Tissue-specific distribution and dynamic changes of 5-hydroxymethylcytosine in mammalian genomes

Shannon Morey Kinney, Hang Gyeong Chin, Romualdas Vaisvila, Jurate Bitinaite, Yu Zheng, Pierre Olivier Estève, Suhua Feng, Hume Stroud, Steven E. Jacobsen, Sriharsa Pradhan

Research output: Contribution to journalArticlepeer-review

148 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)24685-24693
Number of pages9
JournalJournal of Biological Chemistry
Issue number28
StatePublished - Jul 15 2011
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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