Role Of Brahma-related Gene 1 Research Articles

BRG1 Deficiency Promotes Cardiomyocyte Inflammation and Apoptosis by Activating the cGAS-STING Signaling in Diabetic Cardiomyopathy.

Brahma-related gene 1 (BRG1) has been implicated in the repair of DNA double-strand breaks (DSBs). Downregulation of BRG1 impairs DSBs repair leading to accumulation of double-stranded DNA (dsDNA). Currently, the role of BRG1 in diabetic cardiomyopathy (DCM) has not been clarified. In this study, we aimed to explore the function and molecular by which BRG1 regulates DCM using mice and cell models. We found that BRG1 was downregulated in the cardiac tissues of DCM mice and in cardiomyocytes cultured with high glucose and palmitic acid (HG/PA), which was accompanied by accumulation of dsDNA and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. shRNA-mediated Brg1 knockdown aggravated DCM mice cardiac functions, enhanced dsDNA accumulation, cGAS-STING signaling activation, which induced inflammation and apoptosis. In addition, the results were further verified in HG/PA-treated primary neonatal rat cardiomyocytes (NRCMs). Overexpression of BRG1 in NRCMs yielded opposite results. Furthermore, a selective cGAS inhibitor RU.521 or STING inhibitor C-176 partially reversed the BRG1 knockdown-induced inflammation and apoptosis in vitro. In conclusion, our results demonstrate that BRG1 is downregulated during DCM in vivo and in vitro, resulting in cardiomyocyte inflammation and apoptosis due to dsDNA accumulation and cGAS-STING signaling activation. Therefore, targeting the BRG1-cGAS-STING pathway may represent a novel therapeutic strategy for improving cardiac function of patients with DCM.

Redox-sensitive activation of CCL7 by BRG1 in hepatocytes during liver injury

Liver injuries induced by various stimuli share in common an acute inflammatory response, in which circulating macrophages home to the liver parenchyma to participate in the regulation of repair, regeneration, and fibrosis. In the present study we investigated the role of hepatocyte-derived C–C motif ligand 7 (CCL7) in macrophage migration during liver injury focusing on its transcriptional regulation. We report that CCL7 expression was up-regulated in the liver by lipopolysaccharide (LPS) injection (acute liver injury) or methionine-and-choline-deficient (MCD) diet feeding (chronic liver injury) paralleling increased macrophage infiltration. CCL7 expression was also inducible in hepatocytes, but not in hepatic stellate cells or in Kupffer cells, by LPS treatment or exposure to palmitate in vitro. Hepatocyte-specific deletion of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, resulted in a concomitant loss of CCL7 induction and macrophage infiltration in the murine livers. Of interest, BRG1-induced CCL7 transcription and macrophage migration was completely blocked by the antioxidant N-acetylcystine. Further analyses revealed that BRG1 interacted with activator protein 1 (AP-1) to regulate CCL7 transcription in hepatocytes in a redox-sensitive manner mediated in part by casein kinase 2 (CK2)-catalyzed phosphorylation of BRG1. Importantly, a positive correlation between BRG1/CCL7 expression and macrophage infiltration was identified in human liver biopsy specimens. In conclusion, our data unveil a novel role for BRG1 as a redox-sensitive activator of CCL7 transcription.

BRG1 Mediates Nephronectin Activation in Hepatocytes to Promote T Lymphocyte Infiltration in ConA-Induced Hepatitis.

Fulminant hepatitis (FH) is a major cause of acute liver failure. Concanavalin A (ConA) belongs to the lectin family and is frequently used as an inducer of FH in animal models. ConA induced FH is characterized by massive accumulation of T lymphocytes in the liver. A host of chemoattractive substances are known to promote T cell homing to the liver during acute hepatitis. Here we investigated the involvement of Brahma-related gene 1 (BRG1), a chromatin remodeling protein, in FH. BRG1-flox mice were crossed to Alb-Cre mice to generate hepatocyte conditional BRG1 knockout (LKO) mice. The mice were peritoneally injected with a single dose of ConA to induce FH. BRG1 deficiency mitigated ConA-induced FH in mice. Consistently, there were fewer T lymphocyte infiltrates in the LKO livers compared to the wild type (WT) livers paralleling downregulation of T cell specific cytokines. Further analysis revealed that BRG1 deficiency repressed the expression of several chemokines critical for T cell homing including nephronectin (Npnt). BRG1 knockdown blocked the induction of Npnt in hepatocytes and attenuated T lymphocyte migration in vitro, which was reversed by the addition of recombinant nephronectin. Mechanistically, BRG1 interacted with β-catenin to directly bind to the Npnt promoter and activate Npnt transcription. Importantly, a positive correlation between infiltration of CD3+ T lymphocyes and nephronectin expression was detected in human acute hepatitis biopsy specimens. In conclusion, our data identify a novel role for BRG1 as a promoter of T lymphocyte trafficking by activating Npnt transcription in hepatocytes. Targeting the BRG1-Npnt axis may yield novel therapeutic solutions for FH.

