Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.

The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins.In eukaryotes, activation of gene expression involves the ordered assembly of transcriptional regulators, chromatinmodifying enzymes, RNA polymerase II, and associated general transcription factors onto cis-acting elements that are embedded in chromatin. Chromatin-remodeling enzymes play an integral role in gene activation by perturbing chromatin structure and making specific loci permissive for transcription. Molecular analysis of multiple gene activation events suggests that the temporal recruitment of transcription factors and chromatin-remodeling enzymes is gene specific and dictated by the interplay between specific activators and local chromatin structure (1,52,55).Two classes of enzymes have been shown to remodel chromatin structure either by catalyzing covalent modifications of histones or by hydrolyzing ATP to mobilize nucleosomes. Among the latter class of enzymes are the SWI/SNF chromatin-remodeling complexes. A distinguishing feature of this family is the presence of a bromodomain in the ATPase subunit, which promotes interaction with acetylated histones and links the activities of the two classes of chromatin remodelers in the regulation of gene expression (22). SWI/SNF enzymes physically interact with histone acetyltransferases (HATs), histone deacetylases (HDACs), and methyltransferases, showing the potential for coordination of chromatin-remodeling activities (reviewed in reference 53).Mammalian SWI/SNF chromatin-remodeling enzymes are multisubunit complexes that contain either the Brg1 or Brm ATPase subunits and can activate or repress expression of a subset of genes (39, 53). They function in cell cycle control, and some of the subunits are tumor suppressors (49). Diverse SWI/ SNF complexes exist that are distinguished by the particular ATPase, the presence of unique subunits, and tissue-specific ...

We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.

Previous analyses of gene expression in a mouse model of Huntington's disease (R6/2) indicated that an N-terminal fragment of mutant huntingtin causes downregulation of striatal signaling genes and particularly those normally induced by cAMP and retinoic acid. The present study expands the regional and temporal scope of this previous work by assessing whether similar changes occur in other brain regions affected in Huntington's disease and other polyglutamine diseases and by discerning whether gene expression changes precede the appearance of disease signs. Oligonucleotide microarrays were employed to survey the expression of approximately 11,000 mRNAs in the cerebral cortex, cerebellum and striatum of symptomatic R6/2 mice. The number and nature of gene expression changes were similar among these three regions, influenced as expected by regional differences in baseline gene expression. Time-course studies revealed that mRNA changes could only reliably be detected after 4 weeks of age, coincident with development of early pathologic and behavioral changes in these animals. In addition, we discovered that skeletal muscle is also a target of polyglutamine-related perturbations in gene expression, showing changes in mRNAs that are dysregulated in brain and also muscle-specific mRNAs. The complete dataset is available at www.neumetrix.info.

MK-0457 (VX-680

Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle lineage, whereas myogenin mediates terminal differentiation, yet the molecular basis for this distinction is not understood. We show that MyoD can remodel chromatin at binding sites in muscle gene enhancers and activate transcription at previously silent loci. TGF-p, basic-FGF, and sodium butyrate blocked MyoD-mediated chromatin reorganization and the initiation of transcription. In contrast, TGF-3 and sodium butyrate did not block transcription when added after chromatin remodeling had occurred. MyoD and Myf-5 were 10-fold more efficient than myogenin at activating genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the MyoD protein demonstrated that the ability to activate endogenous genes depended on two regions: a region rich in cysteine and histidine residues between the acidic activation domain and the bHLH domain, and a second region in the carboxyl terminus of the protein. Neither region has been shown previously to regulate gene transcription and both have domains that are conserved in the Myf5 protein. Our results establish a mechanism for chromatin modeling in the skeletal muscle lineage and define domains of MyoD, independent of the activation domain, that participate in chromatin reorganization.

