Normal breast tissue 5-hydroxymethylcytosine is enriched at DNA regulatory elements of breast cancer cells

DNA methylation is an established modulator of gene expression in normal mammalian cells. Emerging evidence suggests that the oxidized 5-methylcytosine (5mC) product, 5-hydroxymethylcytosine (5hmC), has distinct biological functions and its dysregulation may contribute to cancer pathogenesis. While 5hmC levels in normal breast tissue have been shown to be elevated relative to several other tissue types, we lack a complete knowledge of the genomic distribution of 5hmC in breast tissue, as well as its potential biological functions. Here, we determined the genome-wide distribution of 5hmC and 5mC using methylation array data from 18 normal breast tissue samples. To disambiguate 5hmC from 5mC, we performed parallel processing of bisulfite and oxidative-bisulfite treated DNA, followed by statistical estimation of the 5mC and 5hmC proportions. Based on the distribution of 5hmC, we identified genomic loci with consistently elevated levels of 5hmC across normal breast tissues. In addition, we showed 5hmC is enriched within transcriptionally active chromatin in breast myoepithelial cells, marked by H3K4 trimethylation. Conversely, 5hmC was depleted within active promoters (H3K9ac-marked DNA) and transcriptionally inactive chromatin (H3K9me3 and H3K27me3-marked DNA). Importantly, the loci with high 5hmC in normal breast tissue were enriched among regulatory elements, including transcription factor binding sites, in transformed normal-like breast MCF10A cells, as well as in multiple breast cancer cell lines. These findings provide a genome-wide distribution of 5hmC in normal breast tissue. Furthermore, these findings identify DNA-regulatory regions in breast cancer cell lines that show statistical enrichment for those sites with elevated 5hmC in normal breast tissue, suggesting deregulation of 5hmC within these elements may contribute to breast carcinogenesis. Citation Format: Owen Michael Wilkins, Kevin C. Johnson, E. Andres Houseman, Jessica E. King, Carmen J. Marsit, Brock C. Christensen. Normal breast tissue 5-hydroxymethylcytosine is enriched at DNA regulatory elements of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5322.