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In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Original publication

DOI

10.1038/nbt.1732

Type

Journal article

Journal

Nat Biotechnol

Publication Date

01/2011

Volume

29

Pages

68 - 72

Keywords

5-Methylcytosine, Animals, Bacteriophage T4, Biotin, Cerebellum, Cytosine, DNA, Genome, Genome, Human, Glucosyltransferases, HEK293 Cells, HeLa Cells, Humans, Mice, Models, Molecular, Molecular Sequence Data, Sequence Analysis, DNA, Staining and Labeling