Human DNA methylation signatures differentiate persistent from resolving MRSA bacteremia.

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is life threatening and occurs in up to 30% of MRSA bacteremia cases despite appropriate antimicrobial therapy. Isolates of MRSA that cause antibiotic-persistent methicillin-resistant S. aureus bacteremia (APMB) typically have in vitro antibiotic susceptibilities equivalent to those causing antibiotic-resolving methicillin-resistant S. aureus bacteremia (ARMB). Thus, persistence reflects host-pathogen interactions occurring uniquely in context of antibiotic therapy in vivo. However, host factors and mechanisms involved in APMB remain unclear. We compared DNA methylomes in circulating immune cells from patients experiencing APMB vs. ARMB. Overall, methylation signatures diverged in the distinct patient cohorts. Differentially methylated sites intensified proximate to transcription factor binding sites, primarily in enhancer regions. In APMB patients, significant hypomethylation was observed in binding sites for CCAAT enhancer binding protein-ß (C/EBPß) and signal transducer/activator of transcription 1 (STAT1). In contrast, hypomethylation in ARMB patients localized to glucocorticoid receptor and histone acetyltransferase p300 binding sites. These distinct methylation signatures were enriched in neutrophils and achieved a mean area under the curve of 0.85 when used to predict APMB using a classification model. These findings validated by targeted bisulfite sequencing (TBS-seq) differentiate epigenotypes in patients experiencing APMB vs. ARMB and suggest a risk stratification strategy for antibiotic persistence in patients treated for MRSA bacteremia.

The methylome of buccal epithelial cells is influenced by age, sex, and physiological properties.

Epigenetic modifications, particularly DNA methylation, have emerged as regulators of gene expression and are implicated in various biological processes and disease states. Understanding the factors influencing the epigenome is essential for unraveling its complexity. In this study, we aimed to identify how the methylome of buccal epithelial cells, a noninvasive and easily accessible tissue, is associated with demographic and health-related variables commonly used in clinical settings, such as age, sex, blood immune composition, hemoglobin levels, and others. We developed a model to assess the association of multiple factors with the human methylome and identify the genomic loci significantly impacted by each trait. We demonstrated that DNA methylation variation is accurately modeled by several factors. We confirmed the well-known impact of age and sex and unveiled novel clinical factors associated with DNA methylation, such as blood neutrophils, hemoglobin, red blood cell distribution width, high-density lipoprotein cholesterol, and urea. Genomic regions significantly associated with these traits were enriched in relevant transcription factors, drugs, and diseases. Among our findings, we showed that neutrophil-impacted loci were involved in neutrophil functionality and maturation. Similarly, hemoglobin-influenced sites were associated with several diseases, including aplastic anemia, and the genomic loci affected by urea were related to congenital anomalies of the kidney and urinary tract. Our findings contribute to a better understanding of the human methylome plasticity and provide insights into novel factors shaping DNA methylation patterns, highlighting their potential clinical implications as biomarkers and the importance of considering these physiological traits in future medical epigenomic investigations.NEW & NOTEWORTHY We have developed a quantitative model to assess how the human methylome is associated with several factors and to identify the genomic loci significantly impacted by each trait. We reported novel health-related factors driving DNA methylation patterns and new site-specific regulations that further elucidate methylome dynamics. Our study contributes to a better understanding of the plasticity of the human methylome and unveils novel physiological traits with a potential role in future medical epigenomic investigations.

Targeted bisulfite sequencing for biomarker discovery.

