Expression Of Brm Research Articles

Hypermethylation of BRM promoter plays oncogenic roles in development of clear cell renal cell carcinoma.

Although the inactivation of BRM plays oncogenic roles in tumorigenesis, regulation mechanism is rarely studied in clear cell renal cell carcinoma (RCC). Thus, we aimed to investigate the mechanism of BRM inactivation and explore the tumor suppressing roles of BRM in the development of clear cell RCC. We verified that hypermethylation of the BRM promoter was correlated with decreased expression of BRM by multi-omics analysis based on the TCGA database. This result was further confirmed in our own tumor tissues. Moreover, BRM inhibited the ability of proliferation and invasion of RCC cells in vitro. Consistent with this, BRM overexpressing virtually inhibited the xenograft tumor growth of ACHN cells in vivo. Finally we found that BRM promoted cell apoptosis and cellular cycle arrest in G2/M. In conclusion, our study confirms that the hypermethylation of BRM promoters plays oncogenic roles by the transcription inhibition of BRM in RCC, and the tumor suppressor gene BRM inhibits RCC cell vitality in vitro and in vivo.

Abstract 4420: Heparan sulfate proteoglycans mediate SW13 oncogenic potential and response to granulin

Abstract The SW13 adrenal adenocarcinoma cell line exists as two epigenetically and phenotypically distinct subtypes. One is a highly proliferative epithelial subtype lacking the expression of key tumor suppressor proteins, Brm and Brg1, the intermediate filament protein vimentin, and the cell cycle check point protein CD44 (SW13-), while the other is a slow growing, mesenchymal-like subtype which expresses all of these proteins (SW13+). Evidence strongly suggests that subtype determination is epigenetic in nature, as HDAC inhibitors can induce an SW13- to SW13+ subtype switch, but the cellular and environmental factors that regulate cellular phenotype have yet to be characterized. We performed a comprehensive microarray analysis and identified heparan sulfate proteoglycan (HSPG) binding as a primary pathway affected between these two subtypes. HSPGs are glycoproteins present in the extracellular matrix that can have tumor-promoting or tumor-suppressing activities depending on the number and sulfation status of attached sugar chains known as glycosaminoglycans (GAGs). Quantitative RT-PCR was then used to confirm the increased expression of the HSPGs, serglycin and glypican-3, as well as the HSPG-binding growth factors granulin and fibroblast growth factors-1 and -2 in SW13+ cells. Furthermore, HDAC inhibition was also shown to increase the expression of these genes in a time-dependent manner. As granulin over-production is associated with cancer progression in a number of human cancers, we were interested in examining the role of this HSPG binding growth factor further. A pro-granulin ELISA showed that SW13+ cells secrete significantly more granulin than SW13- cells. Intriguingly however, treatment with exogenous granulin increases growth rates in the already highly proliferative SW13- subtype, but had no effect on SW13+ growth. We hypothesized that the pleiotropic response of SW13 cells to growth factor treatment may be the result of HSPG post-translational modifications, so we assessed total GAG sulfation was by dimethylmethylene blue assay, and indeed the SW13+ cells were found to have significantly higher levels of total GAG sulfation than SW13- cells. Thus, transcriptional and post-translational modifications of HSPGs may contribute to SW13 phenotype control, and ultimately may affect tumorigenesis. Citation Format: McKale Davis, Elizabeth Hull. Heparan sulfate proteoglycans mediate SW13 oncogenic potential and response to granulin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4420.

Frequent loss of the expression of multiple subunits of the SWI/SNF complex in large cell carcinoma and pleomorphic carcinoma of the lung.

The switch/sucrose non-fermenting (SWI/SNF) complex has recently emerged as a novel tumor suppressor in various human cancers. In the present study, we analyzed the expression of multiple SWI/SNF subunits in primary non-small cell lung cancer (NSCLC). A total of 133 NSCLC, consisting of 25 squamous cell carcinomas (SCC), 70 adenocarcinomas (AD), 16 large cell carcinomas (LC), and 22 pleomorphic carcinomas (PL), were immunohistochemically examined for the expression of BRG1, BRM, BAF47, ARID1A, and ARID1B. The frequency at which reductions in the expression of BRG1 were observed was significantly higher in the LC-PL group (13/38, 34.2%) than in the SCC-AD group (7/95, 7.4%). Similarly, the frequency at which reductions in the expression of BRM were observed was significantly higher in the LC-PL group (17/38, 44.7%) than in the SCC-AD group (14/95, 14.7%). The loss of the expression of ARID1A, ARID1B, and BAF47 was observed only in a fraction of NSCLC cases. Furthermore, the frequency at which the concurrent loss of multiple subunits of the SWI/SNF complex was observed was significantly higher in the LC-PL group (10/38, 26.3%) than in the SCC-AD group (8/95, 8.4%). Collectively, these results indicate that the loss of the SWI/SNF complex was related to dedifferentiation in NSCLC.

