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

John P Thomson,Federico Bolognani,Dirk Schübeler,Colm E Nestor,Edward J Oakeley,Arne Müller,Richard R Meehan,Rémi Terranova,Diana Reinhardt,Jamie A Hackett,Harri Lempiäinen,Jonathan G Moggs

doi:10.1186/gb-2012-13-10-r93

Abstract

BackgroundInduction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital.ResultsExposure 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.ConclusionsCollectively, 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.

Highlights

Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation
Enrichment was validated using quantitative PCR at candidate loci previously identified as being marked by the 5hmC modification [26] (Additional files 2 and 3)
The 28-day 5hmC and 5mC raw data files have been deposited with Gene Expression Omnibus (GEO series number [GSE40540])

Summary

Introduction

Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. We investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. Methylation of the fifth carbon of a cytosine base (5methylcytosine (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]. Recent work investigating epigenetic reprogramming events in the mouse zygote support this hypothesis through the finding that the rapid active demethylation seen in the paternal pro-nuclei is accompanied by an accumulation of genome-wide 5hmC and its derivatives in the absence of cell division [6,32,33]

Methods

Results

Conclusion