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...

Evidence suggests that 4-hydroxylation of RA inside the target cell limits its biological activity and initiates a degradative process of RA leading to its eventual elimination. However, 18-hydroxylation and glucuronidation may also be important steps in this process. In this paper, we describe the cloning and characterization of the first mammalian retinoic acid-inducible retinoic acid-metabolizing cytochrome P450 (hP450RAI), which belongs to a novel class of cytochromes (CYP26). We demonstrate that hP450RAI is responsible for generation of several hydroxylated forms of RA, including 4-OH-RA, 4-oxo-RA, and 18-OH-RA. We also show that hP450RAI mRNA expression is highly induced by RA in certain human tumor cell lines and further show that RA-inducible RA metabolism may correlate with P450RAI expression. We conclude that this enzyme plays a key role in RA metabolism, functioning in a feedback loop where RA levels are controlled in an autoregulatory manner.Regulation of retinoid signaling may be controlled by a number of coordinated mechanisms, including retinoid synthesis, cell-specific expression of retinoid-binding proteins and nuclear receptors, and metabolism of retinoids (for review see Refs. 1-3). The generation of RA 1 from its precursors, retinol and retinaldehyde, and its catabolism to more polar hydroxylated forms such as 4-OH-RA, 4-oxo-RA, and 18-OH-RA are counterbalanced metabolic pathways that regulate RA levels in RAsensitive tissues (4, 5). Cellular retinoic acid-binding proteins may also play a role in establishing this balance by sequestering high levels of RA (6). There is considerable evidence to suggest that 4-OH-, 4-oxo-, and 18-OH-RA are polar intermediates in the catabolism and eventual elimination of RA (5,7,8). Thus both sequestration and metabolism may function to protect RA-sensitive tissues from deleterious concentrations of RA.We have cloned and characterized cDNAs corresponding to a retinoic acid-inducible gene encoding a human cytochrome P450-related hydroxylase (P450RAI) responsible for generation of multiple hydroxylated products of RA. hP450RAI appears to be the human ortholog of the previously characterized zebrafish P450RAI (zP450RAI) (9), indicating that this important cytochrome is highly conserved structurally and functionally across species. We also demonstrate that hP450RAI is inducible by RA in a number of different cell types. We speculate that this enzyme plays a key role in determining the metabolic fate of endogenous retinoids and may also be implicated in the clearance of exogenous retinoids administered therapeutically. MATERIALS AND METHODScDNA Library Screening-A NTERA2-D1 cDNA library (Stratagene) was screened according to the manufacturer's directions. Briefly, 1.0 ϫ 10 Ϫ6 independent plaques were screened using a random-primed, ␣-[ 32 P]dATP-labeled full-length zP450RAI cDNA. Filters were prehybridized for 4 h at 37°C in 50% formamide, 5 ϫ SSPE, 1 ϫ Denhardt's (without bovine serum albumin), 0.2 mg/ml denatured salmon sperm DNA. Hybridization was performed overn...

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, alltrans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.

Tissue-specific transcriptional activators initiate differentiation towards specialized cell types by inducing chromatin modifications permissive for transcription at target loci, through the recruitment of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodelling complex. However, the molecular mechanism that regulates SWI/ SNF nuclear distribution in response to differentiation signals is unknown. We show that the muscle determination factor MyoD and the SWI/SNF subunit BAF60c interact on the regulatory elements of MyoD-target genes in myoblasts, prior to activation of transcription. BAF60c facilitates MyoD binding to target genes and marks the chromatin for signal-dependent recruitment of the SWI/ SNF core to muscle genes. BAF60c phosphorylation on a conserved threonine by differentiation-activated p38a kinase is the signal that promotes incorporation of MyoD-BAF60c into a Brg1-based SWI/SNF complex, which remodels the chromatin and activates transcription of MyoD-target genes. Our data support an unprecedented two-step model by which pre-assembled BAF60c-MyoD complex directs recruitment of SWI/SNF to muscle loci in response to differentiation cues.