Knockdown of Brm and Baf170, Components of Chromatin Remodeling Complex, Facilitates Reprogramming of Somatic Cells.

The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells, esBAF contains Brg1 and Baf155, while their homologs, Brm and Baf170, are present in BAF of somatic cells. In this study, we sought to determine whether Brm and Baf170 play any roles in induced pluripotent stem cell (iPSC) reprogramming by using shRNA-mediated knockdown studies in the mouse model. We found that knocking down Brm during early, mid, and late stages (days 3, 6, and 9 after initial iPSC induction) and knocking down Baf170 during late-stage (day 9) reprogramming improve the numbers of iPSC colonies formed. We further showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed somatic cells (pre-iPSCs). Finally, we found that the expression of Brm and Baf170 during reprogramming was regulated by Jak/Stat3 activity. Taken together, these data suggest that inhibiting somatic BAF improves complete reprogramming by facilitating the activation of the pluripotency circuitry.

345 LOSS OF Brm AND Baf170, COMPONENTS OF THE ATP-DEPENDENT CHROMATIN REMODELING COMPLEX, FACILITATES REPROGRAMMING OF SOMATIC CELL

The SWI/SNF [SWItch/Sucrose NonFermentable; also known as BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodelling complexes are epigenetic modifiers that change the structure of chromatin, and thereby modulate gene transcription. The BAF chromatin remodeling complexes undergo progressive changes in subunit composition during cellular differentiation. In embryonic stem cells (ESC), BAF complex, esBAF, contains Brg1 and Baf155, which are crucial for ESC self-renewal and also facilitate induced pluripotent stem cell (iPSC) reprogramming from somatic cells. Here we sought to determine the roles of somatic BAF components (Brm and Baf170, homologues of Brg1 and Baf155, respectively) in mouse iPSC reprogramming through shRNA-mediated knockdown studies. We found that the expression of Brm and Baf170 were inhibited in reprogramming by Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) activity, which is essential for pluripotency establishment. We further found that knockdown of Brm and Baf170 in mouse somatic cells promotes reprogramming efficiency. Specifically, loss of Brm and Baf170 during early (Days 3 and 6 after initial iPSC induction) and later-stage (Day 9) reprogramming, respectively, improves the numbers of iPSC colonies formed. These also led to significant upregulation of pluripotent genes, including Sox2, Nanog, Fgf5, and Tbx3. Although the somatic Brm and Baf170 are believed to be absent in ESC, the expression levels of Nanog and Tbx3 were increased significantly by knockdown of either Brm or Baf170 in ESC. Finally, we showed that inhibition of these somatic BAF components also promotes complete reprogramming of partially reprogrammed cells (pre-iPSC). These data suggest that inhibiting somatic BAF complex improved complete reprogramming by facilitating the activation of the pluripotency circuitry. A reduced activity of the somatic BAF complex constitutes part of the Stat3 regulated epigenetic changes during pluripotency establishment.

BRM promoter insertion/deletion polymorphisms in hepatocellular carcinoma risk and survival.

