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 ...
The initiation of cellular differentiation involves alterations in gene expression that depend on chromatin changes, at the level of both higher-order structures and individual genes. Consistent with this, chromatin-remodelling enzymes have key roles in differentiation and development. The functions of ATP-dependent chromatin-remodelling enzymes have been studied in several mammalian differentiation pathways, revealing cell-type-specific and gene-specific roles for these proteins that add another layer of precision to the regulation of differentiation. Recent studies have also revealed a role for ATP-dependent remodelling in regulating the balance between proliferation and differentiation, and have uncovered intriguing links between chromatin remodelling and other cellular processes during differentiation, including recombination, genome organization and the cell cycle.
Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes that have been implicated in the regulation of gene expression, cell-cycle control and oncogenesis. MyoD is a muscle-specific regulator able to induce myogenesis in numerous cell types. To ascertain the requirement for chromatin remodeling enzymes in cellular differentiation processes, we examined MyoD-mediated induction of muscle differentiation in fibroblasts expressing dominant-negative versions of the human brahma-related gene-1 (BRG1) or human brahma (BRM), the ATPase subunits of two distinct SWI/SNF enzymes. We find that induction of the myogenic phenotype is completely abrogated in the presence of the mutant enzymes. We further demonstrate that failure to induce muscle-specific gene expression correlates with inhibition of chromatin remodeling in the promoter region of an endogenous muscle-specific gene. Our results demonstrate that SWI/SNF enzymes promote MyoD-mediated muscle differentiation and indicate that these enzymes function by altering chromatin structure in promoter regions of endogenous, differentiation-specific loci.
ATP-dependent chromatin-remodeling complexes are conserved among all eukaryotes and function by altering nucleosome structure to allow cellular regulatory factors access to the DNA. Mammalian SWI-SNF complexes contain either of two highly conserved ATPase subunits: BRG1 or BRM. To identify cellular genes that require mammalian SWI-SNF complexes for the activation of gene expression, we have generated cell lines that inducibly express mutant forms of the BRG1 or BRM ATPases that are unable to bind and hydrolyze ATP. The mutant subunits physically associate with at least two endogenous members of mammalian SWI-SNF complexes, suggesting that nonfunctional, dominant negative complexes may be formed. We determined that expression of the mutant BRG1 or BRM proteins impaired the ability of cells to activate the endogenous stress response gene hsp70 in response to arsenite, a metabolic inhibitor, or cadmium, a heavy metal. Activation of hsp70 by heat stress, however, was unaffected. Activation of the heme oxygenase 1 promoter by arsenite or cadmium and activation of the cadmium-inducible metallothionein promoter also were unaffected by the expression of mutant SWI-SNF components. Analysis of a subset of constitutively expressed genes revealed no or minimal effects on transcript levels. We propose that the requirement for mammalian SWI-SNF complexes in gene activation events will be specific to individual genes and signaling pathways.The packaging of eukaryotic DNA into nucleosomes and higher order chromatin structure presents cells with a significant barrier to DNA utilization and necessitates mechanisms by which chromatin structure can be modified so that transcription can occur. Many multiprotein complexes with the ability to modify chromatin structure have been identified. These include histone acetyltransferases and deacetylases, which directly modify histone tail domains, and a class of energy-dependent enzymes that utilize ATP hydrolysis to alter nucleosome structure (reviewed in references 23, 30, 32, 34, 70, 83, and 84). The ATP-dependent chromatin remodeling complexes are conserved among eukaryotes, they share a related subunit that possesses DNA-stimulated ATPase activity, and each has been demonstrated to alter nucleosome structure in vitro in an ATP-dependent manner. Most of these complexes can be classified into two groups, those containing homologues of the yeast SWI2-SNF2 ATPase subunit, including yeast SWI-SNF (7, 12, 55), human SWI-SNF (hSWI-SNF) (24, 35, 82), yeast RSC (8), and Drosophila BRM complexes (54, 71), and those containing homologues of the Drosophila imitation-switch (ISWI) ATPase gene (16), including yeast ISW1 and ISW2 (76), human RSF (39), and the Drosophila NURF, CHRAC, and ACF complexes (25,75,78). A third group can be defined by Xenopus and human complexes containing the Mi2 protein, a related ATPase found in association with histone deacetylase activity (72,81,87,90).Although members of the ATP-dependent class of chromatin remodelers facilitate alterations in nucleosome structure in ...
