Abstract
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 de novo-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the HoxA 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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5-Methylcytosine / analogs & derivatives
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Animals
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CCAAT-Enhancer-Binding Proteins
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Cell Cycle*
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Cell Proliferation
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Cell Separation
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CpG Islands
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Cytosine / analogs & derivatives*
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Cytosine / chemistry
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DNA (Cytosine-5-)-Methyltransferase 1
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DNA (Cytosine-5-)-Methyltransferases / genetics
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DNA / analysis
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DNA Methylation
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DNA Methyltransferase 3A
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DNA Methyltransferase 3B
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DNA Replication
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Embryonic Stem Cells / cytology*
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Flow Cytometry
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Mice
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Mice, Knockout
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Multigene Family
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Nuclear Proteins / genetics
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Promoter Regions, Genetic
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Protein Structure, Tertiary
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Ubiquitin-Protein Ligases
Substances
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CCAAT-Enhancer-Binding Proteins
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Dnmt3a protein, mouse
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Nuclear Proteins
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5-hydroxymethylcytosine
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5-Methylcytosine
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Cytosine
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DNA
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DNA (Cytosine-5-)-Methyltransferase 1
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DNA (Cytosine-5-)-Methyltransferases
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DNA Methyltransferase 3A
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Dnmt1 protein, mouse
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Ubiquitin-Protein Ligases
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Uhrf1 protein, mouse
Grants and funding
This work was supported by Grant-in-Aids from the Ministry of Education, Science, Sport and Culture to ST, and the Japan Society for the Promotion of Science to ST and IS. IS and MS were supported by a grant from CREST, Japan Science and Technology Agency. JO was supported by the Japan Society for the Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.