Cytosine methylation is one of the best studied epigenetic modifications. In mammals, DNA methylation patterns vary among cells and is mainly found in the CpG context. DNA methylation is involved in important processes during development and differentiation and its dysregulation can lead to or is associated with diseases, such as cancer, loss-of-imprinting syndromes and neurological disorders. It has been also shown that DNA methylation at the cellular, tissue and organism level varies with age. To overcome the costs of Whole-Genome Bisulfite Sequencing, the gold standard method to detect 5-methylcytosines at a single base resolution, DNA methylation arrays have been developed and extensively used. This method allows one to assess the status of a fraction of the CpG sites present in the genome of an organism. In order to combine the relatively low cost of Methylation Arrays and digital signals of bisulfite sequencing, we developed a Targeted Bisulfite Sequencing method that can be applied to biomarker discovery for virtually any phenotype. Here we describe a comprehensive step-by-step protocol to build a DNA methylation-based epigenetic clock.

Epigenome-wide association in adipose tissue from the METSIM cohort.

Most epigenome-wide association studies to date have been conducted in blood. However, metabolic syndrome is mediated by a dysregulation of adiposity and therefore it is critical to study adipose tissue in order to understand the effects of this syndrome on epigenomes. To determine if natural variation in DNA methylation was associated with metabolic syndrome traits, we profiled global methylation levels in subcutaneous abdominal adipose tissue. We measured association between 32 clinical traits related to diabetes and obesity in 201 people from the Metabolic Syndrome in Men cohort. We performed epigenome-wide association studies between DNA methylation levels and traits, and identified associations for 13 clinical traits in 21 loci. We prioritized candidate genes in these loci using expression quantitative trait loci, and identified 18 high confidence candidate genes, including known and novel genes associated with diabetes and obesity traits. Using methylation deconvolution, we examined which cell types may be mediating the associations, and concluded that most of the loci we identified were specific to adipocytes. We determined whether the abundance of cell types varies with metabolic traits, and found that macrophages increased in abundance with the severity of metabolic syndrome traits. Finally, we developed a DNA methylation-based biomarker to assess type 2 diabetes risk in adipose tissue. In conclusion, our results demonstrate that profiling DNA methylation in adipose tissue is a powerful tool for understanding the molecular effects of metabolic syndrome on adipose tissue, and can be used in conjunction with traditional genetic analyses to further characterize this disorder.

Scl binds to primed enhancers in mesoderm to regulate hematopoietic and cardiac fate divergence.

Scl/Tal1 confers hemogenic competence and prevents ectopic cardiomyogenesis in embryonic endothelium by unknown mechanisms. We discovered that Scl binds to hematopoietic and cardiac enhancers that become epigenetically primed in multipotent cardiovascular mesoderm, to regulate the divergence of hematopoietic and cardiac lineages. Scl does not act as a pioneer factor but rather exploits a pre-established epigenetic landscape. As the blood lineage emerges, Scl binding and active epigenetic modifications are sustained in hematopoietic enhancers, whereas cardiac enhancers are decommissioned by removal of active epigenetic marks. Our data suggest that, rather than recruiting corepressors to enhancers, Scl prevents ectopic cardiogenesis by occupying enhancers that cardiac factors, such as Gata4 and Hand1, use for gene activation. Although hematopoietic Gata factors bind with Scl to both activated and repressed genes, they are dispensable for cardiac repression, but necessary for activating genes that enable hematopoietic stem/progenitor cell development. These results suggest that a unique subset of enhancers in lineage-specific genes that are accessible for regulators of opposing fates during the time of the fate decision provide a platform where the divergence of mutually exclusive fates is orchestrated.

Novel features of telomere biology revealed by the absence of telomeric DNA methylation.

