DNA methylation networks underlying mammalian traits

Using DNA methylation profiles (n = 15,456) from 348 mammalian species, we constructed phyloepigenetic trees that bear marked similarities to traditional phylogenetic ones. Using unsupervised clustering across all samples, we identified 55 distinct cytosine modules, of which 30 are related to traits such as maximum life span, adult weight, age, sex, and human mortality risk. Maximum life span is associated with methylation levels in HOXL subclass homeobox genes and developmental processes and is potentially regulated by pluripotency transcription factors. The methylation state of some modules responds to perturbations such as caloric restriction, ablation of growth hormone receptors, consumption of high-fat diets, and expression of Yamanaka factors. This study reveals an intertwined evolution of the genome and epigenome that mediates the biological characteristics and traits of different mammalian species. Description Editor’s summary DNA methylation installs a methyl group to cytosine, placing an epigenetic mark that regulates gene expression. Comparative epigenomics combines epigenetic signatures with phylogenetic relationships to understand species characteristics. Haghani et al. evaluated methylation levels in highly conserved DNA sequences, profiling ~15,000 samples across 348 mammalian species (see the Perspective by de Mendoza). Phylogenetic trees suggest that the divergence of DNA methylation profiles closely mirrors genetic evolution. Species with longer maximum life spans have developed tidier methylation patterns within the genome, characterized by unique peaks and troughs of methylation. Methylation patterns associated with maximum life spans generally differ from those connected to age or interventions that affect mortality risk in mice. These data provide a rich resource of information for fields including evolutionary biology and longevity research. —Di Jiang Phyloepigenetic trees derived from DNA methylation profiles mirror mammalian evolution and are related to traits including maximum life span. INTRODUCTION Comparative epigenomics is an emerging field that combines epigenetic signatures with phylogenetic relationships to elucidate species characteristics such as maximum life span. For this study, we generated cytosine DNA methylation (DNAm) profiles (n = 15,456) from 348 mammalian species using a methylation array platform that targets highly conserved cytosines. RATIONALE Nature has evolved mammalian species of greatly differing life spans. To resolve the relationship of DNAm with maximum life span and phylogeny, we performed a large-scale cross-species unsupervised analysis. Comparative studies in many species enables the identification of epigenetic correlates of maximum life span and other traits. RESULTS We first tested whether DNAm levels in highly conserved cytosines captured phylogenetic relationships among species. We constructed phyloepigenetic trees that paralleled the traditional phylogeny. To avoid potential confounding by different tissue types, we generated tissue-specific phyloepigenetic trees. The high phyloepigenetic-phylogenetic congruence is due to differences in methylation levels and is not confounded by sequence conservation. We then interrogated the extent to which DNA methylation associates with specific biological traits. We used an unsupervised weighted correlation network analysis (WGCNA) to identify clusters of highly correlated CpGs (comethylation modules). WGCNA identified 55 distinct comethylation modules, of which 30 were significantly associated with traits including maximum life span, adult weight, age, sex, human mortality risk, or perturbations that modulate murine life span. Both the epigenome-wide association analysis (EWAS) and eigengene-based analysis identified methylation signatures of maximum life span, and most of these were independent of aging, presumably set at birth, and could be stable predictors of life span at any point in life. Several CpGs that are more highly methylated in long-lived species are located near HOXL subclass homeoboxes and other genes that play a role in morphogenesis and development. Some of these life span–related CpGs are located next to genes that are also implicated in our analysis of upstream regulators (e.g., ASCL1 and SMAD6). CpGs with methylation levels that are inversely related to life span are enriched in transcriptional start site (TSS1) and promoter flanking (PromF4, PromF5) associated chromatin states. Genes located in chromatin state TSS1 are constitutively active and enriched for nucleic acid metabolic processes. This suggests that long-living species evolved mechanisms that maintain low methylation levels in these chromatin states that would favor higher expression levels of genes essential for an organism’s survival. The upstream regulator analysis of the EWAS of life span identified the pluripotency transcription factors OCT4, SOX2, and NANOG. Other factors, such as POLII, CTCF, RAD21, YY1, and TAF1, showed the strongest enrichment for negatively life span–related CpGs. CONCLUSION The phyloepigenetic trees indicate that divergence of DNA methylation profiles closely parallels that of genetics through evolution. Our results demonstrate that DNA methylation is subjected to evolutionary pressures and selection. The publicly available data from our Mammalian Methylation Consortium are a rich source of information for different fields such as evolutionary biology, developmental biology, and aging. DNAm network relates to mammalian phylogeny and traits. (A) Phyloepigenetic tree from the DNAm data generated from blood samples. (B) Unsupervised WGCNA networks identified 55 comethylation modules. (C) EWAS of log-transformed maximum life span. Each dot corresponds to the methylation levels of a highly conserved CpG. Shown is the log (base 10)–transformed P value (y axis) versus the human genome coordinate Hg19 (x axis). (D) Comethylation module correlated with maximum life span of mammals. Eigengene (first principal component of scaled CpGs in the midnightblue module) versus log (base e) transformed maximum life span. Each dot corresponds to a different species.