Abstract
Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.
Highlights
In the vertebrate genome, DNA methylation is the predominant epigenetic modification
Two independent reports demonstrated the presence of another modification, 5-hydroxymethylcytosine (5-hmC), in murine neuronal and embryonic stem cell genomes [13, 14]
Restriction Enzyme MspI Is Blocked by Glucosylated 5-Hydroxymethylcytosine—The restriction enzyme MspI recognizes the sequence CCGG and is able to cleave when the internal cytosine is unmethylated or methylated at the C5 position [17]
Summary
DNA methylation is the predominant epigenetic modification. C, shown is similar validation for locus-specific methylcytosine (5-mC ϩ 5-hmC) detection in fixed amount of pre-mixed DNAs, as shown in A, using HpaII in the presence of -GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine (5-mC ϩ 5-hmC) is plotted.
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