Abstract
Skeletal muscle tissue and muscle stem cells show global hypermethylation with aging. However, it is unknown whether the methylomes of differentiating muscle stem cells overlap that of skeletal muscle tissue and how age modulates this overlap. Using the most recent high coverage DNA methylation arrays (850K CpG sites), we compared the methylomes of young and aged skeletal muscle to that of young and aged muscle stem cells at several time points of differentiation (0, 72 hrs, 7 days and 10 days). In line with the existing literature (450K arrays), aged muscle tissue was hypermethylated compared with young tissue. Hypermethylated genes in aged tissue were enriched for; pathways in cancer (that combined; focal adhesion, MAPK signaling, PI3K-Akt-mTOR signaling, p53 signaling, Jak-STAT signaling, TGF-beta and notch signaling), rap1 signaling, axon guidance and hippo signaling. Aged muscle stem cells also demonstrated a hypermethylated profile, enriched for; axon guidance, adherens junction and calcium signaling pathways, particularly at later timepoints of differentiation and myotube formation, that corresponded with reduced morphological differentiation versus young cells, confirmed by reductions in myoD and myogenin. While young muscle stem cells showed less variability in DNA methylation during differentiation, aged muscle stem cells demonstrated extensive and significant changes in DNA methylation at 7 days of differentiation at distinct CpG sites than those identified with age alone. The differentially methylated genes were enriched for; regulation of localisation, regulation of cell communication and regulation of signaling. We also identified CpG sites whose hypermethylated state was retained in-vitro in muscle stem cells once isolated from tissue in-vivo. These CpGs were located in genes: KIF15, DYRK2, FHL2, MRPS33, ABCA17P. In particular, differential chromosomal region analysis identified 3 regions with 6-8 CpGs in the Homeobox (HOX) transcription factor family of genes that were altered with age in the same direction (hypo or hypermethylated) across both muscle tissue and stem cells. Indeed, HOXD10, HOXD9, HOXD8, HOXA3, HOXC9, HOXB1, HOXB3, HOXC-AS2 and HOXC10 were all significantly differentially hypermethylated in aged tissue. In aged stem cells the same HOX genes (and additionally HOXC-AS3) also displayed the most varied methylation at 7 days of differentiation versus young cells. With HOXD8, HOXC9, HOXB1 and HOXC-AS3 hypermethylated and HOXC10 and HOXC-AS2 hypomethylated. We also determined inverse relationships between mRNA expression and DNA methylation in HOXB1, HOXA3 and HOXC-AS3 in aged cells. Finally, we were able to demonstrate that increased physical activity in young adults was associated with reversing the age-related DNA methylation changes observed in HOXB1, HOXA3 and HOXC10. For the first time, we demonstrated that a large number of HOX genes were differentially epigenetically regulated in aged human skeletal muscle and stem cells and that increased physical activity could reverse the age-related epigenetic changes in the HOX genes.
Footnotes
We now include Table 1 as a Supplemental File. This was missing in the v1 version of the manuscript.
https://drive.google.com/open?id=1yvV8g_KgnYQ0GNy1QrbWTFrsTRXZwsSw