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5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

ABSTRACT: CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with an up to 53% decrease in the frequency of C>T mutations in a CpG context compared to 5mC. Tissue specific 5hmC patterns in brain, kidney and blood correlate with lower regional CpG>T mutation frequency in cancers originating in the respective tissues. Together our data reveal global and opposing effects of the two most common cytosine modifications on the frequency of cancer causing somatic mutations in different cell types.

SUBMITTER: Tomkova M

PROVIDER: S-EPMC4931910 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

Tomkova Marketa M McClellan Michael M Kriaucionis Skirmantas S Schuster-Boeckler Benjamin B

eLife 20160516

CpG dinucleotides are the main mutational hot-spot in most cancers. The characteristic elevated C>T mutation rate in CpG sites has been related to 5-methylcytosine (5mC), an epigenetically modified base which resides in CpGs and plays a role in transcription silencing. In brain nearly a third of 5mCs have recently been found to exist in the form of 5-hydroxymethylcytosine (5hmC), yet the effect of 5hmC on mutational processes is still poorly understood. Here we show that 5hmC is associated with ...[more]

PMID: 27183007

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Project description:BackgroundThe discovery that 5-methylcytosine (5mC) can be oxidized to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation (TET) proteins has prompted wide interest in the potential role of 5hmC in reshaping the mammalian DNA methylation landscape. The gold-standard bisulphite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genome-wide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC. In this study, we provide a comparative analysis on brain DNA using three 5hmC genome-wide approaches, namely whole-genome bisulphite/oxidative bisulphite sequencing (WG Bis/OxBis-seq), Infinium HumanMethylation450 BeadChip arrays coupled with oxidative bisulphite (HM450K Bis/OxBis) and antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq). We also perform loci-specific TET-assisted bisulphite sequencing (TAB-seq) for validation of candidate regions.ResultsWe show that whole-genome single-base resolution approaches are advantaged in providing precise 5hmC values but require high sequencing depth to accurately measure 5hmC, as this modification is commonly in low abundance in mammalian cells. HM450K arrays coupled with oxidative bisulphite provide a cost-effective representation of 5hmC distribution, at CpG sites with 5hmC levels >~10%. However, 5hmC analysis is restricted to the genomic location of the probes, which is an important consideration as 5hmC modification is commonly enriched at enhancer elements. Finally, we show that the widely used hMeDIP-seq method provides an efficient genome-wide profile of 5hmC and shows high correlation with WG Bis/OxBis-seq 5hmC distribution in brain DNA. However, in cell line DNA with low levels of 5hmC, hMeDIP-seq-enriched regions are not detected by WG Bis/OxBis or HM450K, either suggesting misinterpretation of 5hmC calls by hMeDIP or lack of sensitivity of the latter methods.ConclusionsWe highlight both the advantages and caveats of three commonly used genome-wide 5hmC profiling technologies and show that interpretation of 5hmC data can be significantly influenced by the sensitivity of methods used, especially as the levels of 5hmC are low and vary in different cell types and different genomic locations.

Project description:BackgroundDNA methylation, which mainly occurs at CpG dinucleotides, is a dynamic epigenetic regulation mechanism in most eukaryotic genomes. It is already known that methylated CpG dinucleotides can lead to a high rate of C to T mutation at these sites. However, less is known about whether and how the methylation level causes a different mutation rate, especially at the single-base resolution.ResultsIn this study, we used genome-wide single-base resolution methylation data to perform a comprehensive analysis of the mutation rate of methylated cytosines from human embryonic stem cell. Through the analysis of the density of single nucleotide polymorphisms, we first confirmed that the mutation rate in methylated CpG sites is greater than that in unmethylated CpG sites. Then, we showed that among methylated CpG sites, the mutation rate is markedly increased in low-intermediately (20-40% methylation level) to intermediately methylated CpG sites (40-60% methylation level) of the human genome. This mutation pattern was observed regardless of DNA strand direction and the sequence coverage over the site on which the methylation level was calculated. Moreover, this highly non-random mutation pattern was found more apparent in intergenic and intronic regions than in promoter regions and CpG islands. Our investigation suggested this pattern appears primarily in autosomes rather than sex chromosomes. Further analysis based on human-chimpanzee divergence confirmed these observations. Finally, we observed a significant correlation between the methylation level and cytosine allele frequency.ConclusionsOur results showed a high mutation rate in low-intermediately to intermediately methylated CpG sites at different scales, from the categorized genomic region, whole chromosome, to the whole genome level, thereby providing the first supporting evidence of mutation rate variation at human methylated CpG sites using the genome-wide sing-base resolution methylation data.

Dataset Information

5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

Publications

5-hydroxymethylcytosine marks regions with reduced mutation frequency in human DNA.

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