225 Background: MEF2D and HDAC9 regulate the expression of BRM, the catalytic subunit of SWI/SNF (chromatin remodeling complex). MEF2D binding sites are created in the BRM promoter by two germline insertion/deletion polymorphisms (BRM-741, BRM-1321), which mediate epigenetic silencing. We recently identified BRM as a potential drug target and a susceptibility gene for lung and head and neck cancers, while others reported its association with HCC risk in Asians. BRM also correlates with prognosis in lung and esophageal cancers (ASCO 2013; abstracts 11057, 4077). In this analysis, we assessed the effects of the BRM promoter variants on risk and overall survival (OS) in HCC from a North American centre (Toronto). Methods: We conducted a case-control study with 266 histologically confirmed HCC cases and 536 age/sex distribution-matched healthy controls. Survival of the cases was obtained from medical records. Association between the BRM polymorphisms and HCC risk was analyzed by multivariate logistic regression adjusted for age, sex, race, smoking and BMI; their impact on OS was evaluated by Cox proportional hazard regression adjusted for age, sex, treatment intent and Hepatitis B status. Results: Median age was 62 years; 83% were male. Hepatitis B and C infections affected 34% and 31% of cases, respectively. 83% of patients were Child Pugh A at diagnosis, and 66% received initial curative therapy (surgical resection rate 32%). There were 13% deaths at a median follow-up of 24.1 months. There was no significant association between BRM polymorphisms and HCC risk: the adjusted risk odds ratios of the homozygous BRM variants, relative to wild-type, were 0.87 (95% CI: 0.53-1.45; BRM-741) and 1.01 (95% CI: 0.78-1.29; BRM-1321), and 0.94 (95% CI: 0.48-1.86) for the double homozygotes vs. double wild-type. The adjusted hazard ratios for OS were 5.77 (95% CI: 2.9-11.5; BRM-741) and 4.09 (95% CI: 2.2-7.5; BRM-1321) for each variant allele; the double homozygotes were also highly associated with OS compared to double wild-type (p<0.0001). Conclusions: Two functional BRM promoter polymorphisms did not increase the risk of HCC. However, they were strongly associated with OS despite short median follow-up. SPC and RH are co-senior authors.

Proinflammatory Stimuli Engage Brahma Related Gene 1 and Brahma in Endothelial Injury

Endothelial dysfunction inflicted by inflammation is found in a host of cardiovascular pathologies. One hallmark event in this process is the aggregation and adhesion of leukocyte to the vessel wall mediated by the upregulation of adhesion molecules (CAM) in endothelial cells at the transcriptional level. The epigenetic modulator(s) of CAM transactivation and its underlying pathophysiological relevance remain poorly defined. Our goal was to determine the involvement of Brahma related gene 1 (Brg1) and Brahma (Brm) in CAM transactivation and its relevance in the pathogenesis of atherosclerosis. In the present study, we report that proinflammatory stimuli augmented the expression of Brg1 and Brm in vitro in cultured endothelial cells and in vivo in arteries isolated from rodents. Overexpression of Brg1 and Brm promoted while knockdown of Brg1 and Brm abrogated transactivation of adhesion molecules and leukocyte adhesion induced by inflammatory signals. Brg1 and Brm interacted with and were recruited to the CAM promoters by nuclear factor κB/p65. Conversely, depletion of Brg1 and Brm disrupted the kinetics of p65 binding on CAM promoters and crippled CAM activation. Silencing of Brg1 and Brm also altered key epigenetic changes associated with CAM transactivation. Of intrigue, 17β-estradiol antagonized both the expression and activity of Brg1/Brm. Most importantly, endothelial-targeted elimination of Brg1/Brm conferred atheroprotective effects to Apoe(-/-) mice on a Western diet. Our data suggest that Brg1 and Brm integrate various proinflammatory cues into CAM transactivation and endothelial malfunction and, as such, may serve as potential therapeutic targets in treating inflammation-related cardiovascular diseases.

S-nitrosylation of HDAC2 regulates the expression of the chromatin-remodeling factor Brm during radial neuron migration.

Dynamic epigenetic modifications play a key role in mediating the expression of genes required for neuronal development. We previously identified nitric oxide (NO) as a signaling molecule that mediates S-nitrosylation of histone deacetylase 2 (HDAC2) and epigenetic changes in neurons. Here, we show that HDAC2 nitrosylation regulates neuronal radial migration during cortical development. Bead-array analysis performed in the developing cortex revealed that brahma (Brm), a subunit of the ATP-dependent chromatin-remodeling complex BRG/brahma-associated factor, is one of the genes regulated by S-nitrosylation of HDAC2. In the cortex, expression of a mutant form of HDAC2 that cannot be nitrosylated dramatically inhibits Brm expression. Our study identifies NO and HDAC2 nitrosylation as part of a signaling pathway that regulates cortical development and the expression of Brm in neurons.

