Figure 1.
5hmC content is diluted during replication.
A. 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. 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.
Figure 2.
Cell cycle-dependent change in the 5hmC content.
A. 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. The 5hmC content in mESCs sorted by FACS (left panel) was determined (right panel). The values are averages ± SE (n=3). C. 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.
Figure 3.
Dnmt3a and Dnmt3b mainly provide 5mC for the hydroxymethylation in mESCs.
The 5mC, 5hmC, and Tet mRNA contents of J1 parent, Dnmt1 (1-KO), Dnmt3a and Dnmt3b (3-DKO), Dnmt3a (3a-KO), Dnmt3b (3b-KO), and Dnmt1, Dnmt3a and Dnmt3b (TKO) knockout mESCs, and ectopically expressed TAP-tagged Dnmt3a (3a-TAP) or Dnmt3a2 (3a2-TAP) in 3-DKO mESCs were determined. A. The 5mC contents (%) were determined as M.SssI methylation ability from a standard curve (Figure S1A). B. The 5hmC contents were determined from the standard curve obtained on β-GT assaying (Figure S1B). The values are the averages ± SD determined for three independent genomic DNA samples. C. Relative mRNA expression of Tet1, Tet2, and Tet3 was evaluated by semi-quantitative RT-PCR. (-) indicates the product of PCR without the template.
Figure 4.
Enrichment of 5mC and 5hmC in specific promoters.
5hmC (blue bars) and 5mC (red bars) were determined by DNA microarray analysis in the promoters of the Pcdha gene cluster (A), maternal imprinting genes (B), and HoxA gene cluster (C). The abscissas indicate enrichment of 5hmC or 5mC on a log2 scale.
Figure 5.
Dnmt3a and Dnmt3b-dependent 5mC are responsible for the production of 5hmC.
The 5hmC (A) and 5mC (B) contents of J1 (blue bars), Dnmt1 (1-KO, red bars), and Dnmt3a and Dnmt3b (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.
Figure 6.
Dnmt1, Dnmt3a, Dnmt3b, and Tet1 are recruited to 5hmC-enriched promoters.
The occupancy of Dnmt1, Dnmt3a, and Dnmt3b (A), and Tet1, Tet2, and Tet3 (B) was determined by ChIP-qPCR in the promoters of the 5hmC-enriched genes shown in Figure 4. The values are the averages + SD determined for three independent DNA samples.