Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine

Song et al. present the first method for global analysis of 5-hydroxymethylcytosine, a recently identified epigenetic modification in mammalian cells. They use a bacteriophage-derived enzyme to tag the hydroxymethyl group with an azide-modified glucose residue that can be used for affinity purification and sequencing of modified genomic DNA fragments. In contrast to 5-methylcytosine (5-mC), which has been studied extensively1,2,3, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types4,5. 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 recognized4. 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.