MITF-Independent Pro-Survival Role of BRG1-Containing SWI/SNF Complex in Melanoma Cells

Metastasized malignant melanoma has a poor prognosis because of its intrinsic resistance to chemotherapy and radiotherapy. The central role in the melanoma transcriptional network has the transcription factor MITF (microphthalmia-associated transcription factor). It has been shown recently that the expression of MITF and some of its target genes require the SWI/SNF chromatin remodeling complex. Here we demonstrate that survival of melanoma cells requires functional SWI/SNF complex not only by supporting expression of MITF and its targets and but also by activating expression of prosurvival proteins not directly regulated by MITF. Microarray analysis revealed that besides the MITF-driven genes, expression of proteins like osteopontin, IGF1, TGFß2 and survivin, the factors known to be generally associated with progression of tumors and the antiapoptotic properties, were reduced in acute BRG1-depleted 501mel cells. Western blots and RT-PCR confirmed the microarray findings. These proteins have been verified to be expressed independently of MITF, because MITF depletion did not impair their expression. Because these genes are not regulated by MITF, the data suggests that loss of BRG1-based SWI/SNF complexes negatively affects survival pathways beyond the MITF cascade. Immunohistochemistry showed high expression of both BRM and BRG1 in primary melanomas. Exogenous CDK2, osteopontin, or IGF1 each alone partly relieved the block of proliferation imposed by BRG1 depletion, implicating that more factors, besides the MITF target genes, are involved in melanoma cell survival. Together these results demonstrate an essential role of SWI/SNF for the expression of MITF-dependent and MITF-independent prosurvival factors in melanoma cells and suggest that SWI/SNF may be a potential and effective target in melanoma therapy.

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.

Abstract 4609: Epigenetic regulation of BRM

Abstract BRM is part of the SWI/SNF complex and is key anticancer protein which is silenced in variety of tumor types. SWI/SNF is a prerequisite for the function of many different anticancer proteins such as Rb and p53 and it has essential roles in growth control, differentiation, development, cell adhesion, and DNA repair. Consequently, inactivation or impairment of this complex can stimulate tumor development and tumor progression through the abrogation of a number of anticancer mechanisms. Unlike many tumor suppressor proteins, BRM is not mutated, but rather epigenetically suppressed, which is validated by the fact that HDAC inhibitors were the first compounds found to restore BRM expression. Unfortunately, these compounds are too broadly active and in addition to restoring BRM expression, they inactivate it by acetylation. To try to better understand how BRM is epigenetically regulated, we knocked-out each of the known 11 HDACs, using shRNAs in BRM-deficient and BRM-positive cell lines to determine which ones regulate or inactivate BRM. We found that that shRNA knockdown of only HDAC3 or HDAC9 restores both BRM expression and function, while suppression of HDAC2 induces BRM acetylation. Similarly, we tested the impact of over-expression of 25 different Histone Acetyltransferases (HATs) on BRM expression and acetylation. We found ectopic expression of KAT6A, KAT6B, and KAT7 in BRM-deficient cell lines induces BRM whereas ectopic expression of KAT2B and KAT8 causes BRM acetylation in BRM-positive cell lines. We also uncovered that HDAC9 is greatly over-expressed only in BRM-deficient cell lines and its expression is driven by MEF2D and GATA3. Expression and knockdown experiments directed at MEF2D and GATA3 showed that these transcription factors regulate BRM and HDAC9. Similarly, we found that GATA3 expression is only expressed in BRM-deficient cell lines. This work outlines the complex array of proteins which are tied to the expression and regulation of BRM. It is important to note this epigenetic mechanism is not likely specific to BRM, but rather likely regulates a cadre of anticancer and cellular proteins as well. Hence, determining how BRM is aberrantly regulated is the first step in determining how to target BRM and this mechanism for therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4609. doi:1538-7445.AM2012-4609

Abstract 2179: Histopathologic and genetic features of lung adenocarcinomas with loss of chromatin remodeling factors, BRG-1 and BRM

