The Role of 5-hydroxymethylcytosine and DNA Demethylation in Cancer
In mammals DNA methylation commonly occurs at CpG residues (5MeC). Methylation of CpG rich promoter regions is associated with gene repression, whereas methylation of gene bodies is associated with gene activation. One of the unresolved question in the field is what determines which CpG sites are methylated and which CpG sites remain unmethylated during development and conversely what determines the gross alteration of methylation patterns in cancer. The recent discovery of 5-hydroxymethylcytosine (5hMeC) provides a new “twist” to this question and potential dynamic relationship between DNA methylation and gene expression. Due to the structural similarity between 5MeC and 5hMeC, these bases are experimentally almost indistinguishable; in fact the most common techniques for detecting 5MeC, such as enzymatic approaches and bisulphite sequencing cannot distinguish 5hMeC from 5MeC. Consequently, the potential role of 5hMeC has received little attention to date, but notably 5hMeC is proposed to play a key role in demethylation of DNA. Recently a new family of proteins, the TET (Ten-Eleven-Translocation) proteins have been characterized that convert 5MeC to 5hMeC. We find that TET3 is over-expressed in prostate cancer and in this study we now aim to address the presence and function of 5hMeC in the balance between DNA methylation and demethylation in prostate cancer. To interrogate the role of 5hMeC, we have optimized the oxidative bisulphite sequencing technique (Booth et al., Science 2012) to enable nucleotide resolution of 5hMeC. In addition, we have performed an enrichment approach using affinity-based antibodies to specifically enrich for 5hMeC containing DNA, to reveal the genome-wide distribution of 5hMeC relative to 5MeC. Here we present our data and the current challenges of the technologies to map 5hMeC in cancer cells.