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5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

Traumatic brain injury (TBI) is a major cause of mortality and disability worldwide, particularly among individuals under the age of 45. It is a complex, and heterogeneous disease with a multifaceted pathophysiology that remains to be elucidated. Metabolomics has the potential to identify metabolic pathways and unique biochemical profiles associated with TBI. Herein, we employed a longitudinal metabolomics approach to study TBI in a weight drop mouse model to reveal metabolic changes associated with TBI pathogenesis, severity, and secondary injury. Using proton nuclear magnetic resonance ( 1 H NMR) spectroscopy, we biochemically profiled post-mortem brain from mice that suffered mild TBI (N = 25; 13 male and 12 female), severe TBI (N = 24; 11 male and 13 female) and sham controls (N = 16; 11 male and 5 female) at baseline, day 1 and day 7 following the injury. 1 H NMR-based metabolomics, in combination with bioinformatic analyses, highlights a few significant metabolites associated with TBI severity and perturbed metabolism related to the injury. We report that the concentrations of taurine , creatinine , adenine , dimethylamine , histidine , N-Acetyl aspartate , and glucose 1-phosphate are all associated with TBI severity. Longitudinal metabolic observation of brain tissue revealed that mild TBI and severe TBI lead distinct metabolic profile changes. A multi-class model was able to classify the severity of injury as well as time after TBI with estimated 86% accuracy. Further, we identified a high degree of correlation between respective hemisphere metabolic profiles (r > 0.84, p < 0.05, Pearson correlation). This study highlights the metabolic changes associated with underlying TBI severity and secondary injury. While comprehensive, future studies should investigate whether: (a) the biochemical pathways highlighted here are recapitulated in the brain of TBI sufferers and (b) if the panel of biomarkers are also as effective in less invasively harvested biomatrices, for objective and rapid identification of TBI severity and prognosis.

ABSTRACT Knowledge of animal age is essential to wildlife managers for obtaining meaningful and accurate insights into demographic parameters. A common approach to aging wildlife, including bears (Ursus spp.), has been extracting a tooth during physical capture and counting the cementum annuli. Limitations to tooth‐based aging include questionable accuracy and differing results based on the observer and laboratory. DNA methylation‐based epigenetic aging clocks have been developed for many species but not yet for polar bears (Ursus maritimus). We generated DNA methylation data from whole blood samples (n = 109) obtained during live capture operations from polar bears of known age in the Chukchi Sea and southern Beaufort Sea subpopulations. We used these samples to calibrate a species‐specific epigenetic clock to estimate polar bear chronological age from DNA methylation (DNAm) age. The final polar bear clock was highly accurate (r = 0.97) with a median absolute error of approximately 9 months. We applied the polar bear clock to 74 blood samples from live‐captured polar bears with a cementum annuli‐estimated age. Predicted age estimates for these bears ranged from 1.43 to 18.63 years compared to the estimated tooth age range of 3.23–25.27. These epigenetic clocks can be used for polar bear research and management where accurate estimates of age are needed for estimating demographic parameters. We developed a DNA methylation‐based epigenetic aging clock for polar bears (Ursus maritimus) using whole blood samples from polar bears of known age. The final polar bear clock was highly accurate (r = 0.97) with a median absolute error of approximately 9 months.

Circadian rhythmicity governs a remarkable array of fundamental biological functions and is mediated by cyclical transcriptomic and proteomic activities. Epigenetic factors are also involved in this circadian machinery; however, despite extensive efforts, detection and characterization of circadian cytosine modifications at the nucleotide level have remained elusive. In this study, we report that a large proportion of epigenetically variable cytosines show a circadian pattern in their modification status in mice. Importantly, the cytosines with circadian epigenetic oscillations significantly overlap with the cytosines exhibiting age-related changes in their modification status. Our findings suggest that evolutionary advantageous processes such as circadian rhythmicity can also contribute to an organism’s deterioration. While epigenetic factors have been implicated in the circadian rhythm, the detection of circadian cytosine modifications has remained elusive. Here the authors identify a large number of epigenetically variable cytosines that show circadian oscillations in their modification status in mice.

Background Maintenance of physiological circadian rhythm plays a crucial role in human health. Numerous studies have shown that disruption of circadian rhythm may increase risk for malignant, psychiatric, metabolic, and other diseases. Results Extending our recent findings of oscillating cytosine modifications (osc-modCs) in mice, in this study, we show that osc-modCs are also prevalent in human neutrophils. Osc-modCs may play a role in gene regulation, can explain parts of intra- and inter-individual epigenetic variation, and are signatures of aging. Finally, we show that osc-modCs are linked to three complex diseases and provide a new interpretation of cross-sectional epigenome-wide association studies. Conclusions Our findings suggest that loss of balance between cytosine methylation and demethylation during the circadian cycle can be a potential mechanism for complex disease. Additional experiments, however, are required to investigate the possible involvement of confounding effects, such as hidden cellular heterogeneity. Circadian rhythmicity, one of the key adaptations of life forms on Earth, may contribute to frailty later in life.

