The mammalian SWI/SNF chromatin-remodeling complex is essential for the multiple changes in gene expression that occur during differentiation. However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated. The catalytic core of the complex can be either of two closely related ATPases, BRM or BRG1, with the potential that the choice of alternative subunits is a key determinant of specificity. Short hairpin RNAmediated depletion of the ATPases was used to explore their respective roles in the well characterized multistage process of osteoblast differentiation. The results reveal an unexpected role for BRM-specific complexes. Instead of impeding differentiation as was seen with BRG1 depletion, depletion of BRM caused accelerated progression to the differentiation phenotype. Multiple tissue-specific differentiation markers, including the tightly regulated late stage marker osteocalcin, become constitutively up-regulated in BRM-depleted cells. Chromatin immunoprecipitation analysis of the osteocalcin promoter as a model for the behavior of the complexes indicates that the promoter is a direct target of both BRM-and BRG1-containing complexes. BRG1 complexes, which are required for activation, are associated with the promoter well before induction, but the concurrent presence of BRM-specific complexes overrides their activation function. BRM-specific complexes are present only on the repressed promoter and are required for association of the corepressor HDAC1. These findings reveal an unanticipated degree of specialization of function linked with the choice of ATPase and suggest a new paradigm for the roles of the alternative subunits during differentiation.The mammalian SWI/SNF complex is an evolutionarily well conserved ATPase-powered chromatin-remodeling assembly consisting of approximately 10 subunits (see Fig. 1A). This complex (also known as the BAF complex) coordinates the disruption of nucleosomes to permit the binding of various transcription factors, an activity crucial for proper differentiation and development (reviewed in Refs. 1-5).The entity known as the mammalian SWI/SNF complex actually consists of a small series of compositionally distinct assemblies distinguished by the presence of alternative subunits. The choice of ARID family subunit (ARID1A or ARID1B) is a determinant of complexes with generally opposing roles in cell cycle control (Ref. 6 and reviewed in Ref. 7). The complexes also contain either of two closely related alternative ATPases: Brahma (BRM) 3 or Brahma-related gene 1 (BRG1). Although BRM and BRG1 share a high degree of amino acid sequence identity, they are not equally important for development. Brg1-null mice die at a pre-or peri-implantation stage (8), indicating a critical developmental role for BRG1. In contrast, Brm-null mice are viable and fertile, exhibiting only mild abnormalities that include a larger animal size and deregulated cell growth control in derived fibroblasts (9). This study ...

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P< 0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells.

Human SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromosomes and alter gene expression patterns. The activity of the complexes generally promotes tissue-specific gene expression and restricts cell proliferation. The ATPase that drives the complexes, BRG1, is essential for tumor suppression in mice and deficient in a variety of established human tumor cell lines. The complex contains at least 7 other core components, one of which is a large subunit designated p270. p270 RNA is expressed in all normal human tissues examined, but protein expression is severely reduced in at least 2 human tumor lines, C33A and T47D. We show here that loss of p270 in the C33A and T47D cell lines is evident at the RNA level as well as the protein level. The implication that p270 can be informatively screened at the RNA level made a high-efficiency cancer profiling array approach to screening human tumors feasible. Expression was screened in an array containing RNA-derived cDNA from 241 tumor and corresponding matched normal tissues from individual patients. p270 deficiency was observed at a higher overall frequency than BRG1 deficiency, but all tissues were not equally affected. Deficiency of p270 was observed most frequently in carcinomas of the breast and kidney. The results were most striking in kidney, where p270 expression was deficient in 30% of carcinoma samples screened. Key words: p270; SWI/SNF; kidney cancer; breast cancer; tumor suppressionHuman SWI/SNF complexes are transcription-regulating complexes that are essential to normal differentiation and development. They also play a vital role in the control of cell proliferation and suppression of carcinogenesis (reviewed by Klochendler-Yeivin and Yaniv 1 and by Muchardt and Yaniv 2 ). The complexes can vary in composition, but all are able to remodel chromatin structure through ATP-dependent mechanisms. [3][4][5][6][7] ATPase activity, which is DNA-dependent, resides in the SWI2/SNF2 enzyme in yeast and in the SWI2/SNF2-related proteins BRG1 and hBRM in humans. Shortly after BRG1 was cloned on the basis of its homology to the Drosophila and yeast ATPases, 8 it became apparent that BRG1 is occasionally found at low or undetectable levels in common tumor-derived laboratory lines. 2,9,10 This suggestion that loss of BRG1 may be a causative factor in human tumorigenesis is strongly supported by the enhanced tumor susceptibility of BRG1 heterozygous mice; tumors of epithelial origin were observed in the head and inguinal regions of these mice. 11 Additional components of the complex are also likely to be vital for tumor suppression. The hSNF5 complex component (also designated INI1 or BAF47) is lost with high frequency in specific types of human tumors, primarily pediatric rhabdoid tumors, 12-15 and Snf5-deficient mice also develop tumors at an early age. 16,17 BRG1-associated factor-155 (BAF155) is occasionally deficient in tumor cell lines, 10 and BAF155-null mouse embryos die at an early implantation stage, indicating the gene is required for normal developm...

Background:The ARID2 protein is specific for a certain subset of SWI/SNF chromatin-remodeling complexes known as PBAF. Results: ARID2 is essential for gene activation along the osteoblast phenotype. Conclusion: ARID2-containing complexes play a major role in lineage commitment and differentiation. Significance: Manipulating levels of ARID family subunits (of which there are three mutually exclusive alternatives in SWI/ SNF) may influence precursor state and lineage determination without compromising full SWI/SNF function.

A central question in cancer biology is why most tumor susceptibility genes are linked with only limited types of cancer. Human germ-line mutation of the retinoblastoma susceptibility gene Rb1 is closely linked with just retinoblastoma and osteosarcoma, although the gene is universally expressed. Functional analysis of pRB and its close relatives, p107 and p130, has largely focused on their roles in repression of proliferation across all tissue types, but genetic evidence indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Still, potential promoter targets of pRB and its role in normally differentiating osteoblasts remain insufficiently characterized. Here, an early marker of osteoblast differentiation, alkaline phosphatase, is identified as a direct promoter activation target of pRB. One role of pRB on this promoter is to displace the histone lysine demethylase KDM5A, thereby favoring trimethylation of H3K4, a promoter activation mark. A major new aspect of pRB-mediated transcriptional activation revealed in this promoter analysis is its role in recruitment of an activating SWI/SNF chromatin-remodeling complex. SWI/SNF is a critical coordinator of tissue-specific gene expression. In osteoblasts, SWI/SNF complexes containing the BRM ATPase repress osteoblast-specific genes to maintain the precursor state, whereas the alternative ATPase BRG1 distinguishes an activating SWI/SNF complex necessary for RNA polymerase-II recruitment. A switch from BRM to BRG1 on the alkaline phosphatase promoter marks the onset of differentiation and is accomplished in a precise two-step mechanism. Dissociation of BRM-containing SWI/SNF depends on p300, and association of BRG1-containing SWI/SNF depends on pRB. Cancer Res; 70(21); 8282-7. ©2010 AACR.