precipitated with MBD1. The precipitated DNA was quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>, averages ± S. D. being shown. ***, p<0.001. (<b>C</b>) Genome DNA prepared from <i>Dnmt1</i> conditional-knockout ESC expressing either no ectopic Dnmt1 (<i>Dnmt1</i>-F/F), Dnmt1(602–1620), Dnmt1(602–1620) with C1229S clones#1 (602-C1229S #1), or clone #11 (602-C1229S #11) treated without (0) or with OHT for four days (4) or ten days (10) was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel), or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>D</b>) Genome DNA prepared from <i>Dnmt1</i> conditional-knockout ESC expressing either no ectopic Dnmt1 (<i>Dnmt1</i>-F/F), or Dnmt1(602–1620) with C1229S clone #1 (602-C1229S #1) or clone #11 (602-C1229S #11) treated without (blue) or with OHT for four days (red) or ten days (green) was fragmented by sonication, and then precipitated with MBD1. The precipitated DNA was quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>, averages ± S. D. being shown. (<b>E</b>) Genome DNA prepared from parent ESC (J1), TKO ESC (TKO), TKO ESC expressing full-length Dnmt1 (FL), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), full-length Dnmt1 and Dnmt3a2-TAP (FL+Dnmt3a2), or (602–1620) and Dnmt3a2-TAP (602+Dnmt3a2) was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel), or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>F</b>) Genome DNA prepared from J1, TKO ESC (TKO), TKO ESC expressing no ectopic Dnmt1 (TKO), or full-length Dnmt1 (FL), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), full-length Dnmt1 and Dnmt3a2-TAP (FL+Dnmt3a2), or Dnmt1(602–1620) and Dnmt3a2-TAP (602+Dnmt3a2) was fragmented by sonication, precipitated with MBD1, and then analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. The value obtained for TKO was considered as no DNA methylation and subtracted from each measurement. Averages ± S. D. are shown.</p

Localization of Dnmt1 at the replication region in ESC.

<p>ESC (<i>Dnmt1</i>-F/F), and ESC expressing no Dnmt1 (<i>Dnmt1</i>-F/F +OHT), or ectopic full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), Dnmt1(602–1620) (602), or full-length Dnmt1 with the mutation of H168R (PBDm) treated with OHT were labeled with EdU, and detected the EdU (green) and Dnmt1 (red), and the images were merged. White bars indicate 5 μm.</p

DNA methylation analyses of the <i>gag</i> of <i>IAP</i>.

<p>The methylation state of the genome DNA prepared from ESC expressing no Dnmt1 (<i>Dnmt1</i> F/F), or full-length Dnmt1 (FL), oocyte-type Dnmt1 (oocyte), Dnmt1(291–1620) (291), or Dnmt1(602–1620) (602) four or ten days after addition of OHT. The methylation states of the <i>gag</i> of <i>IAP</i> are shown with that of the parent cells before (<i>Dnmt1</i>-F/F) and after the OHT treatment (<i>Dnmt1</i>-F/F+OHT). Each horizontal line indicates the CpGs in one analyzed clone. Each circle indicates one CpG site, methylated (filled circles) or un-methylated (open circles). The percentages of methylation are indicated at the top.</p

DNA methylation analyses of the DMR of three imprinted genes.

<p>The methylation states of the DMRs of <i>Rasgrf1</i>, <i>Peg3</i>, and <i>Kcnq1ot1/Lit</i> are shown with that of the parent cells before and after ten days OHT treatment (+OHT 10d). Each horizontal line indicates the CpGs in one analyzed clone. Each circle indicates one CpG site, methylated (filled circles) or un-methylated (open circles). The percentages of methylation are indicated at the top.</p

DNA methylation of ESC expressing full-length Dnmt1 with a mutation in the PBD.

<p>(<b>A</b>) DNA methylation activity of the recombinant full-length Dnmt1 (FL) and that with the H168R mutation (PBDm) was determined. The specific activities (mol/h/mol Dnmt1) towards hemi-methylated (hm) and un-methylated (um) DNA are shown as averages ± S. D. (n = 3). (<b>B</b>) Genome DNA prepared from the cells before and after deletion of the endogenous <i>Dnmt1</i> gene with OHT for ten days was immuno-blotted with anti-methylated cytosine antibody (5mC, upper panel) or stained with methylene blue (DNA, lower panel). The amounts of DNA are shown at the left. (<b>C</b>) Genome DNA prepared as in B was sonicated and precipitated with MBD1, and then quantitated, averages ± S. D. (n = 3) being shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. The values for the parent genome before and after the OHT-treatment were taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g001" target="_blank">Fig 1D</a>. ***, P<0.001. (<b>D</b>) Genome DNA prepared as in B was analyzed as to the methylation state at the <i>IAP</i> and DMR of three imprinted genes, <i>Rasgrf1</i>, <i>Peg3</i>, and <i>Kcnq1ot1/Lit</i>, in PBDm cells as in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137509#pone.0137509.g004" target="_blank">4</a>. The percentages of methylation are indicated at the top.</p

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

Dnmt1, Dnmt3a, Dnmt3b, and Tet1 are recruited to 5hmC-enriched promoters.

<p>The occupancy of Dnmt1, Dnmt3a, and Dnmt3b (<b>A</b>), and Tet1, Tet2, and Tet3 (<b>B</b>) was determined by ChIP-qPCR in the promoters of the 5hmC-enriched genes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082961#pone-0082961-g004" target="_blank">Figure 4</a>. The values are the averages + SD determined for three independent DNA samples.</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

5hmC content is diluted during replication.

<p><b>A</b>. Hemi-hydroxymethylated DNA (CG/5hmCG) is not a good substrate for Dnmt1. The DNA methylation activity of mouse Dnmt1, Dnmt3a, and Dnmt3b towards 35-bp unmethylated (CG/CG), hemi-methylated (CG/5mCG), or hemi-hydroxymethylated (CG/5hmCG) DNA was determined. <b>B</b>. Gel mobility shift assaying of the SRA domain of mouse Uhrf1. The indicated concentrations of SRA were incubated with either 12-bp CG/5mC, CG/5hmCG, or CG/CG, followed by electrophoresis (left panel). The complex of the SRA and ³²P-labeled CG/5mCG was competed with the indicated amounts of non-labeled CG/5mCG, CG/5hmCG, or CG/CG DNA (right panel). DNA bound to SRA (B) and free DNA (F) are indicated. </p