BRG1 Mutations Research Articles

On the Interaction Between SMARCAL1 and BRG1.

SMARCAL1 and BRG1, both classified as ATP-dependent chromatin remodeling proteins, play a role in double-strand break DNA damage response pathways. Mutations in SMARCAL1 cause Schimke Immuno-osseous Dysplasia (SIOD) while mutations in BRG1 are associated with Coffin-Siris Syndrome (CSS4). In HeLa cells, SMARCAL1 and BRG1 co-regulate the expression of ATM, ATR, and RNAi genes on doxorubicin-induced DNA damage. Both the proteins are found to be simultaneously present on the promoter of these genes. Based on these results we hypothesized that SMARCAL1 and BRG1 interact with each other forming a complex. In this paper, we validate our hypothesis and show that SMARCAL1 and BRG1 do indeed interact with each other both in the absence and presence of doxorubicin. The formation of these complexes is dependent on the ATPase activity of both SMARCAL1 and BRG1. Using deletion constructs, we show that the HARP domains of SMARCAL1 mediate interaction with BRG1 while multiple domains of BRG1 are probably important for binding to SMARCAL1. We also show that SIOD-associated mutants fail to form a complex with BRG1. Similarly, CSS4-associated mutants of BRG1 fail to interact with SMARCAL1, thus, possibly contributing to the failure of the DNA damage response pathway and pathophysiology associated with SIOD and CSS4.

1837P Comprehensive genomic profiling of SMARCA2/4 alterations in Chinese pan-cancer patients (pts) identified by next generation sequencing (NGS)

Genomic alterations in SWI/SNF chromatin remodeling, which is crucially composed with SMARCA4 and SMARCA2 (BRG1 and BRM; B&B), occur in approximately 16% of solid tumors. Mutations in BRG1 (BRG1m) lead to an expression upregulating of functionally opposite BRM gene, which make BRM as a potential therapeutic target in BRG1m pts. However, the genomic features of B&B in solid tumors remain unclear.

Cancer-Associated Gain-of-Function Mutations Activate a SWI/SNF-Family Regulatory Hub

SummarySWI/SNF-family remodelers (BAF/PBAF in mammals) are essential chromatin regulators, and mutations in human BAF/PBAF components are associated with ∼20% of cancers. Cancer-associated missense mutations in human BRG1 (encoding the catalytic ATPase) have been characterized previously as conferring loss-of-function. Here, we show that cancer-associated missense mutations in BRG1, when placed into the orthologous Sth1 ATPase of the yeast RSC remodeler, separate into two categories: loss-of-function enzymes, or instead, gain-of-function enzymes that greatly improve DNA translocation efficiency and nucleosome remodeling in vitro. Our work identifies a structural “hub,” formed by the association of several Sth1 domains, that regulates ATPase activity and DNA translocation efficiency. Remarkably, all gain-of-function cancer-associated mutations and all loss-of-function mutations physically localize to distinct adjacent regions in the hub, which specifically regulate and implement DNA translocation, respectively. In vivo, only gain-of-function cancer-associated mutations conferred precocious chromatin accessibility. Taken together, we provide a structure-function mechanistic basis for cancer-associated hyperactivity.

BRG1 Loss Predisposes Lung Cancers to Replicative Stress and ATR Dependency.

Inactivation of SMARCA4/BRG1, the core ATPase subunit of mammalian SWI/SNF complexes, occurs at very high frequencies in non-small cell lung cancers (NSCLC). There are no targeted therapies for this subset of lung cancers, nor is it known how mutations in BRG1 contribute to lung cancer progression. Using a combination of gain- and loss-of-function approaches, we demonstrate that deletion of BRG1 in lung cancer leads to activation of replication stress responses. Single-molecule assessment of replication fork dynamics in BRG1-deficient cells revealed increased origin firing mediated by the prelicensing protein, CDC6. Quantitative mass spectrometry and coimmunoprecipitation assays showed that BRG1-containing SWI/SNF complexes interact with RPA complexes. Finally, BRG1-deficient lung cancers were sensitive to pharmacologic inhibition of ATR. These findings provide novel mechanistic insight into BRG1-mutant lung cancers and suggest that their dependency on ATR can be leveraged therapeutically and potentially expanded to BRG1-mutant cancers in other tissues. SIGNIFICANCE: These findings indicate that inhibition of ATR is a promising therapy for the 10% of non-small cell lung cancer patients harboring mutations in SMARCA4/BRG1. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3841/F1.large.jpg.

