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

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.     

Parental 5-methylcytosine methylation patterns are stable upon inter-species hybridization of Xiphophorus (Teleostei: Poeciliidae) fish

Cytosine methylation appears to be established as an important DNA base modification involved in regulation of gene expression but is poorly understood from an evolutionary viewpoint. Xiphophorus progeny from inter-species crosses and backcrosses that are utilized in contemporary tumor induction studies were analyzed for cytosine methylation pattern inheritance using Southern blot analyses. Methylation patterns at CCGG sequences of 411 independent chromosomes in three distinct inter-species crosses were analyzed. In every case the non-recurrent parental methylation pattern remained unaltered for each of the genes studied, once introduced into the recurrent parental genetic background. Through F₁ inter-species hybridization and succeeding meiosises leading to first generation (BC₁) and second generation (BC₂) backcross hybrid progeny, we demonstrate that parental species methylation patterns are stable.     

Robust prediction of gene regulation in colorectal cancer tissues from DNA methylation profiles

DNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92<AUC<0.97) as well as in individual patient data (average AUC = 0.82), suggesting a robust interplay between methylation and gene regulation. Validation analysis in other cancerous tissues resulted in lower prediction performances (0.69<AUC<0.90); however, it identified genes that share robust dependencies across cancerous tissues. In conclusion, we present a robust classification approach that predicts the gene-specific regulation through DNA methylation in CRC tissues with possible transition to different cancer entities. Furthermore, we present HMGA1 as consistently associated with methylation across cancers, suggesting a potential candidate for DNA methylation targeting cancer therapy.     

Investigation of the role of epigenetic modification of the rat glucokinase gene in fetal programming

Fetal malnutrition is associated with development of impaired glucose tolerance, diabetes and hypertension in later life in humans and several mammalian species. The mechanisms that underlie this phenomenon of fetal programming are unknown. We hypothesize that adverse effects in utero and early life may influence the basal expression levels of certain genes such that they are re-set with long-term consequences for the organism. An excellent candidate mechanism for this re-setting process is DNA methylation, since post-natal methylation patterns are largely established in utero. We have sought to test this hypothesis by investigating the glucokinase gene (Gck) in rat offspring programmed using a maternal low protein diet model (MLP). Northern blot reveals that fasting levels of Gck expression are reduced after programming, although this distinction disappears after feeding. Bisulphite sequencing of the hepatic Gck promoter indicates a complete absence of methylation at the 12 CpG sites studied in controls and MLP animals. Non-expressing cardiac tissue also showed no DNA methylation in this region, whereas brain and all fetal tissues were fully methylated. These findings are not consistent with the hypothesis that programming results from differential methylation of Gck. However, it remains possible that programming may influence methylation patterns in Gck at a distance from the promoter, or in genes encoding factors that regulate basal Gck expression.     

Gene Regulation in Primates Evolves under Tissue-Specific Selection Pressures

Regulatory changes have long been hypothesized to play an important role in primate evolution. To identify adaptive regulatory changes in humans, we performed a genome-wide survey for genes in which regulation has likely evolved under natural selection. To do so, we used a multi-species microarray to measure gene expression levels in livers, kidneys, and hearts from six humans, chimpanzees, and rhesus macaques. This comparative gene expression data allowed us to identify a large number of genes, as well as specific pathways, whose inter-species expression profiles are consistent with the action of stabilizing or directional selection on gene regulation. Among the latter set, we found an enrichment of genes involved in metabolic pathways, consistent with the hypothesis that shifts in diet underlie many regulatory adaptations in humans. In addition, we found evidence for tissue-specific selection pressures, as well as lower rates of protein evolution for genes in which regulation evolves under natural selection. These observations are consistent with the notion that adaptive circumscribed changes in gene regulation have fewer deleterious pleiotropic effects compared with changes at the protein sequence level.     

Genome-Wide DNA Methylation Patterns and Transcription Analysis in Sheep Muscle

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.     

