title: Muscle Contraction Induces Acute Hydroxymethylation of the Exercise-Responsive GeneNr4a3.
abstract: Exercise training triggers numerous positive adaptations through the regulation of genes controlling muscle structure and function. Epigenetic modifications, including DNA methylation, participate in transcriptional activation by allowing the recruitment of the transcription machinery to gene promoters. Exercise induces dynamic DNA demethylation at gene promoters; however, the contribution of the demethylation precursor hydroxymethylcytosine is unknown. Given the evanescent nature of hydroxymethylcytosine, a muscle contraction model that allows for the collection of samples that are repeatedly stimulated over time is required to determine whether contraction-induced demethylation is preceded by changes in the hydroxymethylcytosine level. Here, we established an acute skeletal muscle contraction model to mimic the effects of acute exercise on gene expression. We used this model to investigate the effect of muscle contraction on DNA demethylation and hydroxymethylation. First, we performed an acute exercise study in healthy humans to identify an exercise-responsive gene that we could study in culture. We identified the nuclear receptor subfamily 4 group A member 3 (Nr4a3) gene with the highest fold-expression increase after acute exercise. We then refined an electrical pulse stimulation (EPS) protocol that could induce expression of theNr4a3gene in C2C12 myotubes. Using targeted bisulfite sequencing, we found that in response to EPS, a region of theNr4a3promoter is rapidly demethylated at 60 min and re-methylated at 120 min. Of interest, hydroxymethylation of the differentially methylated region ofNr4a3promoter after EPS was elevated immediately after EPS, with lowest levels reached at 60 min after EPS. In conclusion, we have established a cell culture-based protocol to mimic the acute transcriptional responses to exercise. Furthermore, we provide insight into the mechanism by which the exercise-responsive geneNr4a3is demethylated after muscle contraction.
authors.affiliation: Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Pattarawan
authors.initials: P
authors.lastname: Pattamaprapanont
authors.affiliation: Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Christian
authors.initials: C
authors.lastname: Garde
authors.affiliation: Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Odile
authors.initials: O
authors.lastname: Fabre
authors.affiliation: Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Romain
authors.initials: R
authors.lastname: Barrès
keywords: DNA methylation; epigenetics; gene expression regulation; muscle contraction; skeletal muscle