Sex-Specific Epigenetic Adaptations to Exercise Training

Physical activity is the most effective intervention to enhance health and prevent chronic diseases, such as obesity, type 2 diabetes, and cardiovascular disease. Studies and consortiums have aimed to understand the underlying molecular mechanisms that bring about a healthier phenotype with exercise training, and growing evidence suggests that epigenetic changes, which are molecular modifications to the DNA, play a large role in regulating exercise training adaptations. DNA methylation is the most widely studied epigenetic modification in exercise training studies, as it has been shown that both acute and maintained exercise (i.e. training) induce changes in the DNA methylome and subsequent gene function in human skeletal muscle. However, to date, studies identifying skeletal muscle epigenetic adaptations to exercise training have not investigated whether there is a sex-specific effect, despite skeletal muscle being one of the tissues with the most sex-biased gene expression. A majority of the animal and human studies that have guided our understanding of the underlying molecular adaptations to exercise training have either included only males or pooled males and females together without considering potential sex differences. However, biological sex has been identified as a confounding variable across many biological disciplines, and sex- specific analysis can be critical to the interpretation, validation, reproducibility and generalizability of research findings [1]. Thus, the overarching aim of this thesis was to investigate the sex-specific epigenome-wide response to exercise training. Sixty-five healthy males and females (females n = 20; males n = 45) from the Gene SMART (Skeletal Muscle Adaptive Response to Training) study completed four weeks of high-intensity interval training (HIIT) to assess sex-specific training-induced DNA methylation changes. This thesis involved adding the female cohort to the already existing male cohort, of which most of the participant data was collected prior to the commencement of this thesis. Participants underwent a four-week control period prior to commencing the training intervention. To determine whether training induced similar changes in physiological fitness in males and females, three measurements were assessed – maximum oxygen consumption (VO2max), peak power output (PP), and lactate threshold (LT) – at three time points (before control, before and after the HIIT intervention). To assess sex differences in DNA methylation and other molecular measurements (fibre type proportions and gene expression), skeletal muscle biopsies were collected at each time point and analysed with the Illumina HumanMethylation EPIC array. In Chapter 3, we have shown that there are 56,813 differentially methylated positions (DMPs) in the autosomes of male and female skeletal muscle at rest (false discovery rate [FDR] < 0.005), using a large scale meta-analysis of three independent cohorts (Gene SMART, FUSION, and GSE38291) comprising 369 individuals. These DMPs were mostly hypomethylated in males (94%), and were annotated to 10,240 differentially methylated regions (DMRs) and 8,420 differentially methylated genes (DMGs). Gene set enrichment analysis (GSEA) revealed enrichment of sex-differential methylation among muscle contraction, anatomical structure, and metabolism related pathways. Overlapping DMGs with genes known to have sex-biased skeletal muscle expression (differentially expressed genes [DEGs] from GTex), revealed a significant enrichment of DEGs among DMGs. We confirmed over-representation of DEGs among DMGs with transcriptomic data in an additional cohort (FUSION) which was also included in the DNA methylation meta-analysis. Lastly, using qPCR, we verified gene expression sex differences of three top genes identified from the differential methylation and expression analysis in an additional cohort included in the DNA methylation meta-analysis (Gene SMART). In Chapter 4, we investigated the underlying biological factors contributing to the observed sex differences in basal skeletal muscle DNA methylation. Using a meta-analysis approach in the Gene SMART and FUSION cohorts, we have shown that type I muscle fibre proportions were associated with DNA methylation at 16% of sex-biased DNA methylation loci. We found that circulating sex hormone levels (estrogen, testosterone, free testosterone, and sex hormone-binding globulin) in the Gene SMART cohort were not associated with differential methylation at the sex-biased DNA methylation loci. Lastly, we identified that the meta-analysis sex-DMPs were enriched for transcription factor binding sites (TFBSs) of 41 transcription factors (TF) , as previously established by uniform processing of multiple ChIP- seq data sets, including sex hormone-related androgen (AR), estrogen (ESR1), and glucocorticoid (NR3C1) receptors. In Chapter 5, after elucidating the basal skeletal muscle DNA methylome sex differences and their biological contributors, we investigated whether there are sex differences in exercise training-induced DNA methylation changes. First, we found that both males and females improved the physiological fitness measurements PP and LT, but not VO2max, in response to the HIIT, with no sex differences in the degree of the responses. We identified 1,261 CpGs whose methylation changed after four weeks of HIIT at a stringent FDR threshold < 0.005. We found no sex-specific DNA methylation changes after four weeks of HIIT (sex- by-training interaction) at a stringent FDR threshold < 0.005. A global examination of all the statistical tests performed genome-wide did not reveal an inflation of near zero p-values, suggesting that males and females do not differ in their epigenetic response to four weeks of HIIT. Given the relatively short training intervention, we then aimed to investigate whether there were sex differences in DNA methylation associated with cardiorespiratory fitness (CRF), an indicator of lifelong physical activity levels. We found 27,987 DMPs associated with CRF (FDR < 0.005), and no sex differences in the association between CRF and DNA methylation. The experimental design and meta-analysis of this thesis provided large-scale epigenome-wide insight on skeletal muscle epigenetic sex differences, and elucidated the role of DNA methylation in exercise training adaptations in both males and females (Chapter 5). It yielded a comprehensive understanding of the profound sex-specific skeletal muscle DNA methylation and transcriptomic profiles (Chapter 3) and the underlying biological factors (Chapter 4) that distinguish male and female skeletal muscle DNA methylomes. Specifically, muscle fibre type proportions were associated with sites displaying sex differences in DNA methylation; nonetheless, the vast majority of loci that exhibit sex-biased DNA methylation differ regardless of sex differences in fibre type proportions. In addition, although circulating hormones were not associated with sex-differential DNA methylation, the enrichment of hormone-responsive TFBSs suggests that hormones underlie a portion of the DNA methylation sex differences in skeletal muscle. However, the influence of other biological factors, such as the sex chromosomes, on the sex differences observed in the autosomal DNA methylome remains to be determined. Lastly, despite the plethora of sex differences in the skeletal muscle DNA methylome at rest, the DNA methylomes of males and females responded similarly to exercise training as well as lifelong physical activity. These novel findings shed light on the epigenetic response of skeletal muscle to exercise training in healthy males and females. Integrating the DNA methylome with downstream -omics, such as transcriptomics, proteomics, and metabolomics, will further elucidate the pathways and networks involved in the skeletal muscle response to exercise training as well as any sex-specific adaptations. Future studies should include males and females in exercise training studies, take sex and other sex-related factors into consideration in study design and analysis, as well as integrate other OMIC layers to better characterise the skeletal muscle response to exercise training in humans.