Abstract We have recently demonstrated that lung adenocarcinoma could be classified into two groups; Group1 is characterized by (1) high expressions of bronchial epithelial markers (TTF-1, MUC1, CK7, and E-cadherin), (2) high phosphorylation of EGFR and MET, (3) frequent mutation or amplification of EGFR, MET and HER2, (4) high expressions of the genes associated with invasion (HER3, MET, Cox-2, laminin β2), and (5) sensitivity to gefitinib and resistance to cisplatin; Group2 is characterized by negative expression of bronchial epithelial markers, no or a little phosphorylation of EGFR and MET, no mutation or amplification of EGFR, MET and HER2, low expressions of many of the genes associated with cancer invasion, and resistance to gefitinib and sensitivity to cisplatin (Matsubara et al, AJP 2010). The activations of receptor tyrosine kinase EGFR and MET would participate in the regulatory network of cancer-associated genes in Group 1. In contrast, rare genetic alterations of EGFR and MET in Group 2 suggest that this group of tumors might have different processes of carcinogenesis. BRG-1 and BRM are two core catalytic ATPase subunits in human SWI/SNF chromatin remodeling complexes, and they have been suggested as tumor suppressors. Now we aimed to reveal the histopathologic/genetic features of lung adenocarcinomas with loss of BRG1 and BRM. METHODS: We analyzed (1) the publicly available data set of 442 lung adenocarcinomas (Shedden et al, Nat Med 2008), and (2) 93 primary lung adenocarcinomas resected in our institution. For the latter set, we analzed the expression of BRG-1 and BRM by immunohistochemistry. RESULTS: Our analysis of the Shedden et al. data revealed that high expressions of BRG1 and BRM were frequently seen in cases with high expressions of bronchial epithelial markers, and that cases with loss of BRG1 or BRM showed significantly poorer prognosis. Among the 93 cases of lung adenocarcinoma in our institution, 11 showed BRG1 loss, and 13 showed BRM loss. BRG1 loss was significantly more frequent in the Group2 (70%) than the Group1 (7%) (p<0.0001), but no significant difference was seen in the frequency of BRM loss between the two groups; 13% in the Group1and 28% in the Group2. Most cases with BRG1 loss were heavy smokers and showed components of solid adenocarcinoma, but lacked lepidic growth pattern. In contrast, cases with BRM loss frequently showed both components of solid adenocarcinoma and adenocarcinoma with lepidic growth pattern. BRG1 loss and EGFR mutations were mutually exclusive, but EGFR mutations were found in about half of the cases with BRM loss. Conclusions: BRG1 loss occurs exclusively in EGFR wild-type tumors and tumors without lepidic growth pattern. BRG1 loss might be associated with carcinogenesis of the Group 2 tumors. In contrast, BRM loss might be involved in the progression of Group1 tumors from adenocarcinoma with lepidic growth into solid adenocarcinomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2179. doi:1538-7445.AM2012-2179

Two novel BRM insertion promoter sequence variants are associated with loss of BRM expression and lung cancer risk

SWI/SNF (SWItch/sucrose non-fermentable) complexes are ATP-dependent chromatin remodeling enzymes critically involved in the regulation of multiple functions, including gene expression, differentiation, development, DNA repair, cell adhesion and cell cycle control. BRM, a key SWI/SNF complex subunit, is silenced in 15-20% of many solid tumors. As BRM-deficient mice develop 10-fold more tumors when exposed to carcinogens, BRM is a strong candidate for a cancer susceptibility gene. In this paper, we show that BRM is regulated by transcription, thus demonstrating that the promoter region is important for BRM expression. We sequenced the BRM promoter region, finding two novel promoter indel polymorphisms, BRM -741 and BRM -1321, that are in linkage disequilibrium (D'≥0.83). The variant insertion alleles of both polymorphisms produce sequence variants that are highly homologous to myocyte enhancer factor-2 (MEF2) transcription factor-binding sites; MEF2 is known to recruit histone deacetylases that silence BRM expression. Each polymorphic BRM insertion variant is found in ~20% of Caucasians, and each correlates strongly with the loss of protein expression of BRM, both in cancer cell lines (P=0.009) and in primary human lung tumor specimens (P=0.015). With such strong functional evidence, we conducted a case-control study of 1199 smokers. We found an increased risk of lung cancer when both BRM homozygous promoter insertion variants were present: adjusted odds ratio of 2.19 (95% confidence interval, 1.40-3.43). Thus, we here demonstrate a strong functional association between these polymorphisms and loss of BRM expression. These polymorphisms thus have the potential to identify a sub-population of smokers at greater lung cancer risk, wherein this risk could be driven by an aberrant SWI/SNF chromatin-remodeling pathway.

