Regenerative myogenesis is the formation of muscle progenitors during the repair of adult skeletal muscle following tissue trauma. In this application, we outline a research program to molecularly define the genetic mechanisms through which Pax7 enforces lineage commitment during regenerative myogenesis. To fully define the Pax7 regulome in primary myoblasts, Pax7 bound to fragmented chromatin will be immunoprecipitated and the DNA isolated and subjected to a subtractive PCR-based procedure to selectively amplify Pax7 binding sites. Direct targets will be identified by a comparative analysis between our existing microarray data, and regulatory regions that recruit Pax7. The function of a select set of prioritized candidate target genes will be investigated using molecular and genetic approaches. Real-time PCR will be used to characterize cell-type and differentiation patterns of expression. Knock down experiments will be performed with RNAi, and full-length cDNAs over-expressed in myoblasts. Lastly, the binding of Pax7 alternative splice forms to candidate promoters will be evaluated. To investigate the hypothesis that Pax7 recruits co-factors to facilitate appropriate regulation of target genes, the ability of Pax7 to direct acetyl-transferase activity will be investigated, and chromatin immunoprecipitation assays will be used to characterize the histone modifications over Pax7 target promoters. A genome wide assessment of histone modifications using microarrays will be conducted. To elucidate Pax7 function, we will isolate and identify protein components of the Pax7 transcriptional complex by mass spectroscopy. These experimental approaches will facilitate a molecular understanding of Pax7 function and the genomic definition of the chromatin domains established by Pax7. Understanding the molecular mechanism regulating gene function during muscle regeneration will provide insights that will potentially lead to new modalities of therapeutic intervention for the treatment of muscle degenerative disease.
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