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...

Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date. In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development. We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly. We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction. We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M. We next tested whether Follistatin suppresses Myostatin activity during muscle development. We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD. However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked. We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner. In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development.

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RPTP is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTP or CRYP␣, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTP are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTP is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTP in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTP. HSPGs are therefore a novel class of heterotypic ligand for cPTP, suggesting that cPTP signaling in axons and growth cones is directly responsive to matrix-associated cues.The complex pattern of neural connectivity established during nervous system development relies on the ability of the axon's motile tip, the growth cone, to receive, transduce, and integrate multiple environmental signals. Protein phosphorylation on tyrosine residues plays a key role in these processes (21, 29). Two major families of enzymes, the protein tyrosine kinases and the protein tyrosine phosphatases (PTPs), control cellular phosphotyrosine levels. These enzymes are found in both cytoplasmic and transmembrane (receptor-like) forms, and the biochemical interactions between them lead to a diversity of cellular behaviors (25,76).Receptor protein tyrosine phosphatases (RPTPs) have recently joined the list of molecules involved in neural development and in particular in axon growth and guidance (reviewed in references 6, 68, 72, and 79). Type 2 RPTPs, containing cell adhesion molecule (CAM)-like extracellular regions, may be particularly well equipped to trigger signals involving cell-cell or cell-extracellular matrix contacts (68). Recent experiments with Drosophila have demonstrated the involvement of the RPTPs DLAR and DPTP69D in motor (19, 20, 50), retinal (27, 57), and midline (73) axon guidance. In leech, a LAR generelated RPTP (HmLAR2) is implicated in Comb cell behavior, specifically in process outgrowth and mutual avoidance by sibling growth cones (2, 28). Several vertebrate RPTPs have been shown to promote neurite outgrowth in cell culture, including cPTP (51), RPTP (23), RPTP (10), and RPTP␦ (82). Moreover, it has recently been shown that RPTP␦ also has a potential guidance function, at least in vitro (74).In mice, gene deficiencies in type 2 RPTPs lead to various abnormalities. LAR deficiency leads to a reduction in size of basal forebrain cholinergic neurons, diminished hippocampal innervation, and defects in o...

SUMMARY In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N-terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a novel reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.