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.     

Sexually divergent DNA methylation patterns with hippocampal aging

DNA methylation is a central regulator of genome function, and altered methylation patterns are indicative of biological aging and mortality. Age‐related cellular, biochemical, and molecular changes in the hippocampus lead to cognitive impairments and greater vulnerability to neurodegenerative disease that varies between the sexes. The role of hippocampal epigenomic changes with aging in these processes is unknown as no genome‐wide analyses of age‐related methylation changes have considered the factor of sex in a controlled animal model. High‐depth, genome‐wide bisulfite sequencing of young (3 month) and old (24 month) male and female mouse hippocampus revealed that while total genomic methylation amounts did not change with aging, specific sites in CG and non‐CG (CH) contexts demonstrated age‐related increases or decreases in methylation that were predominantly sexually divergent. Differential methylation with age for both CG and CH sites was enriched in intergenic and intronic regions and under‐represented in promoters, CG islands, and specific enhancer regions in both sexes, suggesting that certain genomic elements are especially labile with aging, even if the exact genomic loci altered are predominantly sex‐specific. Lifelong sex differences in autosomal methylation at CG and CH sites were also observed. The lack of genome‐wide hypomethylation, sexually divergent aging response, and autosomal sex differences at CG sites was confirmed in human data. These data reveal sex as a previously unappreciated central factor of hippocampal epigenomic changes with aging. In total, these data demonstrate an intricate regulation of DNA methylation with aging by sex, cytosine context, genomic location, and methylation level.     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Insects as innovative models for functional studies of DNA methylation

The emerging field of epigenomics has the potential to bridge the gap between static genomic sequences and complex phenotypes that arise from multigenic, nonlinear and often context-dependent interactions. However, this goal can only be achieved if easily manageable experimental systems are available in which changes in epigenomic settings can be evaluated in the context of the phenotype under investigation. Recent progress in the characterization of insect DNA methylation patterns enables evaluation of the extent to which epigenetic mechanisms contribute to complex phenotypes in easily accessible organisms whose relatively small genomes are not only sparingly methylated, but the methylated sites are also found almost exclusively in gene bodies. The implementation of insect models in the study of DNA methylation will accelerate progress in understanding the functional significance of this important epigenetic mechanism in controlling gene splicing, in environmentally driven reprogramming of gene expression and in adult brain plasticity.     

DNA methylation,an epigenetic mechanism connecting folate to healthy embryonic development and aging

Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our lifetime by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging, and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review, we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation.     

An integrative pan-cancer-wide analysis of epigenetic enzymes reveals universal patterns of epigenomic deregulation in cancer

BackgroundOne of the most important recent findings in cancer genomics is the identification of novel driver mutations which often target genes that regulate genome-wide chromatin and DNA methylation marks. Little is known, however, as to whether these genes exhibit patterns of epigenomic deregulation that transcend cancer types.ResultsHere we conduct an integrative pan-cancer-wide analysis of matched RNA-Seq and DNA methylation data across ten different cancer types. We identify seven tumor suppressor and eleven oncogenic epigenetic enzymes which display patterns of deregulation and association with genome-wide cancer DNA methylation patterns, which are largely independent of cancer type. In doing so, we provide evidence that genome-wide cancer hyper- and hypo- DNA methylation patterns are independent processes, controlled by distinct sets of epigenetic enzyme genes. Using causal network modeling, we predict a number of candidate drivers of cancer DNA hypermethylation and hypomethylation. Finally, we show that the genomic loci whose DNA methylation levels associate most strongly with expression of these putative drivers are highly consistent across cancer types.ConclusionsThis study demonstrates that there exist universal patterns of epigenomic deregulation that transcend cancer types, and that intra-tumor levels of genome-wide DNA hypomethylation and hypermethylation are controlled by distinct processes.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0699-9) contains supplementary material, which is available to authorized users.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

Genetic architecture,epigenetic influence and environment exposure in the pathogenesis of Autism

Autism spectrum disorder(ASD) is a spectral neurodevelopment disorder affecting approximately 1% of the population. ASD is characterized by impairments in reciprocal social interaction, communication deficits and restricted patterns of behavior. Multiple factors, including genetic/genomic, epigenetic/epigenomic and environmental, are thought to be necessary for autism development. Recent reviews have provided further insight into the genetic/genomic basis of ASD. It has long been suspected that epigenetic mechanisms, including DNA methylation, chromatin structures and long non-coding RNAs may play important roles in the pathology of ASD. In addition to genetic/genomic alterations and epigenetic/epigenomic influences, environmental exposures have been widely accepted as an important role in autism etiology, among which immune dysregulation and gastrointestinal microbiota are two prominent ones.     

Human-specific CpG “beacons” identify loci associated with human-specific traits and disease

Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10⁻³) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10⁻³). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.     

