An investigation into exercise-induced modifications to DNA methylation-regulatory enzymes in human peripheral blood mononuclear cells

DNA methylation, an epigenetic modification which can regulate gene transcription independently from alterations to the nucleotide sequence, can be manipulated by lifestyle factors such as diet and exercise, hypothetically reversing aberrant DNA methylation associated with disease pathogenesis. The underlying mechanisms by which these changes occur are currently poorly characterised, however, in vitro data suggest that inflammatory mediators are involved. Furthermore, regular exercise appears to reduce inactivity-associated systemic inflammation, possibly by alterations to the methylome, thereby suggesting a cyclic relationship between exercise, inflammation, and epigenetic modification. The aims of this research programme, therefore, were to: characterise the acute changes that occur to the de novo DNA methyltransferases following exercise in peripheral blood mononuclear cells (PBMCs), and the role of exercise-induced systemic inflammation in this process; investigate how these changes then translate into functional modifications to the methylome; and to determine whether a training programme utilising sedentary individuals manipulates DNA methylation of genes involved in chronic systemic inflammation associated with physical inactivity. Pilot investigations corroborated previous in vitro data that recombinant IL-6 is able to regulate nuclear concentrations of DNMT3A and DNMT3B in PBMCs. In order to isolate the influence of circulating proteins independently from genetic polymorphisms that may influence susceptibility to epigenetic change, cells were stimulated with exercise-conditioned plasma following intense endurance exercise which elicited significant alterations in nuclear concentrations of DNMT3A and DNMT3B. Eccentric exercise, which is typically not associated with elevations in circulating cytokines, did not cause any significant changes in nuclear or cytoplasmic DNMT concentration, or global DNA methylation; this supports the hypothesis that transient systemic elevations in inflammatory cytokines are important regulators of epigenetic modifications associated with exercise. Lack of transcriptional changes in DNMT3A following both exercise training and an acute maximal bout suggests that, in line with in vitro data, that the observed elevations in nuclear DNMT concentration are largely due to cellular relocalisation and not gene expression of this enzyme. It remains to be elucidated whether the training regime, and the subsequent response to an acute maximal bout, is able to elicit differential methylation of IL6, NFκB2, and ASC, however, in vitro stimulation of PBMCs with the cytokines IL-6 and IL-1β did cause significant changes to IL6 promoter methylation, further supporting the role of these proteins in epigenetic regulation. The data presented in this thesis support the postulation that exercise-induced changes to DNA methylation in PBMCs likely occur due to systemic elevations of inflammatory proteins, in particular IL-6, which causes manipulation of de novo DNMT nuclear concentrations due to cellular translocation of the enzymes themselves. While it was not possible to determine whether exercise directly modified gene-specific methylation, in vitro experiments suggest that inflammatory cytokines are able to regulate IL6 promoter methylation in human PBMCs