Cytosine methylation regulates the length and stability of telomeres, which can affect a wide variety of biological features, including cell differentiation, development, or illness. Although it is well established that subtelomeric regions are methylated, the presence of methylated cytosines at telomeres has remained controversial. Here, we have analyzed multiple bisulfite sequencing studies to address the methylation status of Arabidopsis thaliana telomeres. We found that the levels of estimated telomeric DNA methylation varied among studies. Interestingly, we estimated higher levels of telomeric DNA methylation in studies that produced C-rich telomeric strands with lower efficiency. However, these high methylation estimates arose due to experimental limitations of the bisulfite technique. We found a similar phenomenon for mitochondrial DNA: The levels of mitochondrial DNA methylation detected were higher in experiments with lower mitochondrial read production efficiencies. Based on experiments with high telomeric C-rich strand production efficiencies, we concluded that Arabidopsis telomeres are not methylated, which was confirmed by methylation-dependent restriction enzyme analyses. Thus, our studies indicate that telomeres are refractory to de novo DNA methylation by the RNA-directed DNA methylation machinery. This result, together with previously reported data, reveals that subtelomeric DNA methylation controls the homeostasis of telomere length.

Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

Macrophages (Mϕs) of patients with Alzheimer's disease and mild cognitive impairment (MCI) are defective in amyloid-ß1-42 (Aß) phagocytosis and have low resistance to apoptosis by Aß. Omega-3 fatty acids (ω-3s) in vitro and in vivo and the ω-3 mediator, resolvin D1, in vitro increase Aß phagocytosis by Mϕs of patients with MCI. We have investigated the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress by Mϕs in a longitudinal study of fish-derived, ω-3-supplemented patients with MCI. Patients in the apolipoprotein E (ApoE)e3/e3 subgroup over time exhibited an increase of protein kinase RNA-like ER kinase (PERK) expression, Aß phagocytosis, intermediate M1-M2 Mϕ type, and a Mini-Mental State Examination (MMSE) rate of change of +1.8 points per year, whereas patients in the ApoEe3/e4 subgroup showed individually divergent results with an MMSE rate of change of -3.2 points per year. In vitro treatment of Mϕs by fish-derived ω-3 emulsion increased Aß phagocytosis, PERK expression, and UPR RNA signature, and decreased ER stress signature. Augmented genes in the UPR signature included chaperones, lectins, foldases, and N-linked glycosylation enzymes. In summary, fish-derived ω-3s increase cytoprotective genes and decrease proapoptotic genes, improve immune clearance of Aß, and are associated with an improved MMSE rate of change in ApoEe3/e3 vs. ApoEe3/e4 patients.-Olivera-Perez, H. M., Lam, L., Dang, J., Jiang, W., Rodriguez, F., Rigali, E., Weitzman, S., Porter, V., Rubbi, L., Morselli, M., Pellegrini, M., Fiala, M. Omega-3 fatty acids increase the unfolded protein response and improve amyloid-ß phagocytosis by macrophages of patients with mild cognitive impairment.

DNA Methylation Indicates Susceptibility to Isoproterenol-Induced Cardiac Pathology and Is Associated With Chromatin States.

RATIONALE: Only a small portion of the known heritability of cardiovascular diseases, such as heart failure, can be explained based on single-gene mutations. Chromatin structure and regulation provide a substrate through which genetic differences in noncoding regions may affect cellular function and response to disease, but the mechanisms are unknown. OBJECTIVE: We conducted genome-wide measurements of DNA methylation in different strains of mice that are susceptible and resistant to isoproterenol-induced dysfunction to test the hypothesis that this epigenetic mark may play a causal role in the development of heart failure. METHODS AND RESULTS: BALB/cJ and BUB/BnJ mice, determined to be susceptible and resistant to isoproterenol-induced heart failure, respectively, were administered the drug for 3 weeks via osmotic minipump. Reduced representational bisulfite sequencing was then used to compare the differences between the cardiac DNA methylomes in the basal state between strains and then after isoproterenol treatment. Single-base resolution DNA methylation measurements were obtained and revealed a bimodal distribution of methylation in the heart, enriched in lone intergenic CpGs and depleted from CpG islands around genes. Isoproterenol induced global decreases in methylation in both strains; however, the basal methylation pattern between strains shows striking differences that may be predictive of disease progression before environmental stress. The global correlation between promoter methylation and gene expression (as measured by microarray) was modest and revealed itself only with focused analyses of transcription start site and gene body regions (in contrast to when gene methylation was examined in toto). Modules of comethylated genes displayed correlation with other protein-based epigenetic marks, supporting the hypothesis that chromatin modifications act in a combinatorial manner to specify transcriptional phenotypes in the heart. CONCLUSIONS: This study provides the first single-base resolution map of the mammalian cardiac DNA methylome and the first case-control analysis of the changes in DNA methylation with heart failure. The findings demonstrate marked genetic differences in DNA methylation that are associated with disease progression.

Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients.

We have investigated transcriptional and epigenetic differences in peripheral blood mononuclear cells (PBMCs) of monozygotic female twins discordant in the diagnosis of amyotrophic lateral sclerosis (ALS). Exploring DNA methylation differences by reduced representation bisulfite sequencing (RRBS), we determined that, over time, the ALS twin developed higher abundances of the CD14 macrophages and lower abundances of T cells compared to the non-ALS twin. Higher macrophage signature in the ALS twin was also shown by RNA sequencing (RNA-seq). Moreover, the twins differed in the methylome at loci near several genes, including EGFR and TNFRSF11A, and in the pathways related to the tretinoin and H3K27me3 markers. We also tested cytokine production by PBMCs. The ALS twin's PBMCs spontaneously produced IL-6 and TNF-α, whereas PBMCs of the healthy twin produced these cytokines only when stimulated by superoxide dismutase (SOD)-1. These results and flow cytometric detection of CD45 and CD127 suggest the presence of memory T cells in both twins, but effector T cells only in the ALS twin. The ALS twin's PBMC supernatants, but not the healthy twin's, were toxic to rat cortical neurons, and this toxicity was strongly inhibited by an IL-6 receptor antibody (tocilizumab) and less well by TNF-α and IL-1ß antibodies. The putative neurotoxicity of IL-6 and TNF-α is in agreement with a high expression of these cytokines on infiltrating macrophages in the ALS spinal cord. We hypothesize that higher macrophage abundance and increased neurotoxic cytokines have a fundamental role in the phenotype and treatment of certain individuals with ALS.-Lam, L., Chin, L., Halder, R. C., Sagong, B., Famenini, S., Sayre, J., Montoya, D., Rubbi L., Pellegrini, M., Fiala, M. Epigenetic changes in T-cell and monocyte signatures and production of neurotoxic cytokines in ALS patients.

The concerted impact of domestication and transposon insertions on methylation patterns between dogs and grey wolves.

The process of domestication can exert intense trait-targeted selection on genes and regulatory regions. Specifically, rapid shifts in the structure and sequence of genomic regulatory elements could provide an explanation for the extensive, and sometimes extreme, variation in phenotypic traits observed in domesticated species. Here, we explored methylation differences from >24 000 cytosines distributed across the genomes of the domesticated dog (Canis familiaris) and the grey wolf (Canis lupus). PCA and model-based cluster analyses identified two primary groups, domestic vs. wild canids. A scan for significantly differentially methylated sites (DMSs) revealed species-specific patterns at 68 sites after correcting for cell heterogeneity, with weak yet significant hypermethylation typical of purebred dogs when compared to wolves (59% and 58%, P < 0.05, respectively). Additionally, methylation patterns at eight genes significantly deviated from neutrality, with similar trends of hypermethylation in purebred dogs. The majority (>66%) of differentially methylated regions contained or were associated with repetitive elements, indicative of a genotype-mediated trend. However, DMSs were also often linked to functionally relevant genes (e.g. neurotransmitters). Finally, we utilized known genealogical relationships among Yellowstone wolves to survey transmission stability of methylation marks, from which we found a substantial fraction that demonstrated high heritability (both H(2) and h(2 ) > 0.99). These analyses provide a unique epigenetic insight into the molecular consequences of recent selection and radiation of our most ancient domesticated companion, the dog. These findings suggest selection has acted on methylation patterns, providing a new genomic perspective on phenotypic diversification in domesticated species.

Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle.

The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle.

A ncRNA modulates histone modification and mRNA induction in the yeast GAL gene cluster.

The extensively studied yeast GAL1-10 gene cluster is tightly regulated by environmental sugar availability. Unexpectedly, under repressive conditions the 3' region of the GAL10 coding sequence is trimethylated by Set1 on histone H3 K4, normally characteristic of 5' regions of actively transcribed genes. This reflects transcription of a long noncoding RNA (GAL10-ncRNA) that is reciprocal to GAL1 and GAL10 mRNAs and driven by the DNA-binding protein Reb1. Point mutations in predicted Reb1-binding sites abolished Reb1 binding and ncRNA synthesis. The GAL10-ncRNA is transcribed approximately once every 50 min and targeted for degradation by the TRAMP and exosome complexes, resulting in low steady-state levels (approximately one molecule per 14 cells). GAL10-ncRNA transcription recruits the methyltransferase Set2 and histone deacetylation activities in cis, leading to stable changes in chromatin structure. These chromatin modifications act principally through the Rpd3S complex to aid glucose repression of GAL1-10 at physiologically relevant sugar concentrations.

Transcriptomic Modification in the Cerebral Cortex following Noninvasive Brain Stimulation: RNA-Sequencing Approach.

Transcranial direct current stimulation (tDCS) has been shown to modulate neuroplasticity. Beneficial effects are observed in patients with psychiatric disorders and enhancement of brain performance in healthy individuals has been observed following tDCS. However, few studies have attempted to elucidate the underlying molecular mechanisms of tDCS in the brain. This study was conducted to assess the impact of tDCS on gene expression within the rat cerebral cortex. Anodal tDCS was applied at 3 different intensities followed by RNA-sequencing and analysis. In each current intensity, approximately 1,000 genes demonstrated statistically significant differences compared to the sham group. A variety of functional pathways, biological processes, and molecular categories were found to be modified by tDCS. The impact of tDCS on gene expression was dependent on current intensity. Results show that inflammatory pathways, antidepressant-related pathways (GTP signaling, calcium ion binding, and transmembrane/signal peptide pathways), and receptor signaling pathways (serotonergic, adrenergic, GABAergic, dopaminergic, and glutamate) were most affected. Of the gene expression profiles induced by tDCS, some changes were observed across multiple current intensities while other changes were unique to a single stimulation intensity. This study demonstrates that tDCS can modify the expression profile of various genes in the cerebral cortex and that these tDCS-induced alterations are dependent on the current intensity applied.

BayesAge: A maximum likelihood algorithm to predict epigenetic age.

Introduction: DNA methylation, specifically the formation of 5-methylcytosine at the C5 position of cytosine, undergoes reproducible changes as organisms age, establishing it as a significant biomarker in aging studies. Epigenetic clocks, which integrate methylation patterns to predict age, often employ linear models based on penalized regression, yet they encounter challenges in handling missing data, count-based bisulfite sequence data, and interpretation. Methods: To address these limitations, we introduce BayesAge, an extension of the scAge methodology originally designed for single-cell DNA methylation analysis. BayesAge employs maximum likelihood estimation (MLE) for age inference, models count data using binomial distributions, and incorporates LOWESS smoothing to capture non-linear methylation-age dynamics. This approach is tailored for bulk bisulfite sequencing datasets. Results: BayesAge demonstrates superior performance compared to scAge. Notably, its age residuals exhibit no age association, offering a less biased representation of epigenetic age variation across populations. Furthermore, BayesAge facilitates the estimation of error bounds on age inference. When applied to down-sampled data, BayesAge achieves a higher coefficient of determination between predicted and actual ages compared to both scAge and penalized regression. Discussion: BayesAge presents a promising advancement in epigenetic age prediction, addressing key challenges encountered by existing models. By integrating robust statistical techniques and tailored methodologies for count-based data, BayesAge offers improved accuracy and interpretability in predicting age from bulk bisulfite sequencing datasets. Its ability to estimate error bounds enhances the reliability of age inference, thereby contributing to a more comprehensive understanding of epigenetic aging processes.