Circuit diversification in a biofilm regulatory network.

Genotype-phenotype relationships can vary extensively among members of a species. One cause of this variation is circuit diversification, the alteration of gene regulatory relationships among members of a species. Circuit diversification is thought to be a starting point for the circuit divergence or rewiring that occurs during speciation. How widespread is circuit diversification? Here we address this question with the fungal pathogen Candida albicans, which forms biofilms rich in distinctive hyphal cells as a prelude to infection. Our understanding of the biofilm/hyphal regulatory network comes primarily from studies of one clinical isolate, strain SC5314, and its marked derivatives. We used CRISPR-based methods to create mutations of four key biofilm transcription factor genes–BCR1, UME6, BRG1, and EFG1 –in SC5314 and four additional clinical isolates. Phenotypic analysis revealed that mutations in BCR1 or UME6 have variable impact across strains, while mutations in BRG1 or EFG1 had uniformly severe impact. Gene expression, sampled with Nanostring probes and examined comprehensively for EFG1 via RNA-Seq, indicates that regulatory relationships are highly variable among isolates. Our results suggest that genotype-phenotype relationships vary in this strain panel in part because of differences in control of BRG1 by BCR1, a hypothesis that is supported through engineered constitutive expression of BRG1. Overall, the data show that circuit diversification is the rule, not the exception, in this biofilm/hyphal regulatory network.

Targeting of BRM Sensitizes BRG1-Mutant Lung Cancer Cell Lines to Radiotherapy.

Targeting of epigenetic regulators as the chromatin remodeler SWI/SNF is proving to be a promising therapeutic strategy for individualized treatment of cancer patients. Here, we tested whether targeting one of the two mutually exclusive subdomains of the SWI/SNF complex BRM/SMARCA2 can sensitize specifically non-small cell lung carcinoma (NSCLC) cells with mutations in the other subunit BRG1/SMARCA4 toward ionizing radiation (IR). Knockdown of BRM with siRNA or shRNA and its consequences for radiation sensitivity as measured by clonogenic survival and plaque-monolayer control was studied in different NSCLC lines with or without BRG1 mutations and in primary fibroblasts. Furthermore, the effect on double-strand break (DSB) repair markers measured by immunofluorescence staining of 53BP1-, γ-H2AX-, and Rad51-foci was investigated. BRG1-mutated cell lines showed an increased surviving fraction compared with BRG1 proficient cells. Depletion of BRM (i) leads to a decreased proliferation rate and plating efficiency specifically in BRG1-mutated cells, (ii) specifically sensitized BRG1-mutant NSCLC cells toward IR as characterized by a survival reducing factor of 0.63 [95% confidence interval (CI), 0.57-0.69] in the dose range between 2 and 6 Gy, and (iii) decreased the tumor control doses after daily fractionation at 4 Gy in BRG1-mutant NSCLC cell lines A549 and H1299 in minimonolayers by 9.9% ± 1.3% and 13.6% ± 1.8%, respectively. In addition, an increase of residual Rad51-foci at 24 hours after irradiation in BRG1-mutant cells was demonstrated. Therefore, targeting of BRM in combination with radiotherapy is supposed to improve the therapeutic outcome of lung cancer patients harboring BRG1 mutations.The present study shows that the moderate radioresponsiveness of NSCLC cells with BRG1 mutations can be increased upon BRM depletion that is associated with a prolonged Rad51-foci prevalence at DNA DSBs.