BRG1 deficiency in endothelial cells alleviates thioacetamide induced liver fibrosis in mice

Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.

Cognitive functioning in patients treated with electroconvulsive therapy

Liver sinusoidal endothelial cells play a key role maintaining the hepatic homeostasis, the disruption of which is associated with such end-stage liver diseases as hepatocellular carcinoma and cirrhosis. In the present study we investigated the role of brahma-related gene 1 (BRG1), a chromatin remodeling protein, in regulating endothelial transcription and the implication in liver fibrosis. We report that endothelial-specific deletion of BRG1 in mice attenuated liver fibrosis induced by injection with thioacetamide (TAA). Coincidently, alleviation of liver fibrosis as a result of endothelial BRG1 deletion was accompanied by an up-regulation of eNOS activity and NO bioavailability. In cultured endothelial cells, exposure to lipopolysaccharide (LPS) suppressed eNOS activity whereas BRG1 depletion with small interfering RNA restored eNOS-dependent NO production. Further analysis revealed that BRG1 was recruited to the caveolin-1 (CAV1) promoter by Sp1 and activated transcription of CAV1, which in turn inhibited eNOS activity. Mechanistically, BRG1 interacted with the H3K4 trimethyltransferase MLL1 to modulate H3K4 trimethylation surrounding the CAV1 promoter thereby contributing to LPS-induced CAV1 activation. In conclusion, our data unveil a novel role for BRG1 in the regulation of endothelial function and liver fibrosis.

The BRG1/SOX9 axis is critical for acinar cell-derived pancreatic tumorigenesis.

Chromatin remodeler Brahma related gene 1 (BRG1) is silenced in approximately 10% of human pancreatic ductal adenocarcinomas (PDAs). We previously showed that BRG1 inhibits the formation of intraductal pancreatic mucinous neoplasm (IPMN) and that IPMN-derived PDA originated from ductal cells. However, the role of BRG1 in pancreatic intraepithelial neoplasia-derived (PanIN-derived) PDA that originated from acinar cells remains elusive. Here, we found that exclusive elimination of Brg1 in acinar cells of Ptf1a-CreER; KrasG12D; Brg1fl/fl mice impaired the formation of acinar-to-ductal metaplasia (ADM) and PanIN independently of p53 mutation, while PDA formation was inhibited in the presence of p53 mutation. BRG1 bound to regions of the Sox9 promoter to regulate its expression and was critical for recruitment of upstream regulators, including PDX1, to the Sox9 promoter and enhancer in acinar cells. SOX9 expression was downregulated in BRG1-depleted ADMs/PanINs. Notably, Sox9 overexpression canceled this PanIN-attenuated phenotype in KBC mice. Furthermore, Brg1 deletion in established PanIN by using a dual recombinase system resulted in regression of the lesions in mice. Finally, BRG1 expression correlated with SOX9 expression in human PDAs. In summary, BRG1 is critical for PanIN initiation and progression through positive regulation of SOX9. Thus, the BRG1/SOX9 axis is a potential target for PanIN-derived PDA.

Increased Brahma-related Gene 1 Expression Predicts Distant Metastasis and Shorter Survival in Patients with Invasive Ductal Carcinoma of the Breast

