Cost-effective solutions for high-throughput enzymatic DNA methylation sequencing.

Characterizing DNA methylation patterns is important for addressing key questions in evolutionary biology, geroscience, and medical genomics. While costs are decreasing, whole-genome DNA methylation profiling remains prohibitively expensive for most population-scale studies, creating a need for cost-effective, reduced representation approaches (i.e., assays that rely on microarrays, enzyme digests, or sequence capture to target a subset of the genome). Most common whole genome and reduced representation techniques rely on bisulfite conversion, which can damage DNA resulting in DNA loss and sequencing biases. Enzymatic methyl sequencing (EM-seq) was recently proposed to overcome these issues, but thorough benchmarking of EM-seq combined with cost-effective, reduced representation strategies has not yet been performed. To do so, we optimized Targeted Methylation Sequencing protocol (TMS)-which profiles ∼4 million CpG sites-for miniaturization, flexibility, and multispecies use at a cost of ∼$80. First, we tested modifications to increase throughput and reduce cost, including increasing multiplexing, decreasing DNA input, and using enzymatic rather than mechanical fragmentation to prepare DNA. Second, we compared our optimized TMS protocol to commonly used techniques, specifically the Infinium MethylationEPIC BeadChip (n=55 paired samples) and whole genome bisulfite sequencing (n=6 paired samples). In both cases, we found strong agreement between technologies (R² = 0.97 and 0.99, respectively). Third, we tested the optimized TMS protocol in three non-human primate species (rhesus macaques, geladas, and capuchins). We captured a high percentage (mean=77.1%) of targeted CpG sites and produced methylation level estimates that agreed with those generated from reduced representation bisulfite sequencing (R² = 0.98). Finally, we applied our protocol to profile age-associated DNA methylation variation in two subsistence-level populations-the Tsimane of lowland Bolivia and the Orang Asli of Peninsular Malaysia-and found age-methylation patterns that were strikingly similar to those reported in high income cohorts, despite known differences in age-health relationships between lifestyle contexts. Altogether, our optimized TMS protocol will enable cost-effective, population-scale studies of genome-wide DNA methylation levels across human and non-human primate species.

Uncovering methylation-dependent genetic effects on regulatory element function in diverse genomes.

A major goal in evolutionary biology and biomedicine is to understand the complex interactions between genetic variants, the epigenome, and gene expression. However, the causal relationships between these factors remain poorly understood. mSTARR-seq, a methylation-sensitive massively parallel reporter assay, is capable of identifying methylation-dependent regulatory activity at many thousands of genomic regions simultaneously, and allows for the testing of causal relationships between DNA methylation and gene expression on a region-by-region basis. Here, we developed a multiplexed mSTARR-seq protocol to assay naturally occurring human genetic variation from 25 individuals sampled from 10 localities in Europe and Africa. We identified 6,957 regulatory elements in either the unmethylated or methylated state, and this set was enriched for enhancer and promoter annotations, as expected. The expression of 58% of these regulatory elements was modulated by methylation, which was generally associated with decreased RNA expression. Within our set of regulatory elements, we used allele-specific expression analyses to identify 8,020 sites with genetic effects on gene regulation; further, we found that 42.3% of these genetic effects varied between methylated and unmethylated states. Sites exhibiting methylation-dependent genetic effects were enriched for GWAS and EWAS annotations, implicating them in human disease. Compared to datasets that assay DNA from a single European individual, our multiplexed assay uncovers dramatically more genetic effects and methylation-dependent genetic effects, highlighting the importance of including diverse individuals in assays which aim to understand gene regulatory processes.

Immune gene regulation is associated with age and environmental adversity in a nonhuman primate.