The microphthalmia-associated transcription factor (MITF) promotes melanocyte differentiation and cell cycle arrest. Paradoxically, MITF also promotes melanoma survival and proliferation, acting like a lineage survival oncogene. Thus, it is critically important to understand the mechanisms that regulate MITF activity in melanoma cells. SWI/SNF chromatin remodeling enzymes are multiprotein complexes composed of one of two related ATPases, BRG1 or BRM, and 9-12 associated factors (BAFs). We previously determined that BRG1 interacts with MITF to promote melanocyte differentiation. However, it was unclear whether SWI/SNF enzymes regulate the expression of different classes of MITF target genes in melanoma. In this study, we characterized SWI/SNF subunit expression in melanoma cells and observed down-regulation of BRG1 or BRM, but not concomitant loss of both ATPases. Re-introduction of BRG1 in BRG1 deficient SK-MEL5 cells enhanced expression of differentiation specific MITF target genes and resistance to cisplatin. Down-regulation of the single ATPase, BRM, in SK-MEL5 cells inhibited expression of both differentiation specific and pro-proliferative MITF target genes and inhibited tumorigenicity in vitro. Our data suggest that heterogeneous SWI/SNF complexes composed of either the BRG1 or BRM subunit promote expression of distinct and overlapping MITF target genes and that at least one ATPase is required for melanoma tumorigenicity.
Cell cycle arrest is critical for muscle differentiation, and the two processes are closely coordinated but temporally separable. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that have been shown to be required for muscle differentiation in cell culture and have also been reported to be required for Rb-mediated cell cycle arrest. We therefore looked more closely at how SWI/SNF enzymes affect the events that occur during MyoD-induced myogenesis, namely, cell cycle regulation and muscle-specific gene expression, in cells that inducibly express dominant negative versions of Brahma (BRM) and Brahma-related gene 1 (BRG1), the ATPase subunits of two distinct SWI/SNF complexes. Although dominant negative BRM and BRG1 inhibited expression of every muscle-specific regulator and structural gene assayed, there was no effect on MyoD-induced activation of cell cycle regulatory proteins, and thus, cells arrested normally. In particular, in the presence or absence of dominant negative BRM or BRG1, MyoD was able to activate expression of p21, cyclin D3, and Rb, all of which are critical for cell cycle withdrawal in the G 1 /G 0 phase of the cell cycle. These findings suggest that at least one basis for the distinct mechanisms that regulate cessation of cell proliferation and musclespecific gene expression during muscle differentiation is that SWI/SNF-mediated chromatin-remodeling enzymes are required only for the latter.
Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.
The microphthalmia transcription factor (Mitf ) activates melanocyte-specific gene expression, is critical for survival and proliferation of melanocytes during development, and has been described as an oncogene in malignant melanoma. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that play a role in many developmental processes. To determine the requirement for SWI/SNF enzymes in melanocyte differentiation, we introduced Mitf into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, Brahma or Brahma-related gene 1 (BRG1). These dominant negative SWI/SNF components have been shown to inhibit gene activation events that normally require SWI/SNF enzymes. We found that Mitf-mediated activation of a subset of endogenous melanocyte-specific genes required SWI/SNF enzymes but that cell-cycle regulation occurred independently of SWI/SNF function. Activation of tyrosinase-related protein 1, a melanocytespecific gene, correlated with SWI/SNF-dependent changes in chromatin accessibility at the endogenous locus. Both BRG1 and Mitf could be localized to the tyrosinase-related protein 1 and tyrosinase promoters by chromatin immunoprecipitation, whereas immunofluorescence and immunoprecipitation experiments indicate that Mitf and BRG1 co-localized in the nucleus and physically interacted. Together these results suggest that Mitf can recruit SWI/SNF enzymes to melanocyte-specific promoters for the activation of gene expression via induced changes in chromatin structure at endogenous loci.Melanocytes are pigment-producing cells that are developmentally derived from the neural crest and that comprise 1-2% of the epidermis (1). They are also present on the epithelial surfaces of mucous membranes, hair follicles, the cochlea of the inner ear, and both the uvea and conjunctiva of the eye (2, 3). On the skin, they play a photoprotective role by synthesizing and distributing melanin (4). Excessive exposure to UV radiation has been linked to the transformation of cutaneous melanocytes to melanoma, a cancer that has steadily increased in frequency and is difficult to treat (5, 6).Microphthalmia-associated transcription factor (Mitf ) 2 is the "master regulator" of melanocyte differentiation and was elegantly shown to convert fibroblasts into dendritic cells that express melanocyte-specific genes (7). It is important for the commitment, proliferation, and survival of melanocytes during neural crest cell migration, and null mutations of the mouse Mitf gene result in complete absence of melanocytes and lack of pigmentation in the skin, eyes, and inner ear (8, 9). Two human diseases resulting from mutations in the MITF gene are Waardenburg type 2 syndrome and Tietz syndrome, both of which are characterized by pigmentary disturbances and sensineural deafness (8).Mitf is a basic helix-loop-helix leucine zipper transcription factor that binds DNA either as a homodimer or as a heterodimer with TFE3, TFEB, or TFEC to conserved E boxes (CAC(G/A)TG) in the promoters of its target genes, which incl...
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