Abstract
BackgroundHuman skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects.MethodsWe used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model.ResultsWe observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects.ConclusionsOur study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.
Highlights
Human skeletal muscle stem cells are important for muscle regeneration
Changes in the global DNA methylation pattern during differentiation of primary human myoblasts into myotubes of healthy subjects To examine if epigenetic changes take place during human myogenesis, we examined the genome-wide DNA methylation pattern in human myoblasts and myotubes from 14 healthy non-obese subjects using the Infinium HumanMethylation450 BeadChip
Myogenesis was accompanied by dynamic methylation and expression changes of PAX7, all major myogenic regulatory factor (MRF) and myocyte enhancer factor-2 (MEF2) (MYOD, MYF5, MYOG, MYF6, MEF2A, MEF2C, MEF2D), the myosin heavy chain (MHC) and myosin light chain (MLC) families, as well as mitochondrial proteins, thereby demonstrating that the cells were successfully differentiated
Summary
The combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Human skeletal muscle stem cells, called satellite cells, are quiescent, but activated in response to muscle injury [1] and resistance exercise training [2]. Activation of satellite cells represents a muscle repair mechanism. It is possible that novel myogenic factors that regulate differentiation of human myoblasts into myotubes, and muscle cell function, can be discovered. Epigenetic mechanisms such as DNA methylation are known to regulate cell differentiation and cell-specific gene expression, the epigenome and transcriptome of human adult myogenesis is far from defined [6,7,8]
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