The Polycomb group (PcG) genes are required for maintenance of homeotic gene repression during development. Mutations in these genes can be suppressed by mutations in genes of the SWI/SNF family. We have purified a complex, termed PRC1 (Polycomb repressive complex 1), that contains the products of the PcG genes Polycomb, Posterior sex combs, polyhomeotic, Sex combs on midleg, and several other proteins. Preincubation of PRC1 with nucleosomal arrays blocked the ability of these arrays to be remodeled by SWI/SNF. Addition of PRC1 to arrays at the same time as SWI/SNF did not block remodeling. Thus, PRC1 and SWI/SNF might compete with each other for the nucleosomal template. Several different types of repressive complexes, including deacetylases, interact with histone tails. In contrast, PRC1 was active on nucleosomal arrays formed with tailless histones.

Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.[Keywords: Zebrafish; rhabdomyosarcoma; RAS; P53; transgenic] Supplemental material is available at http://www.genesdev.org.

Two known zebrafish dystrophin mutants, sapje and sapje-like (sap c/100 ), represent excellent small-animal models of human muscular dystrophy. Using these dystrophin-null zebrafish, we have screened the Prestwick chemical library for small molecules that modulate the muscle phenotype in these fish. With a quick and easy birefringence assay, we have identified seven small molecules that influence muscle pathology in dystrophin-null zebrafish without restoration of dystrophin expression. Three of seven candidate chemicals restored normal birefringence and increased survival of dystrophin-null fish. One chemical, aminophylline, which is known to be a nonselective phosphodiesterase (PDE) inhibitor, had the greatest ability to restore normal muscle structure and up-regulate the cAMP-dependent PKA pathway in treated dystrophin-deficient fish. Moreover, other PDE inhibitors also reduced the percentage of affected sapje fish. The identification of compounds, especially PDE inhibitors, that moderate the muscle phenotype in these dystrophin-null zebrafish validates the screening protocol described here and may lead to candidate molecules to be used as therapeutic interventions in human muscular dystrophy.phosphodiesterase inhibitor | chemical treatment M uscular dystrophy is a disease in which the muscle forms normally at first but then degenerates faster than it can be repaired. The most common form of muscular dystrophy is Duchenne muscular dystrophy (DMD), representing more than 90% of the diagnosed cases. Mutations in the dystrophin gene were found to be the cause of both DMD and Becker muscular dystrophy (1, 2). Currently, prednisone is the only treatment option available for muscular dystrophy patients in the United States, although there are currently other options through approved clinical trials. Other treatments currently being tested or considered for treating muscular dystrophy include the small molecule PTC124, which promotes read-through of nonsense mutations (3), encouraging muscle development by myostatin down-regulation (4, 5), and the use of oligonucleotides to promote exon skipping to restore dystrophin expression (6).Recently, a number of chemical and drug screens have been published using zebrafish embryos (7-11). It is possible to quickly produce large numbers of mutant offspring that can then be assayed in multiwell plates and treated with different chemicals to determine if disease progression is modulated. Many of these screens have been highly successful in disease modeling (7) and drug screening (8-10), making the zebrafish ideal for highthroughput whole-organism screening of candidate compounds. Chemical compounds of relatively small molecular weight can bind to specific proteins and alter their function, resulting in nonheritable phenotype changes.In addition to their suitability for chemical screens, zebrafish also represent a good model to investigate genes involved in muscle development and degeneration, including human muscular dystrophy (12-18). The orthologs of many dystrophin-glycoprot...

Dystrobrevin is a component of the dystrophin-associated protein complex and has been shown to interact directly with dystrophin, ␣1-syntrophin, and the sarcoglycan complex. The precise role of ␣-dystrobrevin in skeletal muscle has not yet been determined. To study ␣-dystrobrevin's function in skeletal muscle, we used the yeast two-hybrid approach to look for interacting proteins. Three overlapping clones were identified that encoded an intermediate filament protein we subsequently named desmuslin (DMN). Sequence analysis revealed that DMN has a short N-terminal domain, a conserved rod domain, and a long C-terminal domain, all common features of type 6 intermediate filament proteins. A positive interaction between DMN and ␣-dystrobrevin was confirmed with an in vitro coimmunoprecipitation assay. By Northern blot analysis, we find that DMN is expressed mainly in heart and skeletal muscle, although there is some expression in brain. Western blotting detected a 160-kDa protein in heart and skeletal muscle. Immunofluorescent microscopy localizes DMN in a stripe-like pattern in longitudinal sections and in a mosaic pattern in cross sections of skeletal muscle. Electron microscopic analysis shows DMN colocalized with desmin at the Z-lines. Subsequent coimmunoprecipitation experiments confirmed an interaction with desmin. Our findings suggest that DMN may serve as a direct linkage between the extracellular matrix and the Z-discs (through plectin) and may play an important role in maintaining muscle cell integrity.T he severe muscle wasting disorder, Duchenne muscular dystrophy, is caused by abnormalities in the dystrophin gene (1). The dystrophin protein is expressed in heart and skeletal muscle, where it is part of the dystrophin-associated protein complex. Dystrophin's N-terminal domain binds to actin, whereas the WW domain and the total cysteine-rich domain bind to ␤-dystroglycan (2), a component of the dystroglycan subcomplex. This subcomplex links to laminin, a major component of the basal membrane, thereby forming the linkage between an intracellular protein, actin, and the extracellular matrix.A second subcomplex of the dystrophin-associated protein complex includes four transmembrane proteins (␣-, ␤-, ␥-, and ␦-sarcoglycan) (3). Each has been shown to be involved in different forms of limb-girdle muscular dystrophy (LGMD 2D, 2E, 2C, and 2F) (4-8). ␣-Sarcoglycan is a type 1 transmembrane protein and is expressed in heart and skeletal muscle. ␤-, ␥-, and ␦-sarcoglycans are type 2 transmembrane proteins containing a cluster of cysteine residues in their extracellular domains. These four proteins form the sarcoglycan complex, which is thought to be involved in some type of signaling pathway (9).A third subcomplex of the dystrophin-associated protein complex involves ␣-dystrobrevin (10-12) and the syntrophins (␣1, ␤1, and ␤2) (13-15). These intracellular proteins directly bind to dystrophin (16,17). In addition, the N-terminal region of ␣-dystrobrevin associates with the sarcoglycan complex (18). There are at least ...

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.