TNF Inhibits Notch-1 in Skeletal Muscle Cells by Ezh2 and DNA Methylation Mediated Repression: Implications in Duchenne Muscular Dystrophy

Background: Classical NF-kappa B signaling functions as a negative regulator of skeletal myogenesis through potentially multiple mechanisms. The inhibitory actions of TNF alpha on skeletal muscle differentiation are mediated in part through sustained NF-kappa B activity. In dystrophic muscles, NF-kappa B activity is compartmentalized to myofibers to inhibit regeneration by limiting the number of myogenic progenitor cells. This regulation coincides with elevated levels of muscle derived TNF alpha that is also under IKK beta and NF-kappa B control. Methodology/Principal Findings: Based on these findings we speculated that in DMD, TNF alpha secreted from myotubes inhibits regeneration by directly acting on satellite cells. Analysis of several satellite cell regulators revealed that TNF alpha is capable of inhibiting Notch-1 in satellite cells and C2C12 myoblasts, which was also found to be dependent on NF-kappa B. Notch-1 inhibition occurred at the mRNA level suggesting a transcriptional repression mechanism. Unlike its classical mode of action, TNF alpha stimulated the recruitment of Ezh2 and Dnmt-3b to coordinate histone and DNA methylation, respectively. Dnmt-3b recruitment was dependent on Ezh2. Conclusions/Significance: We propose that in dystrophic muscles, elevated levels of TNF alpha and NF-kappa B inhibit the regenerative potential of satellite cells via epigenetic silencing of the Notch-1 gene.