No abstract

The mammalian SWI/SNF-related complexes facilitate gene transcription by remodeling chromatin using the energy of ATP hydrolysis. The recruitment of these complexes to promoters remains poorly understood and may involve histone modifications or direct interactions with site-specific transcription factors or other cofactors. Here we report the isolation of two related but distinct cDNA clones, hOsa1 and hOsa2, that encode the largest subunits of human SWI/SNF. hOsa1 is identical to previously reported BAF250, and hOsa2 shares a high degree of sequence similarity with hOsa1. Mass spectrometric analysis, and immunoblotting with antibodies specific to hOsa1 or hOsa2 demonstrate the presence of both proteins in SWI/SNF-A but not in the related polybromo-BRG1-associated factors complex purified from HeLa cells. Co-precipitation studies indicate that hOsa1 and hOsa2 associate with BRG1 and hBRM through the C-terminal domain of hOsa. We define multiple domains within hBRM and BRG1 that interact with the hOsa C terminus. In cultured mammalian cells, hOsa1 and hOsa2 stimulate transcription by the glucocorticoid, estrogen, and androgen receptors. The glucocorticoid receptor-mediated activation is not observed with the Cterminal domain or with the hOsa2 polypeptide lacking the ARID DNA binding domain. These results suggest that hOsa1 and hOsa2 participate in promoting transcriptional activation by the steroid hormone receptors.Modulation of chromatin structure plays a fundamental role in gene expression because transcription factors must contend with nucleosomes, which are generally thought to inhibit transcription. Genetic and biochemical approaches have led to the discovery of multiple protein complexes that are thought to activate or repress transcription by targeting histones or nucleosomes (reviewed in Refs. 1-5). A recent explosion of research in yeast, fruit flies, and mammals has uncovered at least two classes of complexes, those that enzymatically modify histones and those that remodel nucleosomes in an ATPdependent manner. Cells contain multiple distinct chromatin remodeling complexes, each of which includes a central ATPase subunit. The yeast SWI/SNF complex was the first such complex characterized (reviewed in Refs. 6 and 7). Homologous complexes have since been identified in Drosophila and mammals (8). At least two distinct ATPase subunits, termed hBRM and BRG1, have been identified in mammalian SWI/SNFrelated complexes and are homologous to the yeast SWI2/SNF2 and Drosophila Brahma (Brm) proteins (9 -12). Mammalian cells appear to contain multiple SWI/SNF-related complexes (13) and immunoprecipitation experiments using antibodies specific for hBRM or BRG1 suggest that these subunits are present in distinct complexes (14,15). Purification and characterization of these separate complexes demonstrate differences in activity and subunit composition, implying distinct regulatory roles (15,16). Indeed, recent in vitro transcription studies have shown that PBAF (SWI/SNF-B) is selectively required for activated transc...

(4,6,8,45,48,51,52,55,68,73,77,81,82,90). In addition to stimulation of RNA polymerase II-directed transcription, HBx also stimulates transcription by RNA polymerases I and III (3,43,86,88). HBx is therefore a modest activator of many types of transcription elements and factors (28,29,46,51,54,60,61,90

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.

The largest subunit of the mammalian SWI/SNF-A or BAF (BRG1-associated factor) chromatin-remodelling complex is encoded by two related cDNAs hOsa1/BAF250a and hOsa2/BAF250b that are unique to the BAF complex and absent in the related PBAF (Polybromo BAF). hOsa/BAF250 has been shown to interact with transcriptional activators and bind to DNA suggesting that it acts to target the remodelling complex to chromatin. To better understand the functions of hOsa2, we established inducible stable HeLa cell lines over-expressing FLAG-hOsa2 or a derivative lacking the ARID (AT-rich interactive domain) DNA-binding domain. Immunopurification of complexes containing hOsa2 that was followed by mass spectrometry and immunoblotting demonstrated the presence of BRG1 and known BAFs, but not hOsa1 or hBRM. Deletion of the ARID did not compromise the integrity of the complex. Induction of hOsa2 expression caused impaired cell growth and accumulation of cells in the G0/G1 cell cycle phase. Elevated levels of the p53 and p21 proteins were detected in these cells while c-Myc mRNA and protein levels were found to decrease. Chromatin immunoprecipitation and reporter assays suggested that hOsa2 had a direct effect on c-myc and p21 promoter activity. Thus hOsa2 plays an important role in controlling genes regulating the cell cycle.