Molecular Simulations Discern the Cooperative Binding of Human Brahma-Related Gene 1 Bromodomain and At-Hook Regions in DNA Binding

Human brahma-related gene 1 (Brg1) is a central ATPase in the human SWI/SNF chromatin remodeling complex. It contains a bromodomain, which associates with acetylated nucleosomes and is critical in DNA damage response along with a small AT-hook motif that recognizes DNA and enhances the DNA-binding activity. NMR spectroscopy, CD spectroscopy and isothermal calorimetry studies have been conducted on both the Brg1 bromodomain and the AT-hook interacting with the DNA separately. The results show that the Brg1 bromodomain can bind the DNA through an interaction patch which includes the A helix and the AB and ZA loops while the AT-hook prefers to bind in the minor groove. Information about the mechanism of the simultaneous binding are still unknown and, since Brg1 mutations are involved in several human tumors, understanding the function of DNA-Brg1 complex is of key importance to develop specific inhibitors. Here, we’ve combined molecular docking, classic molecular dynamics, and metadynamics simulations to investigate how the Brg1 bromodomain identifies specific binding sites within the DNA helix, both in the presence and absence of the AT-hook motif. Free energy surface discriminate the different binding modes found for both complexes and show that the most likely configurations adopted primarily involve the AT-hook binding in the minor groove while the A helix is parallel to the DNA. Without the AT-hook, the DNA/Brg complex binds to the DNA through the A helix interactions in a perpendicular orientation with the major groove. Our results suggest that the DNA/Brg1 binding mechanism is different in presence and absence of the AT-hook segment in the Brg1 sequence.

Mechanism of BRG1 silencing in primary cancers.

BRG1 (SMARCA4) is a documented tumor suppressor and a key subunit of the SWI/SNF chromatin remodeling complex that is silenced in many cancer types. Studies have shown that BRG1 is mutated in cancer-derived cell lines, which led to the assertion that BRG1 is also mutated in primary human tumors. However, the sequencing of BRG1-deficient tumors has revealed a paucity of mutations; hence, the cause of BRG1 silencing in tumors remains an enigma. We conducted immunohistochemistry (IHC) on a number of tumor microarrays to characterize the frequency of BRG1 loss in different tumor types. We also analyzed BRG1-deficient tumors by sequencing the genomic DNA and the mRNA. We then tested if BRG1 expression could be induced in BRG1-negative cell lines (i.e., that lack mutations in BRG1) after the application of several different epigenetic agents, including drugs that inhibit the AKT pathway. We found that a subset of BRG1-negative cell lines also demonstrated aberrant splicing of BRG1, and in at least 30% of BRG1-deficient tumors, BRG1 expression appeared to be suppressed due to aberrant BRG1 splicing. As the majority of BRG1-deficient tumors lack mutations or splicing defects that could drive BRG1 loss of expression, this suggests that other mechanisms underlie BRG1 silencing. To this end, we analyzed 3 BRG1-deficient nonmutated cancer cell lines and found that BRG1 was inducible in these cell lines upon inhibition of the AKT pathway. We show that the loss of BRG1 is associated with the loss of E-cadherin and up-regulation of Vimentin in primary tumors, which explains why BRG1 loss is associated with a poor prognosis in multiple tumor types.

EZH2 inhibition sensitizes BRG1 and EGFR mutant lung tumours to TopoII inhibitors.

SUMMARYNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide1. Chemotherapies such as the topoisomerase II inhibitor (TopoIIi) etoposide effectively reduce disease in a minority of NSCLC patients2,3; therefore, alternative drug targets, including epigenetic enzymes, are under consideration for therapeutic intervention4. A promising potential epigenetic target is the methyltransferase EZH2, which in the context of the Polycomb Repressive Complex 2 (PRC2) is well known to tri-methylate Histone H3 at lysine 27 (H3K27me3) and elicit gene silencing5. Here, we demonstrate that EZH2 inhibition (EZH2i) had differential effects on TopoIIi response of NSCLCs in vitro and in vivo. EGFR and BRG1 mutations were genetic biomarkers that predicted enhanced sensitivity to TopoIIi in response to EZH2i. BRG1 loss-of-function mutant tumors responded to EZH2i with increased S phase, anaphase bridging, apoptosis, and TopoIIi sensitivity. Conversely, EGFR and BRG1 wild-type tumors up-regulated BRG1 in response to EZH2i and ultimately became more resistant to TopoIIi. EGFR gain-of-function mutant tumors were also sensitive to dual EZH2i and TopoIIi, due to genetic antagonism between EGFR and BRG1. These findings suggest an exciting opportunity for precision medicine in the genetically complex disease of NSCLC.