Phenotypic aging is ubiquitous across mammalian species, suggesting shared underlying mechanisms of aging. Aging is linked to molecular changes to DNA methylation and gene expression, and environmental factors, such as severe external challenges or adversities, can moderate these age-related changes. Yet, it remains unclear whether environmental adversities affect gene regulation via the same molecular pathways as chronological, or 'primary', aging. Investigating molecular aging in naturalistic animal populations can fill this gap by providing insight into shared molecular mechanisms of aging and the effects of a greater diversity of environmental adversities - particularly those that can be challenging to study in humans or laboratory organisms. Here, we characterised molecular aging - specifically, CpG methylation - in a sample of free-ranging rhesus macaques living off the coast of Puerto Rico (n samples = 571, n individuals = 499), which endured a major hurricane during our study. Age was associated with methylation at 78,661 sites (31% of all sites tested). Age-associated hypermethylation occurred more frequently in areas of active gene regulation, while hypomethylation was enriched in regions that show less activity in immune cells, suggesting these regions may become de-repressed in older individuals. Age-associated hypomethylation also co-occurred with increased chromatin accessibility while hypermethylation showed the opposite trend, hinting at a coordinated, multi-level loss of epigenetic stability during aging. We detected 32,048 CpG sites significantly associated with exposure to a hurricane, and these sites overlapped age-associated sites, most strongly in regulatory regions and most weakly in quiescent regions. Together, our results suggest that environmental adversity may contribute to aging-related molecular phenotypes in regions of active gene transcription, but that primary aging has specific signatures in non-regulatory regions.

Diet composition impacts eQTL discovery across multiple tissues in baboons.

Understanding how genetic variation impacts gene expression is a major goal of genomics; however, only a fraction of disease-associated loci have been demonstrated to impact gene expression when cells are in an unperturbed "steady state." In this issue of Cell Genomics, Lin et al.1 investigate how exposure to a particular cellular context (i.e., a high-cholesterol, high-fat diet) can enhance our ability to identify new regulatory variants through longitudinal sampling of three tissue types in the baboon.

The biology of aging in a social world: Insights from free-ranging rhesus macaques.

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques (Macaca mulatta). We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes.

Early life adversity has sex-dependent effects on survival across the lifespan in rhesus macaques.

Exposure to adversity during early life is linked to lasting detrimental effects on evolutionary fitness across many taxa. However, due to the challenges of collecting longitudinal data, especially in species where one sex disperses, direct evidence from long-lived species remains relatively scarce. Here we test the effects of early life adversity on male and female longevity in a free-ranging population of rhesus macaques (Macaca mulatta) at Cayo Santiago, Puerto Rico. We leveraged six decades of data to quantify the relative importance of ten forms of early life adversity for 6,599 macaques (3,230 male, 3,369 female), with a smaller sample size (N=299) for one form of adversity (maternal social isolation) which required high-resolution behavioral data. We found that individuals who experienced more early life adversity died earlier than those who experienced less adversity. Mortality risk was highest during early life, defined as birth to four years old, suggesting acute survival effects of adversity, but heightened mortality risk was also present in macaques who survived to adulthood. Females and males were affected differently by some forms of adversity, and these differences might be driven by varying energetic demands, female philopatry, and male dispersal. By leveraging data on thousands of macaques collected over decades, our results show that the fitness consequences of early life adversity are not uniform across individuals but vary as a function of the type of adversity, timing, and social context, and thus contribute to our limited but growing understanding of the evolution of early life sensitivities in long-lived species.

Natural Animal Populations as Model Systems for Understanding Early Life Adversity Effects on Aging.

Adverse experiences in early life are associated with aging-related disease risk and mortality across many species. In humans, confounding factors, as well as the difficulty of directly measuring experiences and outcomes from birth till death, make it challenging to identify how early life adversity impacts aging and health. These challenges can be mitigated, in part, through the study of non-human animals, which are exposed to parallel forms of adversity and can age similarly to humans. Furthermore, studying the links between early life adversity and aging in natural populations of non-human animals provides an excellent opportunity to better understand the social and ecological pressures that shaped the evolution of early life sensitivities. Here, we highlight ongoing and future research directions that we believe will most effectively contribute to our understanding of the evolution of early life sensitivities and their repercussions.

The biology of aging in a social world:insights from free-ranging rhesus macaques.

Social adversity can increase the age-associated risk of disease and death, yet the biological mechanisms that link social adversities to aging remain poorly understood. Long-term naturalistic studies of nonhuman animals are crucial for integrating observations of social behavior throughout an individual's life with detailed anatomical, physiological, and molecular measurements. Here, we synthesize the body of research from one such naturalistic study system, Cayo Santiago Island, which is home to the world's longest continuously monitored free-ranging population of rhesus macaques. We review recent studies of age-related variation in morphology, gene regulation, microbiome composition, and immune function. We also discuss ecological and social modifiers of age-markers in this population. In particular, we summarize how a major natural disaster, Hurricane Maria, affected rhesus macaque physiology and social structure and highlight the context-dependent and domain-specific nature of aging modifiers. Finally, we conclude by providing directions for future study, on Cayo Santiago and elsewhere, that will further our understanding of aging across different domains and how social adversity modifies aging processes.

