Alveolar rhabdomyosarcoma-associated proteins PAX3/FOXO1A and PAX7/FOXO1A suppress the transcriptional activity of MyoD-target genes in muscle stem cells.

Rhabdomyosarcoma (RMS) is the commonest soft-tissue sarcoma in childhood and is characterized by expression of myogenic proteins, including the transcription factors MyoD and myogenin. There are two main subgroups, embryonal RMS and alveolar RMS (ARMS). Most ARMS are associated with chromosomal translocations that have breakpoints in introns of either PAX3 or PAX7, and FOXO1A. These translocations create chimeric transcription factors termed PAX3/FOXO1A and PAX7/FOXO1A respectively. Upon ectopic PAX3/FOXO1A expression, together with other genetic manipulation in mice, both differentiating myoblasts and satellite cells (the resident stem cells of postnatal muscle) can give rise to tumours with ARMS characteristics. As PAX3 and PAX7 are part of transcriptional networks that regulate muscle stem cell function in utero and during early postnatal life, PAX3/FOXO1A and PAX7/FOXO1A may subvert normal PAX3 and PAX7 functions. Here we examined how PAX3/FOXO1A and PAX7/FOXO1A affect myogenesis in satellite cells. PAX3/FOXO1A or PAX7/FOXO1A inhibited myogenin expression and prevented terminal differentiation in murine satellite cells: the same effect as dominant-negative (DN) Pax3 or Pax7 constructs. The transcription of MyoD-target genes myogenin and muscle creatine kinase were suppressed by PAX3/FOXO1A or PAX7/FOXO1A in C2C12 myogenic cells again as seen with Pax3/7DN. PAX3/FOXO1A or PAX7/FOXO1A did not inhibit the transcriptional activity of MyoD by perturbing MyoD expression, localization, phosphorylation or interaction with E-proteins. Chromatin immunoprecipitation on the myogenin promoter showed that PAX3/FOXO1A or PAX7/FOXO1A did not prevent MyoD from binding. However, PAX3/FOXO1A or PAX7/FOXO1A reduced occupation of the myogenin promoter by RNA polymerase II and decreased acetylation of histone H4, but did not directly bind to the myogenin promoter. Together, these observations reveal that PAX3/FOXO1A and PAX7/FOXO1A act to prevent myogenic differentiation via suppression of the transcriptional activation of MyoD-target genes.

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells.

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.