The response to influenza vaccination is associated with DNA methylation-driven regulation of T cell innate antiviral pathways.

Background: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell type composition. Results: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. Conclusions: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Longitudinal analysis of influenza vaccination implicates regulation of RIG-I signaling by DNA methylation.

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

Intrauterine calorie restriction affects placental DNA methylation and gene expression.

Maternal nutrient restriction causes the development of adult onset chronic diseases in the intrauterine growth restricted (IUGR) fetus. Investigations in mice have shown that either protein or calorie restriction during pregnancy leads to glucose intolerance, increased fat mass, and hypercholesterolemia in adult male offspring. Some of these phenotypes are shown to persist in successive generations. The molecular mechanisms underlying IUGR remain unclear. The placenta is a critical organ for mediating changes in the environment and the development of embryos. To shed light on molecular mechanisms that might affect placental responses to differing environments we examined placentas from mice that had been exposed to different diets. We measured gene expression and whole genome DNA methylation in both male and female placentas of mice exposed to either caloric restriction or ad libitum diets. We observed several differentially expressed pathways associated with IUGR phenotypes and, most importantly, a significant decrease in the overall methylation between these groups as well as sex-specific effects that are more pronounced in males. In addition, a set of significantly differentially methylated genes that are enriched for known imprinted genes were identified, suggesting that imprinted loci may be particularly susceptible to diet effects. Lastly, we identified several differentially methylated microRNAs that target genes associated with immunological, metabolic, gastrointestinal, cardiovascular, and neurological chronic diseases, as well as genes responsible for transplacental nutrient transfer and fetal development.

Epigenetic aging in cows is accelerated by milk production.

DNA methylation has proven to be the most promising age-predictive biomarker in mammals resulting in the emergence of 'epigenetic clocks' that describe the relationship between methylation levels and age. Using Targeted bisulfite Sequencing, we evaluated blood DNA-methylation data from 96 domesticated cows (Bos Taurus) of which 88 were adults and 8 were calves. This allowed us to measure DNA methylation across three thousand regions in the genome that were conserved across mammals. The significant association of age with the changes in DNA methylation enabled us to construct an epigenetic clock that predicts the age of cows to within nine months. We also investigated whether factors exist that moderate the association between epigenetic age and actual age and found that milk production levels significantly increase the rate of epigenetic ageing, suggesting that the stress of excessive milk production might be accelerating epigenetic ageing in cows.

The effects of age, sex, weight, and breed on canid methylomes.

Unlike genomes, which are static throughout the lifespan of an organism, DNA methylomes are dynamic. To study these dynamics, we developed quantitative models that measure the effect of multiple factors on DNA methylomes including, age, sex, weight, and genetics. We conducted our study in canids, which prove to be an ideal species to assess epigenetic moderators due to their extreme variability in size and well-characterized genetic structure. We collected buccal swabs from 217 canids (207 domestic dogs and 10 grey wolves) and used targeted bisulphite sequencing to measure methylomes. We also measured genotypes at over one thousand single nucleotide polymorphisms (SNPs). As expected, we found that DNA methylomes are strongly associated with age, enabling the construction of epigenetic clocks. However, we also identify novel associations between methylomes and sex, weight, and sterilization status, leading to accurate models that predict these factors. Methylomes are also affected by genetics, and we observe multiple associations between SNP loci and methylated CpGs. Finally, we show that several factors moderate the relationship between epigenetic ages and real ages, such as body weight, which increases epigenetic ageing. In conclusion, we demonstrate that the plasticity of DNA methylomes is impacted by myriad genetics and physiological factors, and that DNA methylation biomarkers are accurate predictors of age, sex and sterilization status.