Relationship between genome-wide and MHC class I and II genetic diversity and complementarity in a nonhuman primate.

Although mate choice is expected to favor partners with advantageous genetic properties, the relative importance of genome-wide characteristics, such as overall heterozygosity or kinship, versus specific loci, is unknown. To disentangle genome-wide and locus-specific targets of mate choice, we must first understand congruence in global and local variation within the same individual. This study compares genetic diversity, both absolute and relative to other individuals (i.e., complementarity), assessed across the genome to that found at the major histocompatibility complex (MHC), a hyper-variable gene family integral to immune system function and implicated in mate choice across species. Using DNA from 22 captive olive baboons (Papio anubis), we conducted double digest restriction site-associated DNA sequencing to estimate genome-wide heterozygosity and kinship, and sequenced two class I and two class II MHC loci. We found that genome-wide diversity was not associated with MHC diversity, and that diversity at class I MHC loci was not correlated with diversity at class II loci. Additionally, kinship was a significant predictor of the number of MHC alleles shared between dyads at class II loci. Our results provide further evidence of the strong selective pressures maintaining genetic diversity at the MHC in comparison to other randomly selected sites throughout the genome. Furthermore, our results indicate that class II MHC disassortative mate choice may mediate inbreeding avoidance in this population. Our study suggests that mate choice favoring genome-wide genetic diversity is not always synonymous with mate choice favoring MHC diversity, and highlights the importance of controlling for kinship when investigating MHC-associated mate choice.

Equivocal evidence for a link between megalencephaly-related genes and primate brain size evolution.

A large brain is a defining feature of modern humans, and much work has been dedicated to exploring the molecular underpinnings of this trait. Although numerous studies have focused on genes associated with human microcephaly, no studies have explicitly focused on genes associated with megalencephaly. Here, we investigate 16 candidate genes that have been linked to megalencephaly to determine if: (1) megalencephaly-associated genes evolved under positive selection across primates; and (2) selection pressure on megalencephaly-associated genes is linked to primate brain size. We found evidence for positive selection for only one gene, OFD1, with 1.8% of the sites estimated to have dN/dS values greater than 1; however, we did not detect a relationship between selection pressure on this gene and brain size across species, suggesting that selection for changes to non-brain size traits drove evolutionary changes to this gene. In fact, our primary analyses did not identify significant associations between selection pressure and brain size for any candidate genes. While we did detect positive associations for two genes (GPC3 and TBC1D7) when two phyletic dwarfs (i.e., species that underwent recent evolutionary decreases in brain size) were excluded, these associations did not withstand FDR correction. Overall, these results suggest that sequence alterations to megalencephaly-associated genes may have played little to no role in primate brain size evolution, possibly due to the highly pleiotropic effects of these genes. Future comparative studies of gene expression levels may provide further insights. This study enhances our understanding of the genetic underpinnings of brain size evolution in primates and identifies candidate genes that merit further exploration.

Alpha male status and availability of conceptive females are associated with high glucocorticoid concentrations in high-ranking male rhesus macaques (Macaca mulatta) during the mating season.

The relationship between male mating opportunities, stress, and glucocorticoid concentrations is complicated by the fact that physiological stress and glucocorticoid concentrations can be influenced by dominance rank, group size, and the stability of the male dominance hierarchy, along with ecological factors. We studied the three highest-ranking males in nine different social groups within the same free-ranging population of rhesus macaques on Cayo Santiago, Puerto Rico, during the mating season, to examine variation in glucocorticoid concentrations in relation to number of females that conceived each month, alpha status, number of adult males in a group, and male rank hierarchy stability. We found that glucocorticoid concentrations were highest in the early mating season period when more females conceived in each group and declined linearly as the mating season progressed and the number of conceptive females decreased. Alpha males had significantly higher mean monthly glucocorticoid concentrations than other high-ranking males throughout the study period. Male age, number of adult males in a group, and hierarchy stability were not significantly associated with glucocorticoid concentrations. Our findings suggest that alpha males may experience significantly higher levels of physiological stress than their immediate subordinates and that this stress coincides with the period of the mating season when most conceptions occur.