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Epigenetic marks in gametes modulate developmental programming after fertilization. Spermatozoa from obese men exhibit distinct epigenetic signatures compared to lean men, however, whether epigenetic differences are concentrated in a sub-population of spermatozoa or spread across the ejaculate population is unknown. Here, by using whole-genome single-cell bisulfite sequencing on 87 motile spermatozoa from 8 individuals (4 lean and 4 obese), we found that spermatozoa within single ejaculates are highly heterogeneous and contain subsets of spermatozoa with marked imprinting defects. Comparing lean and obese subjects, we discovered methylation differences across two large CpG dense regions located near PPM1D and LINC01237. These findings confirm that sperm DNA methylation is altered in human obesity and indicate that single ejaculates contain subpopulations of spermatozoa carrying distinct DNA methylation patterns. Distinct epigenetic patterns of spermatozoa within an ejaculate may result in different intergenerational effects and therefore influence strategies aiming to prevent epigenetic-related disorders in the offspring.

In response to physical exercise and diet, skeletal muscle adapts to energetic demands through large transcriptional changes. This remodelling is associated with changes in skeletal muscle DNA methylation which may participate in the metabolic adaptation to extracellular stimuli. Yet, the mechanisms by which muscle-borne DNA methylation machinery responds to diet and exercise and impacts muscle function are unknown. Here, we investigated the function of de novo DNA methylation in fully differentiated skeletal muscle. We generated muscle-specific DNA methyltransferase 3A (DNMT3A) knockout mice (mD3AKO) and investigated the impact of DNMT3A ablation on skeletal muscle DNA methylation, exercise capacity and energy metabolism. Loss of DNMT3A reduced DNA methylation in skeletal muscle over multiple genomic contexts and altered the transcription of genes known to be influenced by DNA methylation, but did not affect exercise capacity and whole-body energy metabolism compared to wild type mice. Loss of DNMT3A did not alter skeletal muscle mitochondrial function or the transcriptional response to exercise however did influence the expression of genes involved in muscle development. These data suggest that DNMT3A does not have a large role in the function of mature skeletal muscle although a role in muscle development and differentiation is likely., Author summary Skeletal muscle is a plastic tissue able to adapt to environmental stimuli such as exercise and diet in order to respond to energetic demand. One of the ways in which skeletal muscle can rapidly react to these stimuli is DNA methylation. This is when chemical groups are attached to DNA, potentially influencing the transcription of genes. We investigated the function of DNA methylation in skeletal muscle by generating mice that lacked one of the main enzymes responsible for de novo DNA methylation, DNA methyltransferase 3A (DNMT3A), specifically in muscle. We found that loss of DNMT3A reduced DNA methylation in muscle however this did not lead to differences in exercise capacity or energy metabolism. This suggests that DNMT3a is not involved in the adaptation of skeletal muscle to diet or exercise.

Objectives Regular physical exercise improves health by reducing the risk of a plethora of chronic disorders. We hypothesized that endurance exercise training remodels the activity of gene enhancers in skeletal muscle and that this remodeling contributes to the beneficial effects of exercise on human health. Methods and results By studying changes in histone modifications, we mapped the genome-wide positions and activities of enhancers in skeletal muscle biopsies collected from young sedentary men before and after 6 weeks of endurance exercise. We identified extensive remodeling of enhancer activities after exercise training, with a large subset of the remodeled enhancers located in the proximity of genes transcriptionally regulated after exercise. By overlapping the position of enhancers with genetic variants, we identified an enrichment of disease-associated genetic variants within the exercise-remodeled enhancers. Conclusion Our data provide evidence of a functional link between epigenetic rewiring of enhancers to control their activity after exercise training and the modulation of disease risk in humans., Highlights • Exercise training changes in skeletal muscle gene expression is enriched for secreted factors. • The activity of skeletal muscle enhancers undergoes substantial remodeling after exercise training. • Skeletal muscle enhancer activity and gene transcription are strongly associated. • Exercise training-remodeled enhancer regions are enriched for GWAS SNPs associated with human traits and diseases.