ABSTRACT Senescent cells emerge with aging and injury. The contribution of senescent cells to DNA methylation age (DNAmAGE) in vivo is uncertain. Furthermore, stem cell therapy can mediate “rejuvenation”, but how tissue regeneration controlled by resident stem cells affects whole tissue DNAmAGE is unclear. We assessed DNAmAGE with or without senolytics (BI01) in aged male mice (24–25 months) 35 days following muscle healing (BaCl2‐induced regeneration versus non‐injured). Young injured mice (5–6 months) without senolytics were comparators. DNAmAGE was decelerated by up to 68% after injury in aged muscle. DNAmAGE was modestly but further significantly decelerated by injury recovery with senolytics. ~1/4 of measured CpGs were altered by injury then recovery regardless of senolytics in aged muscle. Specific methylation changes caused by senolytics included differential regulation of Col, Hdac, Hox, and Wnt genes, which likely contributed to improved regeneration. Altered extracellular matrix remodeling using histological analysis aligned with the methylomic findings with senolytics. Without senolytics, regeneration had a contrasting effect in young mice and tended not to influence or modestly accelerate DNAmAGE. Comparing young to old injury recovery without senolytics using methylome‐transcriptome integration, we found a more coordinated molecular profile in young and differential regulation of genes implicated in muscle stem cell performance: Axin2, Egr1, Fzd4, Meg3, and Spry1. Muscle injury and senescent cells affect DNAmAGE and aging influences the transcriptomic‐methylomic landscape after resident stem cell‐driven tissue reformation. Our data have implications for understanding muscle plasticity with aging and developing therapies aimed at collagen remodeling and senescence. Injury then recovery markedly rewires the DNA methylome in aged skeletal muscle.The addition of senolytics during muscle regeneration decelerates DNAmAGE more than regeneration alone as well as targets collagen remodeling and stem cell function‐related genes. Regeneration in young adult mice has a less pronounced effect on the methylome‐transcriptome landscape than in aged skeletal muscle, but still elicits a distinct molecular profile versus aged skeletal muscle after injury.

Background: Despite extensive efforts, significant gaps remain in our understanding of Alzheimer’s disease (AD) pathophysiology. Novel approaches using circulating cell-free DNA (cfDNA) have the potential to revolutionize our understanding of neurodegenerative disorders. Methods: We performed DNA methylation profiling of cfDNA from AD patients and compared them to cognitively normal controls. Six Artificial Intelligence (AI) platforms were utilized for the diagnosis of AD while enrichment analysis was used to elucidate the pathogenesis of AD. Results: A total of 3684 CpGs were significantly (adj. p-value < 0.05) differentially methylated in AD versus controls. All six AI algorithms achieved high predictive accuracy (AUC = 0.949–0.998) in an independent test group. As an example, Deep Learning (DL) achieved an AUC (95% CI) = 0.99 (0.95–1.0), with 94.5% sensitivity and specificity. Conclusion: We describe numerous epigenetically altered genes which were previously reported to be differentially expressed in the brain of AD sufferers. Genes identified by AI to be the best predictors of AD were either known to be expressed in the brain or have been previously linked to AD. We highlight enrichment in the Calcium signaling pathway, Glutamatergic synapse, Hedgehog signaling pathway, Axon guidance and Olfactory transduction in AD sufferers. To the best of our knowledge, this is the first reported genome-wide DNA methylation study using cfDNA to detect AD.

Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system that exhibits significant variation in the stage of differentiation and cell composition of tumors. Global loss of DNA methylation and genomic 5-hydroxymethylcytosine (5hmC) is a hallmark of human cancers. Here, we used our recently developed single-base resolution approaches, hmTOP-seq and uTOP-seq, for construction of 5hmC maps and identification of large partially methylated domains (PMDs) in different NB cell subpopulations. The 5hmC profiles revealed distinct signatures characteristic to different cell lineages and stages of malignant transformation of NB cells in a conventional and oxygen-depleted environment, which often occurs in tumors. The analysis of the cell-type-specific PMD distribution highlighted differences in global genome organization among NB cells that were ascribed to the same lineage identity by transcriptomic networks. Collectively, we demonstrated a high informativeness of the integrative epigenomic and transcriptomic research and large-scale genome structure in investigating the mechanisms that regulate cell identities and developmental stages of NB cells. Such multiomics analysis, as compared with mutational studies, open new ways for identification of novel disease-associated features which bring prognostic and therapeutic value in treating this aggressive pediatric disease.

Dynamic patterns of cytosine-5 methylation and successive hydroxylation are part of epigenetic regulation in eukaryotes, including humans, which contributes to normal phenotypic variation and disease risk. Here we present an approach for the mapping of unmodified regions of the genome, which we call the unmethylome. Our technique is based on DNA methyltransferase-directed transfer of activated groups and covalent biotin tagging of unmodified CpG sites followed by affinity enrichment and interrogation on tiling microarrays or next generation sequencing. Control experiments and pilot studies of human genomic DNA from cultured cells and tissues demonstrate that, along with providing a unique cross-section through the chemical landscape of the epigenome, the methyltransferase-directed transfer of activated groups-based approach offers high precision and robustness as compared with existing affinity-based techniques. Chemical modifications of CpG dinucleotides form part of the epigenetic code and various methods for the detection of modified CpG sites exist. Here Kriukiene and colleagues report a complementary method that allows the profiling of unmodified CpG sites within the genome, which they call the 'unmethylome'.

The impact of environmental factors on epigenetic changes is well established, and cellular function is determined not only by the genome but also by interacting partners such as metabolites. Given the significant impact of metabolism on disease progression, exploring the interaction between the metabolome and epigenome may offer new insights into Huntington’s disease (HD) diagnosis and treatment. Using fourteen post-mortem HD cases and fourteen control subjects, we performed metabolomic profiling of human postmortem brain tissue (striatum and frontal lobe), and we performed DNA methylome profiling using the same frontal lobe tissue. Along with finding several perturbed metabolites and differentially methylated loci, Aminoacyl-tRNA biosynthesis (adj p-value = 0.0098) was the most significantly perturbed metabolic pathway with which two CpGs of the SEPSECS gene were correlated. This study improves our understanding of molecular biomarker connections and, importantly, increases our knowledge of metabolic alterations driving HD progression.