P44 Expression of calcium/calcineurin pathway-regulated transcription factor NFATc1 and chromatin-remodelling genes BRG1 and BRM in invasive breast cancer

Background: NFATc1 is a transcription factor activated by the calcium/calcineurin pathway, which regulates several physiological processes. Evidence has also shown a role for NFAT transcriptions factors in oncogenesis. BRG1 and BRM are chromatinremodelling genes that are also regulated by calcium homoeostasis. Diminished expression of BRG1 is associated with poor prognosis in breast cancer, non-small-cell lung cancer, colorectal cancer, and prostate cancer. To understand the biological relationship between these two pathways, we examined the expression pattern of NFATc1, BRG1, and BRM in invasive breast cancer. Methods: Paraffin blocks of 150 cases of invasive breast cancer were retrieved from Kaiser Permanente Medical Centre database. Monoclonal antibodies against NFATc1, BRG1, and BRM were used, and expression pattern was determined as no expression, weak (1+ staining intensity), moderate (2+), and high expression (3+). Pearson Chi-Squared test was used for statistical analysis. Findings: NFATc1 was expressed in approximately 22% of the 150 cases of invasive breast cancer, whereas BRG1 was expressed in 56.7% and BRM in 52%. Both nuclear and cytoplasmic expression of NFATc1 was detected, with nuclear expression as the predominant feature. BRM1/BRM are predominantly expressed in the nucleus. Most cases expressing NFATc1 showed 2+ moderate intensity expression patterns. Statistical analysis showed that expression of NFATc1 was highly correlated with the expression of both BRG1 and BRM. Interpretation: Our preliminary data shows that NFATc1 is expressed in a subset of BRG1/BRM-positive invasive breast cancers. This correlation of two calcium homoeostasis-regulated pathways provides insight into the oncogenesis of breast cancer. Funding: Kaiser Hospital Foundation Community Benefit Grant. The author declared no conflicts of interest.

Expression of BRG1 and BRM proteins in prostatic cancer

To study the effect of BRG1 and BRM, the catalytic subunits expressed by SWI/SNF, in benign and malignant prostatic tissues and to correlate the BRG1/BRM expression with the development and progression of prostatic cancer. The expression levels of the BRG1 and BRM proteins in benign and malignant prostatic tissues were studied using semi-quantitative immunohisto-chemistry. The results correlated with various clinical and pathologic parameters. The average immuno-reactive score for BRG1 expression in prostatic cancer tissues was significantly higher than that in benign prostatic tissues (57+/-9.8 and 19+/-4.1, respectively, P = 0.000 17). The difference was more obvious in the high-grade cancer. On the other hand, BRM expression exhibited a heterogeneous pattern. The average immuno-reactive score for BRM expression was lower in cancer tissues than in benign tissues (112+/-17 and 151+/-19, respectively, P = 0.0047). BRG1 and BRM demonstrated a reciprocal expression pattern in benign and malignant tissues. The average immuno-reactive score for BRG1 expression was higher in the cancer cases with a larger tumor volume than in the cases with a smaller tumor volume (P = 0.0112). The expression of BRG1 and BRM correlates with the development of prostatic cancer. Increased BRG1 expression may have certain implications in tumor progression.

Differentiation-specific expression of chromatin remodeling factor BRM

The chromatin remodeling complex SWI/SNF exclusively contains BRG1 or BRM as an ATPase subunit, but the physiological relevance of these ATPases is not yet clear. Here, we studied the developmental regulation of the brm gene. A differentiation-specific up-regulation of BRM expression was observed with P19, F9 and C2C12 cells as with neural cells and hepatocytes. The promoter region of brm contains two putative binding sites for CCAAT enhancer binding protein β (C/EBPβ). Each site partially overlapped with binding sites for GATAs. C/EBPβ stimulated the transcriptional activity of the brm promoter but GATA2 and 3 down-regulated the expression. C/EBPβ bound to the proximal sequences of the promoter in differentiated cells and was replaced by GATAs which bound to the overlapping sites in the undifferentiated state. C/EBPβ and GATAs may developmentally regulate the expression of brm by mutually exclusive binding.

Aberrant expression of SWI/SNF catalytic subunits BRG1/BRM is associated with tumor development and increased invasiveness in prostate cancers

