Muscle Contraction Induces Acute Hydroxymethylation of the Exercise-Responsive Gene Nr4a3

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 the Nr4a3 gene in C2C12 myotubes. Using targeted bisulfite sequencing, we found that in response to EPS, a region of the Nr4a3 promoter is rapidly demethylated at 60 min and re-methylated at 120 min. Of interest, hydroxymethylation of the differentially methylated region of Nr4a3 promoter 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 gene Nr4a3 is demethylated after muscle contraction.

GREM1 is epigenetically reprogrammed in muscle cells after exercise training and controls myogenesis and metabolism

“…Since hypomethylation is classically described to be associated with increased transcription, these data suggested that DNA methylation after exercise allows for increased mRNA expression. Overall hypomethylation as a way to enable gene expression is consistent with the global increase in gene expression reported in human skeletal muscle after exercise 56,57 . Our methylated DNA sequencing analysis identified a DMR localized in the intron of FMN1 gene but closer to the transcription start site of the Grem1 gene.…”

In vitro experimental models for examining the skeletal muscle cell biology of exercise: the possibilities, challenges and future developments

“…It is likely that timedependent activation also likely occurs following cessation of exercise-like treatment in vitro. Indeed, serial sample harvests have demonstrated that PFI-induced changes in IL-6 mRNA expression [25], and EPS-induced GLUT4 recycling [83], IL-6 mRNA expression [7], PPARδ mRNA expression [93] and myotube hypertrophy [119] exhibit significant time effects over the period following treatment cessation. However, most studies harvest samples at a single time point following treatment cessation, which can vary across studies, meaning the time-course for changes following each of these treatments is not well characterised.…”

“…Since exposure to high glucose concentrations (i.e. 25 mM) can induce insulin resistance in skeletal muscle myotubes [42,53,125], studies that have cultured myotubes in hyperglycaemic conditions prior to and during the respective exercise-like treatment (for example, EPS-based [7,26,52,75,93,126], AICAR-based [88,94,110] and caffeinebased [113,115] models) may have been conducted under disease-inducing conditions. Therefore, acknowledging whether the effects of these exercise-like treatments have been investigated in the context of health (e.g.…”

Transcriptomic Profiling of Skeletal Muscle Adaptations to Exercise and Inactivity

“…The top 5 up-and downregulated genes for each perturbation are presented in Figure 3A. Changes in NR4A3 expression have been described after acute aerobic exercise [12][13][14] , although its specific role in orchestrating exercise-adaptations in skeletal muscle remains unclear. The transcriptional regulator MAFF (MAF BZIP Transcription Factor F) and the stress responsive protein GADD45G (Growth Arrest And DNA Damage Inducible Gamma) have yet to be studied in the context of exercise.…”