Time-restricted feeding (TRF) improves metabolism independent of dietary macronutrient composition or energy restriction. To elucidate mechanisms underpinning the effects of short-term TRF, we investigated skeletal muscle and serum metabolic and transcriptomic profiles from 11 men with overweight/obesity after TRF (8 h day

Exercise training improves skeletal muscle function, notably through tissue regeneration by muscle stem cells. Here, we hypothesized that exercise training reprograms the epigenome of muscle cell, which could account for better muscle function. Genome-wide DNA methylation of myotube cultures established from middle-aged obese men before and after endurance exercise training identified a differentially methylated region (DMR) located downstream ofGremlin 1(GREM1), which was associated with increasedGREM1expression. GREM1 expression was lower in muscle satellite cells from obese, compared to lean mice, and exercise training restored GREM1 levels to those of control animals. We show that GREM1 regulates muscle differentiation through the negative control of satellite cell self-renewal, and that GREM1 controls muscle lineage commitment and lipid oxidation through the AMPK pathway. Our study identifies novel functions of GREM1 and reveals an epigenetic mechanism by which exercise training reprograms muscle stem cells to improve skeletal muscle function.

Obesity and type 2 diabetes (T2D) are metabolic disorders influenced by lifestyle and genetic factors that are characterized by insulin resistance in skeletal muscle, a prominent site of glucose disposal. Numerous genetic variants have been associated with obesity and T2D, of which the majority are located in non-coding DNA regions. This suggests that most variants mediate their effect by altering the activity of gene-regulatory elements, including enhancers. Here, we map skeletal muscle genomic enhancer elements that are dynamically regulated after exposure to the free fatty acid palmitate or the inflammatory cytokine TNFα. By overlapping enhancer positions with the location of disease-associated genetic variants, and resolving long-range chromatin interactions between enhancers and gene promoters, we identify target genes involved in metabolic dysfunction in skeletal muscle. The majority of these genes also associate with altered whole-body metabolic phenotypes in the murine BXD genetic reference population. Thus, our combined genomic investigations identified genes that are involved in skeletal muscle metabolism., Obesity and type 2 diabetes (T2D) are metabolic disorders characterized by insulin resistance in skeletal muscle. Here, the authors map skeletal muscle enhancer elements dynamically regulated after exposure to free fatty acid palmitate or inflammatory cytokine TNFα and identify target genes involved in metabolic dysfunction in skeletal muscle.

Deterioration of the adipogenic potential of preadipocytes may contribute to adipose tissue dysfunction in obesity and type 2 diabetes (T2D). Here, we hypothesized that extracellular factors in obesity epigenetically reprogram adipogenesis potential and metabolic function of preadipocytes. The transcriptomic profile of visceral adipose tissue preadipocytes collected from Lean, Obese and Obese with T2D was assessed throughout in vitro differentiation using RNA sequencing. Reduced Representation Bisulfite Sequencing was used to establish the genome-wide DNA methylation profile of human preadipocytes and 3T3-L1 preadipocytes treated by the inflammatory cytokine Tumour Necrosis Factor-α (TNF-α) or palmitate. While preadipocytes from all obese subjects (Obese+Obese T2D), compared to those of Lean, were transcriptionally different in response to differentiation in culture, preadipocytes from Obese T2D showed impaired insulin signalling and a further transcriptomic shift towards altered adipocyte function. Cultures with a lower expression magnitude of adipogenic genes throughout differentiation (PLIN1, CIDEC, FABP4, ADIPOQ, LPL, PDK4, APOE, LIPE, FABP3, LEP, RBP4 and CD36) were associated with DNA methylation remodelling at genes controlling insulin sensitivity and adipocytokine signalling pathways. Prior incubation of 3T3-L1 preadipocytes with TNF-α or palmitate markedly altered insulin responsiveness and metabolic function in the differentiated adipocytes, and remodelled DNA methylation and gene expression at specific genes, notably related to PPAR signalling. Our findings that preadipocytes retain the memory of the donor in culture and can be reprogrammed by extracellular factors support a mechanism by which adipocyte precursors are epigenetically reprogrammed in vivo. Epigenetic reprogramming of preadipocytes represents a mechanism by which metabolic function of visceral adipose tissue may be affected in the long term by past exposure to obesity- or T2D-specific factors.

Exposure to ionizing radiation increases the risk of chronic metabolic disorders such as insulin resistance and type 2 diabetes later in life. We hypothesized that irradiation reprograms the epigenome of metabolic progenitor cells, which could account for impaired metabolism after cancer treatment. C57Bl/6 mice were treated with a single dose of irradiation and subjected to high-fat diet (HFD). RNA sequencing and reduced representation bisulfite sequencing were used to create transcriptomic and epigenomic profiles of preadipocytes and skeletal muscle satellite cells collected from irradiated mice. Mice subjected to total body irradiation showed alterations in glucose metabolism and, when challenged with HFD, marked hyperinsulinemia. Insulin signaling was chronically disrupted in skeletal muscle and adipose progenitor cells collected from irradiated mice and differentiated in culture. Epigenomic profiling of skeletal muscle and adipose progenitor cells from irradiated animals revealed substantial DNA methylation changes, notably for genes regulating the cell cycle, glucose/lipid metabolism, and expression of epigenetic modifiers. Our results show that total body irradiation alters intracellular signaling and epigenetic pathways regulating cell proliferation and differentiation of skeletal muscle and adipose progenitor cells and provide a possible mechanism by which irradiation used in cancer treatment increases the risk for metabolic disease later in life.

