Satellite cells purified from adult skeletal muscle can participate extensively in muscle regeneration and can also re-populate the satellite cell pool, suggesting that they have direct therapeutic potential for treating degenerative muscle diseases1,2. The paired-box transcription factor Pax7 is required for satellite cells to generate committed myogenic progenitors3. In this study we undertook a multilevel approach to define the role of Pax7 in satellite cell function. Using comparative microarray analysis, we identified several novel and strongly regulated targets; in particular, we identified Myf5 as a gene whose expression was regulated by Pax7. Using siRNA, fluorescence-activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP) studies we confirmed that Myf5 is directly regulated by Pax7 in myoblasts derived from satellite cells. Tandem affinity purification (TAP) and mass spectrometry were used to purify Pax7 together with its co-factors. This revealed that Pax7 associates with the Wdr5-Ash2L-MLL2 histone methyltransferase (HMT) complex that directs methylation of histone H3 lysine 4 (H3K4, refs 4-10). Binding of the Pax7-HMT complex to Myf5 resulted in H3K4 tri-methylation of surrounding chromatin. Thus, Pax7 induces chromatin modifications that stimulate transcriptional activation of target genes to regulate entry into the myogenic developmental programme.Satellite cells arise from a population of muscle progenitor cells that originate in the central domain of the dermomyotome. These progenitors express the paired-box transcription factors Pax3 and Pax7 (refs 11,12), and although neither their emergence nor their maintenance requires Pax3 function13, recent studies have demonstrated that Pax7 is uniquely indispensable for these cells 14. In the absence of Pax7, satellite cells die and thus fail to re-populate their niche11,14,15. Pax7, is therefore essential for the formation and maintenance of a population of functional satellite cells. Analysis of the physiological functions of Pax7 has been hindered by relatively weak trans-activation properties resulting from cis-repression16. Consequently, the mechanisms by which Pax7 activates downstream target genes remain unclear. To address this problem, we used a comparative microarray approach to globally identify Pax7 myogenic targets and we propose that the regulation of those genes by Pax7 is intimately related to the protein complexes with which it interacts.Pools of C2C12 myoblasts were transfected with retrovirus expressing either mouse Pax7-FLAG, or a control virus expressing only the puromycin resistance gene (Puro). As expected, persistent Pax7 expression resulted in the maintenance of a proliferative phenotype and 3Correspondence should be addressed to M.A.R. (e-mail: mrudnicki@ohri.ca). * These authors contributed equally to this work. Supplementary Information, S1). Total RNA was harvested to generate probes for hybridization to Affymetrix GeneChip microarrays; 43 genes were upregulated by Pax7-FLAG, including several that exh...

SUMMARY Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome wide binding-site analysis we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrates distinct attributes of Pax7 function and provides mechanistic insight into the non-redundancy of Pax3 and Pax7 in muscle development.

Muscle stem cells comprise different populations of stem and progenitor cells found in embryonic and adult tissues. A number of signaling and transcriptional networks are responsible for specification and survival of these cell populations and regulation of their behavior during growth and regeneration. Muscle progenitor cells are mostly derived from the somites of developing embryos, while satellite cells are the progenitor cells responsible for the majority of postnatal growth and adult muscle regeneration. In resting muscle, these stem cells are quiescent, but reenter the cell cycle during their activation, whereby they undergo decisions to self-renew, proliferate, or differentiate and fuse into multinucleated myofibers to repair damaged muscle. Regulation of muscle stem cell activity is under the precise control of a number of extrinsic signaling pathways and active transcriptional networks that dictate their behavior, fate, and regenerative potential. Here, we review the networks responsible for these different aspects of muscle stem cell biology and discuss prevalent parallels between mechanisms regulating the activity of embryonic muscle progenitor cells and adult satellite cells.