Dynamics of DNA Methylation in Recent Human and Great Ape Evolution

DNA methylation is an epigenetic modification involved in regulatory processes such as cell differentiation during development, X-chromosome inactivation, genomic imprinting and susceptibility to complex disease. However, the dynamics of DNA methylation changes between humans and their closest relatives are still poorly understood. We performed a comparative analysis of CpG methylation patterns between 9 humans and 23 primate samples including all species of great apes (chimpanzee, bonobo, gorilla and orangutan) using Illumina Methylation450 bead arrays. Our analysis identified ∼800 genes with significantly altered methylation patterns among the great apes, including ∼170 genes with a methylation pattern unique to human. Some of these are known to be involved in developmental and neurological features, suggesting that epigenetic changes have been frequent during recent human and primate evolution. We identified a significant positive relationship between the rate of coding variation and alterations of methylation at the promoter level, indicative of co-occurrence between evolution of protein sequence and gene regulation. In contrast, and supporting the idea that many phenotypic differences between humans and great apes are not due to amino acid differences, our analysis also identified 184 genes that are perfectly conserved at protein level between human and chimpanzee, yet show significant epigenetic differences between these two species. We conclude that epigenetic alterations are an important force during primate evolution and have been under-explored in evolutionary comparative genomics.     

Tissue-Specific Effects of Genetic and Epigenetic Variation on Gene Regulation and Splicing

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans’ lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.     

Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

The evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human (Homo sapiens) and six chimpanzee (Pan troglodytes) induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.     

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.     

DNA methylation in health, disease, and cancer

The spatial arrangement and three-dimensional structure of DNA in the nucleus is controlled through the interdigitation of DNA binding proteins such as histones and their modifiers, the Polycomb-Trithorax proteins, and the DNA methyltransferase enzymes. DNA methylation forms the foundation of chromatin and is crucial to epigenetic gene regulation in mammals. Disease pathogenesis mediated through infectious agents, inflammation, aging, or genetic damage often involves changes in gene expression. In particular, cellular transformation coincides with multiple changes in chromatin architecture, many of which appear to affect genome integrity and gene expression. Infectious agents, such as viruses directly affect genome structure and induce methylation of particular sequences to suppress host immune responses. Hyperproliferative tissues such as those in the gastrointestinal tract and colon have been shown to gradually acquire aberrant promoter hypermethylation. Here we review recent findings on altered DNA methylation in human disease, with particular focus on cancer and the increasingly large number of genes subject to tumor-specific promoter hypermethylation and the possible role of aberrant methylation in tumor development.     

Epigenetics: differential DNA methylation in mammalian somatic tissues

Epigenetics refers to heritable phenotypic alterations in the absence of DNA sequence changes, and DNA methylation is one of the extensively studied epigenetic alterations. DNA methylation is an evolutionally conserved mechanism to regulate gene expression in mammals. Because DNA methylation is preserved during DNA replication it can be inherited. Thus, DNA methylation could be a major mechanism by which to produce semi-stable changes in gene expression in somatic tissues. Although it remains controversial whether germ-line DNA methylation in mammalian genomes is stably heritable, frequent tissue-specific and disease-specific de novo methylation events are observed during somatic cell development/differentiation. In this minireview, we discuss the use of restriction landmark genomic scanning, together with in silico analysis, to identify differentially methylated regions in the mammalian genome. We then present a rough overview of quantitative DNA methylation patterns at 4600 NotI sites and more than 150 differentially methylated regions in several C57BL/6J mouse tissues. Comparative analysis between mice and humans suggests that some, but not all, tissue-specific differentially methylated regions are conserved. A deeper understanding of cell-type-specific differences in DNA methylation might lead to a better illustration of the mechanisms behind tissue-specific differentiation in mammals.     

CpG island methylation pattern in different human tissues and its correlation with gene expression

The study on DNA methylation pattern in different human tissues attracts increasing interest nowadays, but a systematic analysis of CpG island methylation pattern between both somatic tissues and gametocyte is still lacking. In this work, we analyzed the CpG island methylation data of sperm and other 11 somatic tissues from Human Epigenome Project, and found that the CpG island methylation profiles are highly correlated between somatic tissues, while the methylation profile in sperm is quite distinct. Furthermore, we observed that in the six tissues investigated, there is no obvious correlation between the methylation level of promoter CpG islands and corresponding gene expression across different tissues.