Abstract 5186: Reduced expression of BRM in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is the fifth most common malignancy in men and the eighth most common in women worldwide. The eukaryotic genome is organized into highly ordered chromatin structures in the nucleus. Chromatin creates a barrier to nuclear processes, such as transcription, by obstructing the access of the transcriptional machinery and gene-specific regulators to recognition sequences within target promoters. The SWI/SNF protein complex, an ATPase-dependent chromatin remodeling enzyme, mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics. SWI/SNF is a large multiprotein complex that contains either BRG1 or brahma (BRM) as the catalytic ATPase. Each SWI/SNF complex thus incorporates one of two possible ATPases, BRG1 or BRM. Aims: To examine alterations of BRG1 and BRM in HCC. Methods: We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumors, and conducted further searches for mutations in BRG1 and BRM genes. Results: Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumors respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumors, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumors, compared to non-tumor tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival. Conclusion: Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear. Citation Format: Kohichiroh Yasui, Yasuyuki Gen, Akira Tomie, Tomoko Kitaichi, Yoshito Itoh. Reduced expression of BRM in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2014-5186

Gene Silencing Associated with SWI/SNF Complex Loss during NSCLC Development

The SWI/SNF chromatin-remodeling complex regulates gene expression and alters chromatin structures in an ATP-dependent manner. Recent sequencing efforts have shown mutations in BRG1 (SMARCA4), one of two mutually exclusive ATPase subunits in the complex, in a significant number of human lung tumor cell lines and primary non-small cell lung carcinoma (NSCLC) clinical specimens. To determine how BRG1 loss fuels tumor progression in NSCLC, molecular profiling was performed after restoration of BRG1 expression or treatment with a histone deacetylase inhibitor or a DNA methyltransferase (DNMT) inhibitor in a BRG1-deficient NSCLC cells. Importantly, validation studies from multiple cell lines revealed that BRG1 reexpression led to substantial changes in the expression of CDH1, CDH3, EHF, and RRAD that commonly undergo silencing by other epigenetic mechanisms during NSCLC development. Furthermore, treatment with DNMT inhibitors did not restore expression of these transcripts, indicating that this common mechanism of gene silencing did not account for their loss of expression. Collectively, BRG1 loss is an important mechanism for the epigenetic silencing of target genes during NSCLC development. Inactivation of the SWI/SNF complex provides a novel mechanism to induce gene silencing during NSCLC development.

Lung cancer with loss of BRG1/BRM, shows epithelial mesenchymal transition phenotype and distinct histologic and genetic features

BRG1 and BRM, two core catalytic subunits in SWI/SNF chromatin remodeling complexes, have been suggested as tumor suppressors, yet their roles in carcinogenesis are unclear. Here, we present evidence that loss of BRG1 and BRM is involved in the progression of lung adenocarcinomas. Analysis of 15 lung cancer cell lines indicated that BRG1 mutations correlated with loss of BRG1 expression and that loss of BRG1 and BRM expression was frequent in E-cadherin-low and vimentin-high cell lines. Immunohistochemical analysis of 93 primary lung adenocarcinomas showed loss of BRG1 and BRM in 11 (12%) and 16 (17%) cases, respectively. Loss of expression of BRG1 and BRM was frequent in solid predominant adenocarcinomas and tumors with low thyroid transcription factor-1 (TTF-1, master regulator of lung) and low cytokeratin7 and E-cadherin (two markers for bronchial epithelial differentiation). Loss of BRG1 was correlated with the absence of lepidic growth patterns and was mutually exclusive of epidermal growth factor receptor (EGFR) mutations. In contrast, loss of BRM was found concomitant with lepidic growth patterns and EGFR mutations. Finally, we analyzed the publicly available dataset of 442 cases and found that loss of BRG1 and BRM was frequent in E-cadherin-low, TTF-1-low, and vimentin-high cases and correlated with poor prognosis. We conclude that loss of either or both BRG1 and BRM is involved in the progression of lung adenocarcinoma into solid predominant tumors with features of epithelial mesenchymal transition and loss of the bronchial epithelial phenotype. BRG1 loss was specifically involved in the progression of EGFR wild-type, but not EGFR-mutant tumors.

