Abstract
Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.
Highlights
Cytosine methylation at CpG dinucleotides is a key epigenetic mark with an essential role in regulating gene expression and other cellular and whole body phenotypes
Comparison with previous reports implicates common mechanisms for regulation of cytosine methylation that are highly conserved across species
Of the,40 million CpG dinucleotides covered by mapped reads on either strand in either strain, we focus here on the 10,614,445 CpG dinucleotides that were sequenced at a coverage depth of at least 56 strand-specific reads across a minimum of three animals per strain (Figure S1)
Summary
Cytosine methylation at CpG dinucleotides is a key epigenetic mark with an essential role in regulating gene expression and other cellular and whole body phenotypes. While the molecular mechanisms for de novo and maintenance methylation of CpG cytosines are well established [1,2,3], allele-specific influences on CpG methylation have been documented [4,5,6,7,8,9,10,11,12], and association of genotype and epigenotype has been shown in plants recently [13], the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation in mammals are poorly characterised. Isogenic inbred lines provide a powerful platform for establishing relationships between the germline genome and downstream phenotypes. We have generated extensive genetic, genomic and physiological resources in our studies of the SHR, including expression datasets [14,15]
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