Objectives Chronic and high consumption of fat constitutes an environmental stress that leads to metabolic diseases. We hypothesized that high-fat diet (HFD) transgenerationally remodels the epigenome of spermatozoa and metabolism of the offspring. Methods F0-male rats fed either HFD or chow diet for 12 weeks were mated with chow-fed dams to generate F1 and F2 offspring. Motile spermatozoa were isolated from F0 and F1 breeders to determine DNA methylation and small non-coding RNA (sncRNA) expression pattern by deep sequencing. Results Newborn offspring of HFD-fed fathers had reduced body weight and pancreatic beta-cell mass. Adult female, but not male, offspring of HFD-fed fathers were glucose intolerant and resistant to HFD-induced weight gain. This phenotype was perpetuated in the F2 progeny, indicating transgenerational epigenetic inheritance. The epigenome of spermatozoa from HFD-fed F0 and their F1 male offspring showed common DNA methylation and small non-coding RNA expression signatures. Altered expression of sperm miRNA let-7c was passed down to metabolic tissues of the offspring, inducing a transcriptomic shift of the let-7c predicted targets. Conclusion Our results provide insight into mechanisms by which HFD transgenerationally reprograms the epigenome of sperm cells, thereby affecting metabolic tissues of offspring throughout two generations., Highlights • Body weight and glucose metabolism are altered in F1 and F2 offspring of F0-HFD fathers. • High-fat diet reprograms the epigenome of sperm cells. • Spermatozoa from F0-HFD fathers and F1 offspring share common epigenetic signatures. • Expression of let-7c is changed in sperm of founders and in the adipose tissue of the offspring.

Obesity is a heritable disorder, with children of obese fathers at higher risk of developing obesity. Environmental factors epigenetically influence somatic tissues, but the contribution of these factors to the establishment of epigenetic patterns in human gametes is unknown. Here, we hypothesized that weight loss remodels the epigenetic signature of spermatozoa in human obesity. Comprehensive profiling of the epigenome of sperm from lean and obese men showed similar histone positioning, but small non-coding RNA expression and DNA methylation patterns were markedly different. In a separate cohort of morbidly obese men, surgery-induced weight loss was associated with a dramatic remodeling of sperm DNA methylation, notably at genetic locations implicated in the central control of appetite. Our data provide evidence that the epigenome of human spermatozoa dynamically changes under environmental pressure and offers insight into how obesity may propagate metabolic dysfunction to the next generation.

AIM: To determine the genomic mechanisms by which adipose tissue responds to acute and chronic exercise.METHODS: We profiled the transcriptomic and epigenetic response to acute exercise in human adipose tissue collected before and after endurance training.RESULTS: Although acute exercises were performed at same relative intensities, the magnitude of transcriptomic changes after acute exercise was reduced by endurance training. DNA methylation remodeling induced by acute exercise was more prominent in trained versus untrained state. We found an overlap between gene expression and DNA methylation changes after acute exercise for 32 genes pre-training and six post-training, notably at adipocyte-specific genes.CONCLUSION: Training status differentially affects the epigenetic and transcriptomic response to acute exercise in human adipose tissue.

Methylation of nuclear genes encoding mitochondrial proteins participates in the regulation of mitochondria function. The existence of cytosine methylation in the mitochondrial genome is debated. To investigate whether mitochondrial DNA (mtDNA) is methylated, we used both targeted- and whole mitochondrial genome bisulfite sequencing in cell lines and muscle tissue from mouse and human origin. While unconverted cytosines were detected in some portion of the mitochondrial genome, their abundance was inversely associated to the sequencing depth, indicating that sequencing analysis can bias the estimation of mtDNA methylation levels. In intact mtDNA, few cytosines remained 100% unconverted. However, removal of supercoiled structures of mtDNA with the restriction enzymeBamHIprior to bisulfite sequencing decreased cytosine unconversion rate to