BRG1 mutations found in human cancer cell lines inactivate Rb‐mediated cell‐cycle arrest

Eukaryotic organisms package DNA into chromatin for compact storage in the cell nucleus. However, this process promotes transcriptional repression of genes. To overcome the transcriptional repression, chromatin remodeling complexes have evolved that alter the configuration of chromatin packaging of DNA into nucleosomes by histones. The SWI/SNF chromatin remodeling complex uses energy from ATP hydrolysis to reposition nucleosomes and make DNA accessible to transcription factors. Recent studies showing mutations of BRG1, one of two mutually exclusive ATPase subunits, in human tumor cell lines and primary tissue samples have implicated a role for its loss in cancer development. While most of the mutations lead to complete loss of BRG1 protein expression, others result in single amino acid substitutions. To better understand the role of these BRG1 point mutations in cancer development, we characterized SWI/SNF function in human tumor cell lines with these mutations in the absence of BRM expression, the other ATPase component. We found that the mutant BRG1 proteins still interacted with the core complex members and appeared at the promoters of target genes. However, while these mutations did not affect CD44 and CDH1 expression, known targets of the SWI/SNF complex, they did abrogate Rb-mediated cell-cycle arrest. Therefore, our results implicate that these mutations disrupt the de novo chromatin remodeling activity of the complex without affecting the status of existing nucleosome positioning.

Abstract 4022: Inactivation of the tumor suppressor BRG1 in lung cancer: Influence on nuclear receptors and cell differentiation

Abstract BRG1, encoding an ATPase of the SWI/SNF chromatin remodelling complex, is genetically inactivated in a variety of human solid tumors, especially non-small cell lung cancer, thus, constituting a bona fide tumor suppressor gene. However, the mechanisms that mediate the tumor suppressor function of BRG1 are not completely elucidated. We previously reported that lung tumors with amplification at any of the MYC family of genes lacked BRG1 mutations, suggesting a functional relationship between both proteins. Using isogenic lung cancer cell lines, that stably and conditionally express wild type or mutant BRG1, we found that wtBRG1 induced the up-regulation of MYC-target genes. This was accompanied by a reduction in MYC levels. Interestingly, we observed an overlap in the gene expression pattern after restoring wtBRG1 in lung cancer cells and gene expression in normal lung, indicating that BRG1 is promoting cell differentiation. In addition, the effects of BRG1 activity in lung cancer cells, i.e. changes in morphology and in gene expression depended on the presence of specific vitamins/hormones. Exposition to these compounds conferred lung cancer cells specific morphological features that were compatible with cell differentiation, only after restoring wtBRG1. Finally, using lung orthotopic mouse models we demonstrated that BRG1 was able to cause significant tumor shrinkage and delay tumor progression. Taken together our data support a tight functional relationship between BRG1 and MYC by which inactivation of BRG1 in cancer cells leads to de-regulation of MYC activity and allow refractoriness to nuclear receptor-mediated external signals impairing the control important cellular processes, such as cell differentiation, proliferation and response to inflammation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4022. doi:10.1158/1538-7445.AM2011-4022

Abstract 1134: Analysis of the tumor supressor role of BRG1 in lung cancer

Abstract The BRG1 protein features a bromodomain and helicase/ATPase activity and is a central component of the SWI/SNF chromatin remodeling complex. At present, the tumor suppressor role of the BRG1 gene, also called SMARCA4, is strongly supported by several genetic and functional observations. First of all, somatic and homozygous genetic inactivation of BRG1 has been observed in a variety of cancer cell lines and studies in lung cancer cells have estimated the frequency of BRG1 inactivation in about thirty per cent of this tumor type. Here we have cloned two BRG1 mutations, lacking the ATPase domain, found in lung cancer cell lines. One of these is a missense change in a highly conserved aminoacid within the ATPase domain (W764R) identified in the NCI-H2126 cell line while the other is an in-frame deletion (E668-Q758 del) identified in the NCI-H1703 cell line. We have studied the capability of these mutations to affect the biological function of BRG1 and compared the efficiency of the wild type BRG1 and that of the two mutants in reducing cell growth in cancer cells. To determine the effect of these mutations on the activity of BRG1 we took advantage of a transcription reporter assay in the human lung carcinoma cell line (NCI-H1299) that lacks wild type BRG1 due to a homozygous mutation. In this system, the simultaneous transfection of the BRG1 constructs and a construct carrying the MMTV-Luciferase reporter plasmid and a GR (glucocorticoid) expression plasmid allows the determination of the contribution of BRG1 to the hormone-dependent regulation of MMTV transcription. Transfection of H1299 cells with these constructs resulted in an eightfold increase of MMTV transcription in the presence of hormone when the cells were transfected with the wild type BRG1 as compared as to the cells transfected with either the mutants or the empty vectors. In addition, we observed that overexpression of wild type BRG1, but not the mutant proteins, in several lung cancer cell lines lead to cell growth suppression. Thus, our present data support an important role of BRG1 tumor suppressor gene in cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1134.