The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here, we show that p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression.Supplemental material is available at http://www.genesdev.org. Studies of transcriptional regulation at individual promoters have led to the general model that ordered recruitment of a combination of factors achieves gene-specific transcription (Cosma 2002). Global genomic analysis extends this model to show how complex transcriptional regulatory networks can emerge from the combinatorial regulation of individual genes and has identified classes of simple regulatory motifs, such as feed-forward loops and regulatory cascades ( Fig. 1A) (Lee et al. 2002;Milo et al. 2002;Shen-Orr et al. 2002). A current opportunity for developmental biology is to use these two approaches, promoter-specific molecular biology and systems network biology, to reveal the molecular events that temporally pattern multistage gene expression programs during development and cell differentiation.The differentiation of skeletal muscle is a powerful system for studying the molecular regulation of a multistaged program of cell differentiation. Vertebrate myogenesis is regulated by the bHLH transcription factor MyoD, and its paralogs Myogenin, Myf-5, and MRF4. These act by heterodimerizing with E-proteins and binding CAnnTG recognition sites (Blackwell and Weintraub 1990). Genetic experiments have shown that MyoD or Myf-5 act as lineage-determination factors and Myogenin mediates terminal differentiation (for review, see Arnold and Braun 1996). When expressed in nonmuscle cell types in vitro, each of these factors is sufficient to drive differentiation into skeletal muscle and allows the process to be studied in molecular detail (Weintraub et al. 1989;Choi et al. 1990).Studies of myogenesis have revealed a predictable temporal pattern of gene expression both in vivo and in vitro (Lin et al. 1994;Zhao et al. 2002). In our previous study, we characterized the timing of gene expression associated with muscle differentiation in a model system consisting of mouse embryonic fibroblasts (MEFs) expressing an inducible MyoD-Estrogen Receptor fusion protein (MyoD-ER), which allows synchronized skeletal muscle differentiation (Hollenberg et al. 1993;Bergstrom et al. 2002). Microarray analysis demonstrated that MyoD activity altered the expression of ∼5% of a...

The involvement of c-Myc in cellular proliferation or apoptosis has been linked to differential cyclin gene expression. We observed that in both proliferating cells and cells undergoing apoptosis, cyclin A (but not B. C, D1, and E) mRNA level was elevated in unsynchronized Myc-overexpressing cells when compared with parental Ratla fibroblasts. We further demonstrated that Zn2+-inducible cyclin A expression was sufficient to cause apoptosis When Myc-induced apoptosis was blocked by coexpression ofBc1-2, the levels ofcyclin C, D1, and E mRNAs were also elevated. Thus, while apoptosis induced by c-Myc is associated with an elevated cyclin A mRNA level, protection from apoptouis by coexpressed Bc1-2 is associated with a complementary increase in cycin C, D1, and E mRNAs.c-Myc is a helix-loop-helix leucine-zipper transcriptional factor that participates in opposite cellular fates of proliferation or programmed cell death (apoptosis) (1-4). Under normal cell culture conditions (i.e., high serum), overexpression of c-Myc causes cells to proliferate. Upon serum starvation or growth factor withdrawal, however, certain cells that overexpress c-Myc undergo apoptosis instead of growth arrest (1, 2). Although the domains of c-Myc protein required for neoplastic transformation and apoptosis are the same, the molecular events underlying these divergent cellular fates remain unknown (2-4).Recent studies on the interleukin 2-dependent BAF-B03 pre-B cell line indicate that c-Myc alters cyclin gene expression and affects the transition of cells into the G2 phase (5). Inducible c-Myc activity in a Myc-estrogen receptor chimeric system augments cyclin A and E gene expression and stimulates mitogenesis in rat fibroblasts (6). We sought to define further the possible connections between c-Myc, cyclin gene expression and cell fates. Specifically, we hypothesized that cell death might result from constitutive imbalanced cyclin gene expression induced by c-Myc in the setting of growth factor withdrawal (1, 2, 7). In addition, because Bcl-2 could counteract c-Myc-induced apoptosis (8, 9), the Bcl-2 effect may also be manifested through altered cycin gene expression. (10). Polypeptides from one-tenth of total cell lysates from each 100 x 20 mm plate were resolved by SDS/10%o PAGE and subjected to immunoblot analysis with either anti-Myc 9E10 monoclonal antibody (1:100 dilution) (2) or anti-Bcl-2 monoclonal antibody (1:100 dilution) (Dako). After incubation of blots with a secondary goat anti-mouse horseradish peroxidase-conjugated antibody (Bio-Rad) (1:10,000 dilution), reactive polypeptides were detected by the enhanced chemiluminescence (ECL) system (Amersham). Luminograms from the same blot developed with either antiMyc or anti-Bcl-2 primary antibodies were superimposed and shown with molecular weight standards for clarity of presentation. MATERIALS AND METHODSExpression of Zn2+-inducible human cyclin A in cell lines was detected 24 hr after induction with various final concentrations of ZnSO4. Total cell lysates were prepared,...