Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome

Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters.Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.Methylation of the fifth carbon of a cytosine base (5-methylcytosine (5mC)) in the dinucleotide sequence CpG is a well-established epigenetic modification of vertebrate DNA thought to have important roles in the preservation of genomic integrity, allele-specific expression of imprinted genes, maintenance of X-chromosome inactivation in females, tissue-specific gene regulation and long-term silencing of genes and retrotransposable elements [1,2]. Until recently, incorporation of a methyl group was thought to be the only form of direct DNA modification in the mammalian genome. However, landmark studies by two groups in 2009 re-discovered the modified base 5-hydroxymethylcytosine (5hmC) in mouse purkinje cells and granule neurons [3,4], a mark initially found over 50 years ago in T2 phage [5]. Shortly after this work, it was shown that a group of enzymes belonging to the TET family (TET1,2 & 3) of Fe(II) and α-KG-dependent dioxygenases utilize molecular oxygen to transfer a hydroxyl group to 5mC to form 5hmC [4,6-9]. In human cancers, the TET genes were found to exhibit a substantial reduction in their expression level