Previous studies have demonstrated aberrant Brahma-related gene 1 (BRG1) expression in various tumor types. Increased BRG1 expression has recently been shown to correlate with aggressive oncogenic behavior in many different types of human cancer. However, the role of BRG1 in breast cancer development and progression is not fully understood. We evaluated BRG1 expression in 224 patients with invasive ductal carcinoma (IDC) of the breast using tissue microarray samples and immunohistochemistry. We also investigated whether BRG1 expression status is associated with clinicopathological characteristics and outcomes of patients with IDC. Among the 224 patients with IDC, 37.5% (84/224) exhibited high BRG1 expression. IDC exhibited significantly higher BRG1 expression compared to ductal carcinoma in situ (p=0.009) and normal breast tissue (p=0.005). High BRG1 expression in IDC significantly correlated with higher histological grade (p=0.035) and presence of distant metastasis (p=0.002). Furthermore, high BRG1 expression was an independent factor for predicting distant metastasis (relative risk=4.079; p=0.007). In addition, high BRG1 expression predicted shorter overall (p=0.011) and recurrence-free (p=0.003) survival in patients with IDC. In particular, BRG1 had a significant prognostic value in predicting recurrence-free survival of patients with IDC with lymph node metastasis or stage III disease. BRG1 is involved in the progression and metastasis of breast cancer and can serve as a novel biomarker predictive of distant metastasis and patient outcomes.

BRG1 overexpression in smooth muscle cells promotes the development of thoracic aortic dissection.

BackgroundHere we investigated Brahma-related gene 1 (BRG1) expression in aortic smooth muscle cells (SMCs) and its role in the regulation of the pathological changes in aortic SMCs of thoracic arotic dissection (TAD).MethodsBRG1, matrix metalloproteinase 2 (MMP2), and MMP9 mRNA and protein expression in human aortic specimens were examined by qPCR and western blot, respectively. The percentage of apoptotic and contractile SMCs in aortic specimens were determined by TUNEL assay and α-SMA immunohistochemical staining, respectively. The role of BRG1 in MMP2 and MMP9 expression, cell apoptosis, and phenotype transition in aortic SMCs were investigated using a human aortic SMC line via adenovirus mediated gene transfer. MMPs mRNA and protein levels were analyzed by qPCR and western blot, respectively. The percentage of apoptotic and contractile cells were determined through flow cytometry analysis.ResultsThe expression level of BRG1 in the aortic walls (adventitia-removed) was significantly higher in the TAD than the normal group. BRG1 expression was positively correlated to expression of MMP2 and MMP9 and SMC apoptosis, but was negatively correlated to the percentage of contractile aortic SMCs in TAD specimens. In human aortic SMC line, BRG1 transfection led to significant upregulation of MMP2 and MMP9 expression and a concomitant increase in SMC apoptosis as well as a decrease in the percentage of contractile phenotype of cells.ConclusionsBRG1 is significantly upregulated in the aortic SMCs of TAD, and its overexpression might promote the development of TAD by increasing MMP2 and MMP9 expression, inducing SMC apoptosis and the transition from contractile to synthetic phenotype.

Alterations of the SWI/SNF chromatin remodelling subunit‐BRG1 and BRM in hepatocellular carcinoma

The SWI/SNF chromatin remodelling complex, which contains either brahma-related gene-1 (BRG1) or brahma (BRM) as the catalytic ATPase, functions as a master regulator of gene expression. To examine alterations of BRG1 and BRM in hepatocellular carcinoma (HCC). 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 tumours, and conducted further searches for mutations in BRG1 and BRM genes. 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 tumours respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumours, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumours, compared to non-tumour 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. 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.

135 Discovery and Validation of a Novel DNA Methylation Biomarker for Colorectal Cancer With Application to Blood Testing

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

134 Brahma-Related Gene 1 (BRG1) as a Novel Epigenetic Modulator of Gene Dysregulation in Early Colorectal Carcinogenesis: Implications for Chemoprevention

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

136 Live-Cell Imaging of Growing Intestinal Crypts Reveals Dynamic Relationships

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

Isolation of an scFv targeting BRG1 using phage display with characterization by AFM

Remodeling of chromatin is a vitally important event in processes such as transcription and replication. Brahma-related gene 1 (BRG1) protein is the major ATPase subunit in the human Swi/Snf complex (hSwi/Snf), an important example of the family of enzymes that carry out such remodeling events. We have used a recently developed technique, recognition imaging, to better understand the role of BRG1 in remodeling chromatin. In such experiments, a specific antibody against BRG1 is needed. However, we have found that the commercially available polyclonal (CAP) antibodies interact non-specifically with nucleosomes, making it impossible to identify hSwi/Snf (BRG1) in their presence. Here antibody phage display technology is employed for development of an antibody specifically targeting BRG1. The Tomlinson I and J single chain variable fragment (scFv) libraries were used for successful isolation of an anti-BRG1 scFv. We demonstrate that the scFv binds more strongly and with less nonspecific interactions than the CAP antibody. This work lays the groundwork for future studies involving chromatin remodeling.