Brahma gene (BRM) and Brahma-related gene 1 (BRG1) are major components with ATPase enzymatic activities in the nucleosome remodeling SWI/SNF complex, and their expression pattern in human prostate cancers is unknown. We analyzed a published cDNA microarray data set of prostate cancers for the expression of SWI/SNF genes, and then we evaluated the expression levels of BRG1 and BRM proteins with a semi-quantitative immunohistochemistry (IHC) approach in a pairwise manner of malignant versus benign tissues from individual prostate cancers. The correlation of BRG1/BRM expression with clinical parameters was analyzed. Microarray data showed an aberrant expression of BRG1 and BRM but not SNF5/INI1 genes in different stages of the disease course. In immunochemistry studies, BRG1 expression was significantly higher in malignant tissues compared to their benign compartments, and this difference was more profound in high-grade cancers. Although BRM expression showed a heterogeneous pattern, the average level of BRM expression was lower in malignant tissues than that in benign tissues. More interestingly, BRG1 and BRM expression showed a reciprocal pattern in both benign and malignant tissues of individual cases. In malignant tissues, higher BRG1 but not BRM expression levels were associated with larger volume of tumor mass. Increased expression of BRG1 but not BRM protein was observed in invasive cancer cells. Consistently, overexpression of exogenous wild-type BRG1 and BRM but not mutant BRG1 enhanced cancer cell invasion in an in vitro cell invasion assay. We provide the first evidence that aberrant expression of BRG1 and BRM genes is associated with disease development and progression in prostate cancers and increased BRG1 expression may promote tumor growth and invasion.

Mammalian Chromatin Remodeling Complex SWI/SNF Is Essential for Enhanced Expression of the Albumin Gene during Liver Development

The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.

The Brm gene suppressed at the post-transcriptional level in various human cell lines is inducible by transient HDAC inhibitor treatment, which exhibits antioncogenic potential

The mammalian SWI/SNF chromatin remodeling complex is composed of more than 10 protein subunits, and plays important roles in epigenetic regulation. Each complex includes a single BRG1 or Brm molecule as the catalytic subunit. We previously reported that loss of Brm, but not BRG1, causes transcriptional gene silencing of murine leukemia virus-based retrovirus vectors. To understand the biological function and biogenesis of Brm protein, we examined seven cell lines derived from various human tumors that do not produce Brm protein. We show here that these Brm-deficient cell lines transcribe the Brm genes efficiently as detected by nuclear run-on transcription assay, whereas Brm mRNA and Brm hnRNA were undetectable by reverse transcription-polymerase chain reaction analysis. These results indicate that expression of Brm is strongly and promptly suppressed at the post-transcriptional level, through processing and transport of the primary transcript or through stability of mature Brm mRNA. This suppression was attenuated by transient treatment of these cell lines with HDAC inhibitors probably through indirect mechanism. Importantly, all of the treated cells showed prolonged induction of Brm expression after the removal of HDAC inhibitors, and acquired the ability to maintain retroviral gene expression. These results indicate that these Brm-deficient human tumor cell lines carry a functional Brm gene. Treatment with HDAC inhibitors or introduction of exogenous Brm into Brm-deficient cell lines significantly reduced the oncogenic potential as assessed by colony-forming activity in soft agar or invasion into collagen gel, indicating that, like BRG1, Brm is involved in tumor suppression.

Role of an mSin3A-Swi/Snf chromatin remodeling complex in the feedback repression of bile acid biosynthesis by SHP.

The orphan receptor SHP interacts with many nuclear receptors and inhibits their transcriptional activities. SHP is central to feedback repression of cholesterol 7alpha hydroxylase gene (CYP7A1) expression by bile acids, which is critical for maintaining cholesterol homeostasis. Using CYP7A1 as a model system, we studied the molecular mechanisms of SHP repression at the level of native chromatin. Chromatin immunoprecipitation studies showed that mSin3A and a Swi/Snf complex containing Brm as a central ATPase were recruited to the promoter. This recruitment was associated with chromatin remodeling after bile acid treatment that was blunted by inhibition of the endogenous Swi/Snf function by dominant-negative ATPase mutants. Biochemical studies indicated that SHP was associated with the mSin3A-Swi/Snf complex by direct interaction with Brm and mSin3A through its repression domain. Expression of Brm, but not an ATPase mutant, inhibited CYP7A1 promoter activity and further enhanced SHP-mediated repression. Bile acid-induced recruitment of mSin3A/Brm, chromatin remodeling, and concomitant repression of endogenous CYP7A1 expression were impaired when SHP expression was inhibited by SHP small interfering RNA. Our results suggest that SHP mediates recruitment of mSin3A-Swi/Snf to the CYP7A1 promoter, resulting in chromatin remodeling and gene repression, which may also be a mechanism for the repression by SHP of genes activated by many nuclear receptors. Our study establishes the first link between a Swi/Snf complex and regulation of cholesterol metabolism.