Global MEF2 Target Gene Analysis in Skeletal and Cardiac Muscle
Wales, Stephanie Elizabeth
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A loss of muscle mass or function occurs in many genetic and acquired pathologies such as heart disease, sarcopenia and cachexia which are predominantly found among the rapidly increasing elderly population. Developing effective treatments relies on understanding the genetic networks that control these disease pathways. Transcription factors occupy an essential position as regulators of gene expression. Myocyte enhancer factor 2 (MEF2) is an important transcription factor in striated muscle development in the embryo, skeletal muscle maintenance in the adult and cardiomyocyte survival and hypertrophy in the progression to heart failure. We sought to identify common MEF2 target genes in these two types of striated muscles using chromatin immunoprecipitation and next generation sequencing (ChIP-seq) and transcriptome profiling (RNA-seq). Using a cell culture model of skeletal muscle (C2C12) and primary cardiomyocytes we found 294 common MEF2A binding sites within both cell types. Individually MEF2A was recruited to approximately 2700 and 1600 DNA sequences in skeletal and cardiac muscle, respectively. Two genes were chosen for further study: DUSP6 and Hspb7. DUSP6, an ERK1/2 specific phosphatase, was negatively regulated by MEF2 in a p38MAPK dependent manner in striated muscle. Furthermore siRNA mediated gene silencing showed that MEF2D in particular was responsible for repressing DUSP6 during C2C12 myoblast differentiation. Using a p38 pharmacological inhibitor (SB 203580) we observed that MEF2D must be phosphorylated by p38 to repress DUSP6. This established a unique model whereby MAPK signaling results in repression of a MAPK phosphatase. The second MEF2 target gene studied was Hspb7, a small heat shock protein that is highly expressed in striated muscle. Using a combination of bioinformatic and biochemical analysis we found that AP-1 can inhibit Hspb7 transcription, in contrast to MEF2 which activates it. Additionally, the glucocorticoid receptor (GR) regulates Hspb7 in a manner dependent on the presence of MEF2. We also demonstrate an in vivo role for Hspb7 in autophagy which has significant implications in skeletal muscle wasting. Overall we found that MEF2A regulates distinct gene networks in skeletal and cardiac muscle, yet important shared target genes such as DUSP6 and Hspb7 also illustrate that MEF2A regulates some common gene programs that are critical to striated muscle health.