Genome-wide abundance of 5-hydroxymethylcytosine in breast tissue reveals unique function in dynamic gene regulation and carcinogenesis

5-hydroxymethylcytosine (5hmC) is generated by oxidation of 5-methylcytosine (5mC), however little is understood regarding the distribution and functions of 5hmC in mammalian cells. We determined the genome-wide distribution of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC is differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. In independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Our findings provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue and demonstrate that 5hmC is positioned to contribute to gene regulatory functions which protect against carcinogenesis.