Divergent whole-genome methylation maps of human and chimpanzee brains reveal epigenetic basis of human regulatory evolution

Abstract

DNA methylation is a pervasive epigenetic DNA modification that strongly affects chromatin regulation and gene expression. To date, it remains largely unknown how patterns of DNA methylation differ between closely related species and whether such differences contribute to species-specific phenotypes. To investigate these questions, we generated nucleotide-resolution whole-genome methylation maps of the prefrontal cortex of multiple humans and chimpanzees. Levels and patterns of DNA methylation vary across individuals within species according to the age and the sex of the individuals. We also found extensive species-level divergence in patterns of DNA methylation and that hundreds of genes exhibit significantly lower levels of promoter methylation in the human brain than in the chimpanzee brain. Furthermore, we investigated the functional consequences of methylation differences in humans and chimpanzees by integrating data on gene expression generated with next-generation sequencing methods, and we found a strong relationship between differential methylation and gene expression. Finally, we found that differentially methylated genes are strikingly enriched with loci associated with neurological disorders, psychological disorders, and cancers. Our results demonstrate that differential DNA methylation might be an important molecular mechanism driving gene-expression divergence between human and chimpanzee brains and might potentially contribute to the evolution of disease vulnerabilities. Thus, comparative studies of humans and chimpanzees stand to identify key epigenomic modifications underlying the evolution of human-specific traits.

Figure 1

Differences in DNA-Methylation Levels among Human Tissues and Genomic Features (A) Proportional representation of genome-wide DNA-methylation levels for individual CG dinucleotides in the human prefrontal cortex (brain), ESCs, neonatal fibroblasts, and PBMCs. (B) Same analyses as in (A) but for CH dinucleotide context (H = A, T, or C). (C) Mean methylation levels in each tissue for gene promoters (CG context, n = 18,416; CH context, n = 18,584), gene bodies (CG context, n = 18,477; CH context, n = 18,656), and transposable elements (CG context, n = 1,837,431; CH context, n = 2,989,765). Horizontal lines indicate global means of methylation levels for individual CG sites (main panel) or CH sites (inset). Error bars indicate 95% confidence intervals of the mean.

Figure 2

Between- and Within-Species Variation of Genomic DNA Methylation in Human and Chimpanzee Prefrontal Cortex Regions Principal-component analyses of (A) promoters and (B) gene bodies of human-chimpanzee orthologs demonstrate that the patterns of DNA methylation are distinct between humans and chimpanzees. For promoters, the first principal component, which explains 46.1% of variation, distinguishes samples from human and chimpanzees. The second principal component, explaining 27.7% of total variation, separates two human samples from the third one. For gene bodies, the first principal component (explaining 42.8% of total variation) separates the third human from the rest, whereas the second principal component (explaining 22.6% of total variation) separates the human and chimpanzee brains. Hierarchical clustering analyses of (C) promoters and (D) gene bodies demonstrate that the overall levels of methylation are lower in human brains than in the chimpanzee brains. The youngest human individual (H3) exhibits the most distinctive pattern of DNA methylation. The error bars indicate 95% confidence intervals of the mean.

Figure 3

Patterns of DNA Methylation in Genic Regions Influence Gene Expression Density plots of (A) promoter and (B) gene-body DNA methylation from humans and chimpanzees. Promoter DNA methylation exhibits distinctive “bimodal” patterns previously observed. In comparison, gene bodies of both species are heavily methylated (B). DNA-methylation-level differences, measured as the mean of human methylation levels minus the mean of chimpanzee methylation levels, show that promoters particularly exhibit lower levels of DNA methylation in the human brain than in the chimpanzee brain (C). In contrast, gene bodies show similar levels of DNA methylation between species (D).

Figure 4

DNA Methylation Is Negatively Correlated with Gene-Expression Level in Both Promoters and Gene Bodies in the Prefrontal Cortex Integrating levels of DNA methylation with levels of gene expression measured by digital gene-expression profiling, we observe a negative correlation between human gene-expression level and both (A) human promoter methylation (Spearman’s correlation coefficient r = −0.24, p < 10⁻¹⁵) and (B) human gene-body methylation (r = −0.18, p < 10⁻¹⁵). The x axis represents increasing levels of gene expression from left to right. We also observe a negative correlation between chimpanzee gene-expression level and both (C) chimpanzee promoter methylation (r = −0.19, p < 10⁻¹⁵) and (D) chimpanzee gene-body methylation (r = −0.20, p < 10⁻¹⁵).

Figure 5

Differences in Promoter Methylation Are Associated with Differences in Gene Expression between the Human and Chimpanzee Prefrontal Cortex (A) The proportion of genes with higher or lower expression values in humans, as compared to chimpanzees, in the prefrontal cortex. Each bar represents a class of genes and is based on the number of standard deviations from the mean of methylation measures in human versus chimpanzee promoters in the prefrontal cortex. (B) Selected genes with hypomethylated promoters in humans, hypermethylated promoters in chimpanzees, and significantly higher expression in humans than in chimpanzees. Error bars represent 95% confidence intervals of the mean (n = 6). Gamma-aminobutyric acid (GABA) A receptor, beta 1 (GABRB1 [MIM 137190]) is involved in neurotransmission of the CNS. Clock homolog (mouse) (CLOCK [MIM 601851]) encodes a transcription factor essential to the circadian rhythm. SCN8A facilitates the generation of action potentials in neurons and other cells. Growth hormone receptor (GHR [MIM 600946]) is integral to activating insulin-like growth-factor production, leading to growth. IGFBP7 regulates insulin-like growth-factor availability and receptor binding.