Frequent BRG1/SMARCA4-inactivating mutations in human lung cancer cell lines

Components of the SWI/SNF chromatin-remodeling complex, such as INI1, are inactivated in human cancer and, thus, act as tumor suppressors. Here we screened for mutations the entire coding sequence of BRG1 (SMARCA4), which encodes the ATPase of the complex, in 59 lung cancer cell lines of the most common histopathological types. Mutations were detected in 24% of the cancer cell lines, many of them in cells commonly used for lung cancer research. All mutations were homozygous and most predicted truncated proteins. The alterations were significantly more frequent in the non-small-cell lung cancer (NSCLC) type (13/37, 35%) as compared to the small-cell lung cancer (SCLC) type (1/19, 5%) (P<0.05; Fisher's Exact test) and BRG1 was the fourth most frequently altered gene in NSCLC cell lines. BRG1 mutations coexisted with mutations/deletions at KRAS, LKB1, NRAS, P16, and P53. However, alterations at BRG1 always occurred in the absence of MYC amplification, suggesting a common role in lung cancer development. In conclusion, our data strongly support that BRG1 is a bona fide tumor suppressor and a major factor in lung tumorigenesis.

Mutation analysis of the BRG1 gene in prostate cancer clinical samples

Previous studies in hereditary and sporadic prostate cancer have indicated the existence of a tumor suppressor gene in chromosomal region 19p13. The BRG1 gene in this region is one of the possible candidates, based on both the frequency of inactivating mutations in human cancer cell lines, including the prostate cancer cell line DU145, and its functional properties. To our knowledge, no studies have been done to evaluate possible involvement of the BRG1 gene in clinical prostate cancer. To accomplish this, we carried out a complete mutation analysis of all 35 BRG1 exons in tumor and constitutional DNA samples from 21 prostate cancer patients. We report the absence of somatic mutations in the panel of samples employed, but the existence of five germline single nucleotide polymorphisms (SNPs) in CpG islands of the BRG1 gene, among them, three novel ones. In conclusion, the study excludes the presence of common BRG1 mutations in prostate cancer.

Compensation of BRG-1 Function by Brm: INSIGHT INTO THE ROLE OF THE CORE SWI·SNF SUBUNITS IN RETINOBLASTOMA TUMOR SUPPRESSOR SIGNALING

The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity.

Sniffing Out a Job for BRCA1

BIOMEDICINE The discovery that mutations in the BRCA1 gene predispose individuals to an increased risk of developing breast or ovarian cancer has prompted the search for what the normal BRCA1 protein does in the cell. BRCA1 has structural motifs characteristic of transcription factors, and it increases the expression of certain genes including the master tumor suppressor gene, p53 . The finding by Bochar et al. that BRCA1 is a crucial component of SWI/SNF, an 18-protein complex that remodels chromatin, now suggests how BRCA1 might regulate gene transcription. Within the SWI/SNF complex, BRCA1 binds to a DNA-dependent ATPase called BRG1, the catalytic component that is essential for chromatin remodeling. If BRG1 is defective, BRCA1 is no longer able to switch on transcription of p53-responsive genes. Intriguingly, BRG1 binds to BRCA1 within the region encoded by exon 11, the section of the BRCA1 gene that harbors many of the predisposing cancer mutations identified so far. Now the search is on for mutations in BRG1 and other SWI/SNF proteins that also might predispose to cancer. — OMS Cell 102 , 257 (2000).