Epigenetic changes in B lymphocytes associated with house dust mite allergic asthma

Although there is no doubt about the influence of the genetic background in the onset of the allergic diseases, Epigenome-Wide Association Studies are needed to elucidate the possible relationship between allergic diseases and epigenomic dysregulation. In this study we aimed to analyze the epigenetic patterns, in terms of DNA methylation, of three well-characterized populations of house dust mite allergic subjects, aspirin-intolerant asthmatics and controls. As a first, genome-wide phase, we used the HELP assay to study the methylation patterns in CD19 (+) B lymphocytes in these populations, and found that there are reproducible epigenetic differences at limited numbers of loci distinguishing the groups, corroborated by bisulphite MassArray in a second validation phase of an expanded 40 subject group. These validated epigenetic changes occur at loci characterized as important for the immune response. One such locus is a new candidate gene, CYP26A1, which shows differential methylation patterns and expression levels between groups. Our results suggest that epigenomic dysregulation may contribute to the susceptibility to allergic diseases, showing for the first time differences in DNA methylation between allergic and non-allergic healthy subjects, both globally and at specific loci. These observations indicate that the epigenome may offer new pathophysiological insights and therapeutic targets in atopic diseases.     

Human-specific CpG “beacons” identify loci associated with human-specific traits and disease

Regulatory change has long been hypothesized to drive the delineation of the human phenotype from other closely related primates. Here we provide evidence that CpG dinucleotides play a special role in this process. CpGs enable epigenome variability via DNA methylation, and this epigenetic mark functions as a regulatory mechanism. Therefore, species-specific CpGs may influence species-specific regulation. We report non-polymorphic species-specific CpG dinucleotides (termed “CpG beacons”) as a distinct genomic feature associated with CpG island (CGI) evolution, human traits and disease. Using an inter-primate comparison, we identified 21 extreme CpG beacon clusters (≥ 20/kb peaks, empirical p < 1.0 × 10^?3) in humans, which include associations with four monogenic developmental and neurological disease related genes (Benjamini-Hochberg corrected p = 6.03 × 10^?3). We also demonstrate that beacon-mediated CpG density gain in CGIs correlates with reduced methylation in these species in orthologous CGIs over time, via human, chimpanzee and macaque MeDIP-seq. Therefore mapping into both the genomic and epigenomic space the identified CpG beacon clusters define points of intersection where a substantial two-way interaction between genetic sequence and epigenetic state has occurred. Taken together, our data support a model for CpG beacons to contribute to CGI evolution from genesis to tissue-specific to constitutively active CGIs.     

Epigenetic changes in B lymphocytes associated with house dust mite allergic asthma

Although there is no doubt about the influence of the genetic background in the onset of the allergic diseases, Epigenome-Wide Association Studies are needed to elucidate the possible relationship between allergic diseases and epigenomic dysregulation. In this study we aimed to analyze the epigenetic patterns, in terms of DNA methylation, of three well-characterized populations of house dust mite allergic subjects, aspirin-intolerant asthmatics and controls. As a first, genome-wide phase, we used the HELP assay to study the methylation patterns in CD19⁺ B lymphocytes in these populations, and found that there are reproducible epigenetic differences at limited numbers of loci distinguishing the groups, corroborated by bisulphite MassArray in a second validation phase of an expanded 40 subject group. These validated epigenetic changes occur at loci characterized as important for the immune response. One such locus is a new candidate gene, CYP26A1, which shows differential methylation patterns and expression levels between groups. Our results suggest that epigenomic dysregulation may contribute to the susceptibility to allergic diseases, showing for the first time differences in DNA methylation between allergic and non-allergic healthy subjects, both globally and at specific loci. These observations indicate that the epigenome may offer new pathophysiological insights and therapeutic targets in atopic diseases.     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

DNA methylation variation along the cancer epigenome and the identification of novel epigenetic driver events

While large-scale studies applying various statistical approaches have identified hundreds of mutated driver genes across various cancer types, the contribution of epigenetic changes to cancer remains more enigmatic. This is partly due to the fact that certain regions of the cancer genome, due to their genomic and epigenomic properties, are more prone to dysregulated DNA methylation than others. Thus, it has been difficult to distinguish which promoter methylation changes are really driving carcinogenesis from those that are mostly just a reflection of their genomic location. By developing a novel method that corrects for epigenetic covariates, we reveal a small, concise set of potential epigenetic driver events. Interestingly, those changes suggest different modes of epigenetic carcinogenesis: first, we observe recurrent inactivation of known cancer genes across tumour types suggesting a higher convergence on common tumour suppressor pathways than previously anticipated. Second, in prostate cancer, a cancer type with few recurrently mutated genes, we demonstrate how the epigenome primes tumours towards higher tolerance of other aberrations.     

Aberrant epigenetic landscape in cancer: how cellular identity goes awry

Appropriate patterns of DNA methylation and histone modifications are required to assure cell identity, and their deregulation can contribute to human diseases, such as cancer. Our aim here is to provide an overview of how epigenetic factors, including genomic DNA methylation, histone modifications, and microRNA regulation, contribute to normal development, paying special attention to their role in regulating tissue-specific genes. In addition, we summarize how these epigenetic patterns go awry during human cancer development. The possibility of "resetting" the abnormal cancer epigenome by applying pharmacological or genetic strategies is also discussed.