Abstract
It has long been known that methylated cytosines deaminate at higher rates than unmodified cytosines and constitute mutational hotspots in mammalian genomes. The repertoire of naturally occurring cytosine modifications, however, extends beyond 5-methylcytosine to include its oxidation derivatives, notably 5-hydroxymethylcytosine. The effects of these modifications on sequence evolution are unknown. Here, we combine base-resolution maps of methyl- and hydroxymethylcytosine in human and mouse with population genomic, divergence and somatic mutation data to show that hydroxymethylated and methylated cytosines show distinct patterns of variation and evolution. Surprisingly, hydroxymethylated sites are consistently associated with elevated C to G transversion rates at the level of segregating polymorphisms, fixed substitutions, and somatic mutations in tumors. Controlling for multiple potential confounders, we find derived C to G SNPs to be 1.43-fold (1.22-fold) more common at hydroxymethylated sites compared to methylated sites in human (mouse). Increased C to G rates are evident across diverse functional and sequence contexts and, in cancer genomes, correlate with the expression of Tet enzymes and specific components of the mismatch repair pathway (MSH2, MSH6, and MBD4). Based on these and other observations we suggest that hydroxymethylation is associated with a distinct mutational burden and that the mismatch repair pathway is implicated in causing elevated transversion rates at hydroxymethylated cytosines.
Highlights
In mammalian genomes, most cytosines that occur in a CpG context are methylated. 5-methylcytosines (5mCs) at CpG dinucleotides exhibit mutation rates an order of magnitude above that of unmodified cytosines, a consequence both of their greater propensity to deaminate and error-prone repair of the resulting thymine [1]
It has become clear that the repertoire of naturally occurring cytosine modifications in mammals extends beyond 5mC to include a series of modifications derived from successive rounds of 5mC oxidation: 5-hydroxymethylcytosine (5hmC), 5formylcytosine (5fC), and 5-carboxylcytosine (5caC) [8,9]. 5fC and 5caC have been found to occur at low frequencies in genome-wide studies in human and mouse (,0.01–0.0001% of cytosines [10]), consistent with being rapidly converted intermediates in an active demethylation pathway that involves cumulative oxidation of 5mC by Tet enzymes and the eventual removal of 5fC or 5caC via base excision repair (BER) [11]
In the context of the high mutational burden of 5mC and considering that 5hmC can be present as a stable epigenetic mark, we wondered whether methylated and hydroxymethylated sites might be associated with distinct patterns of sequence evolution, perhaps as a consequence of divergent mutational biases
Summary
Most cytosines that occur in a CpG context are methylated. 5-methylcytosines (5mCs) at CpG dinucleotides exhibit mutation rates an order of magnitude above that of unmodified cytosines, a consequence both of their greater propensity to deaminate and error-prone repair of the resulting thymine [1]. 5-methylcytosines (5mCs) at CpG dinucleotides exhibit mutation rates an order of magnitude above that of unmodified cytosines, a consequence both of their greater propensity to deaminate and error-prone repair of the resulting thymine [1]. This mutational liability is evident in higher levels of single nucleotide polymorphisms (SNPs) segregating at CpGs in mammalian populations [2,3,4], higher rates of divergence between species at these sites [5,6], and higher somatic mutation rates in many cancer genomes compared to other nucleotide contexts [7]. In mammalian systems, repair of 5hmU:G mismatches (derived from 5hmC deamination) by the glycosylases TDG and SMUG1 is less error-prone than dealing with 5mC-derived T:G
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