PMC

BRCA1 provides an example of co-occurrence between protein sequence evolution and gene regulation. The BRCA1 gene shows changes in DNA methylation in a regulatory region upstream of the TSS and a K_A/K_I ratio of 0.69 between human and chimpanzee.

Heat maps showing genus specific differentially methylated CpG sites. We found 2,284 human-specific differentially methylated CpGs, 1,245 specific to Pan species, 1,374 specific to Gorilla species and 5,501 changes specific to Pongo species. Each vertical line represents a single CpG, with each row showing the β-value obtained in each individual tested.

(A) Methylation changes correlate with DNA sequence changes. x-axis shows the number of nucleotide substitutions between two species per kb, y-axis shows the changes in methylation based on β-values. (B) Neighbor-joining tree based on methylation data from probes with a perfect match in all reference genomes (31,853 autosomal CpGs). Bootstrap values (1,000 permutations) are shown for each node.

We performed a comparison of alterations in promoter methylation with (A) the frequency of amino-acid alterations and (B) the relative rate of coding to non-coding variation with genes (K_A/K_I) between human and chimpanzee. Using both metrics we observed a significant association between the rate of protein evolution and epigenetic regulatory changes. P-values are based on 1,000 permutations (Differentially methylated genes, n = 745; genes without significant changes in methylation, n = 6,507).

Differentially methylated sites we identified using whole blood of human and chimpanzee were compared to a previous study that used the Illumina HumanMethylation27 DNA Analysis BeadChip to study liver, kidney and heart tissue in an independent population of humans and chimpanzees . We found a significant trend for sites that are differentially methylated in blood to also show higher human-chimpanzee divergence in these other tissues (yellow box plot, n = 457) suggesting a conservation across other somatic tissues compared to non-differentially methylated sites in blood (grey box plot, n = 7,942).

Each data point represents the mean β-value of the group and whiskers show 2 standard deviations above and below the mean. (A) ARTN is a neurotrophic factor and it shows hypermethylation of 3 CpG sites associated with the long isoform specifically in human. (B) COL2A1 shows hypermethylation of 4 CpG sites at the promoter specifically in human. This gene encodes the alpha-1 chain of type II collagen, which is found primarily in cartilage, the inner ear and the vitreous humor of the eye. (C) PGAM2 shows hypomethylation of CpG sites at the promoter specifically in human. PGAM2 is an enzyme involved in the glycolitic pathway, mutations in which are associated with muscle cramping and intolerance for strenuous exercise. (D) GABBR1 shows a complex methylation pattern in which human and gorilla shows similar pattern of methylation, orangutan shows relative hypomethylation, while chimpanzees and bonobos show increased methylation levels at TSS and intermediate levels associated with the short isoform of this gene. GABBR1 is a neuronal receptor involved in synaptic inhibition, slow wave sleep, muscle relaxation and sensitivity to pain.

(A) Distribution of 99,191 CpG sites interrogated in all great ape species. Left: Gene-centric functional distribution of methylation changes : 1,500 bp upstream of gene TSSs, 200 bp upstream of TSSs, 5′UTR, 1^st exon, gene body, 3′ UTR and intergenic. Right: CpG-island centric distribution: CpG island, shore (±2 kb flanking CpG islands), shelf (2–4 kb from CpG islands). (B) A non-random distribution of methylation changes in recent primate evolution. We observe an excess of differential CpG methylation within the first 1,500 bp upstream of gene TSSs, gene bodies, intergenic regions, shore regions flanking CpG islands and non-CpG island regions. In contrast DNA methylation tends to be relatively conserved close to gene transcription start sites (−200 bp of TSS to 1^st exon), and in the body of CpG islands. Each bar shows the relative enrichment for differential methylation versus that expected under a null distribution. * denotes p<0.0001 (permutation test). (C) Density plot showing the distribution of methylation levels of differentially methylated sites compared to that in the rest of the genome. Sites of evolutionary change among great apes have a significantly different distribution (p = 2.2×10⁻¹⁶ Kolmogorov-Smirnov test).