BRG1 Protein Research Articles

Abstract 2995: CDK9 phosphorylates BRG1 chromatin remodeler

Abstract Cyclin-dependent kinase 9 (CDK9) belongs to the class of CDKs involved in transcription regulation along with CDK7, 8, 10-13. Earlier works have established that CDK9 is the catalytic subunit of P-TEFb, and a transcriptional activator. CDK9 inhibition holds promise for patients belonging to Chronic Lymphocytic Leukemia (CLL) and BRD4-NUT-rearranged NUT midline carcinoma (NMC) groups. It is therefore fundamentally important to understand CDK9 mediated gene regulation. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is known to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors. Additionally, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. It is however not known whether CDK9 has any transcriptional repression activity. Earlier work from our lab demonstrated that long-term CDK9 inhibition leads to the activation of BRG1, an ATP-dependent nucleosome-remodeling complex from the SWI/SNF family of proteins. We therefore hypothesized that CDK9 directly phosphorylates BRG1 and regulates its activity. Using Co-immunoprecipitation (Co-IP) assay we discovered that CDK9 associates with BRG1 endogenously in colon cancer cells. We tested the association of these proteins more rigorously by transiently over-expressing either FLAG-tagged or GFP-tagged CDK9 in HEK293T cells and performed immunoprecipitation using BRG1 antibody. CDK9 was found to co-precipitate with BRG1. We validated this via reciprocal Co-IPs. We performed an in vitro kinase assay using purified CDK9 and BRG1 proteins to demonstrate that CDK9 directly phosphorylates BRG1 and that inhibition of CDK9 leads to reduced phosphorylation of BRG1. We validated these results in vivo in colon cancer cells. Furthermore, our LC-MS/MS data lead to the identification of novel phosphorylation sites in BRG1 following CDK9 inhibition. These findings provide a potential mechanism for the transcriptional repressor activity associated with CDK9 and may be relevant for developing therapies targeting this transcriptional regulator. Citation Format: Somnath Pandey, Hanghang Zhang, Carlos A. Barrero, Salim Merali, Xavier Graña, Jean-Pierre Issa. CDK9 phosphorylates BRG1 chromatin remodeler [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2995.

SWI/SNF Chromatin-remodeling Complex Status in SMARCB1/INI1-preserved Epithelioid Sarcoma.

The SWI/SNF chromatin-remodeling complex, which is composed of evolutionarily conserved core subunits such as SMARCB1/INI1 (INI1), SMARCA4/BRG1 (BRG1), SMARCC1/BAF155 (BAF155), and SMARCC2/BAF170 (BAF170), can be viewed as the prototype of an epigenetic regulator of gene expression that is involved in tumor suppression. Epithelioid sarcoma, which classified as a tumor of uncertain differentiation, shows an almost complete loss of INI1. However, some cases of epithelioid sarcoma have preserved INI1, and the clinicopathologic features of these cases are uncertain. To date, there has been no investigation focused on the SWI/SNF chromatin-remodeling complex in INI1-preserved epithelioid sarcoma cases. First, an investigation of INI1 immunoexpression statuses in 60 formalin-fixed paraffin-embedded epithelioid sarcoma specimens (proximal type, 29 cases; conventional type, 31 cases) was performed. In the available INI1-preserved epithelioid sarcoma cases, we analyzed the BRG1, BAF155, and BAF170 protein expressions. INI1 preservation was observed in 6 of 29 (21%) proximal-type and 2 of 31 (6%) conventional-type epithelioid sarcoma cases. Six cases of INI1-preserved epithelioid sarcomas of proximal type were available for further immunohistochemical study. One proximal type showed loss of BAF170, and 2 proximal-type cases revealed loss of BRG1 with preservation of the other remaining core subunit proteins. One proximal-type case showed a mosaic pattern of BRG1 and loss of BAF155. However, in the remaining 2 proximal-type cases, all core subunit proteins were preserved. Overall, these results suggest that loss of expression of SWI/SNF chromatin-remodeling complex proteins has an important role in tumorigenesis. The remaining 2 INI1-preserved epithelioid sarcoma cases may have had other abnormalities causing dysfunction of SWI/SNF chromatin remodeling.

INI1 negative hepatoblastoma, a vanishing entity representing malignant rhabdoid tumor

Malignant rhabdoid tumors (MRT) represent a distinct group of aggressive tumors usually occurring in infancy involving a variety of anatomic locations including the kidney, brain, soft tissue and liver. The molecular hallmark of these tumors is the presence of deletions and/or mutations of SMARCB1 or rarely SMARCA4 that lead to the absence of INI1 or BRG1 proteins, core components of the SWI/SNF chromatin remodeling complex.MRT of the liver are rare tumors that comprise less than 5% of malignant pediatric liver tumors. They are currently diagnosed on the basis of documenting both INI1 negative immunoreactivity and rhabdoid morphology. In the absence of molecular testing, MRT lacking classic rhabdoid morphology are currently classified as small cell undifferentiated hepatoblastoma (HB). Here we report a case of MRT initially classified as a INI1 negative small cell HB. Whole Exome Sequencing with Transcriptome subsequently showed homozygous deletion of SMARCB1 on chromosome 22q11.23 with evidence of loss of expression of the gene, establishing the diagnosis of MRT. As molecular testing of these tumors is performed on a more routine basis, this case illustrates that the INI negative small cell undifferentiated variant of HB actually represents MRT, a tumor unrelated to HB.

1720P - Downregulation of BRCA1 protein in clear cell renal cellular carcinoma

Background: Around 75% of renal cancer in adult kidney is clear cell renal cellular carcinoma (ccRCC). This type of cancer is characterized by lipids overacumulation and mutations in VHL (around 90% cases), BAP1 and PBRM1 genes as well as stabilization of HIF1α transcription factor. Additionally, metabolic switch to aerobic glycolysis and aberration in TCA cycle was observed in ccRCC independently on the stage of the disease. Moreover, the mTOR pathway hyperactivation and downregulation of AMPK pathway featured the ccRCC. This type of cancer is highly resistant to classical chemotherapy. BRCA1 is tumor suppressor gene, mutation in this gene is associated with breast and ovarian cancer. BRCA1 is a protein involved in DNA repair and apoptosis also interacts with BRG1 – core subunit of SWI/SNF chromatin remodeling complex. BRCA1 is transcribed from bidirectional promoter together with NBR2 – lncRNA. Interestingly, NBR2 interacts with AMPK and is downregulated in ccRCC. CTCF is a protein which binds Topologicaly Associated Domains, and CTCF binding site was found in BRCA1/NBR2 promoter region. Methods: Immunohistochemistry (IHC) on paraffin embedded clinical samples for BRCA1 and BRG1 core subunit of SWI/SNF complex, comparative transcriptomic study, co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) method were used in this work. Results: In this study we found downregulation of BRCA1 and BRG1 proteins in ccRCC patient samples independently on stage of the disease. Interestingly, downregulation of BRG1 was more severe in samples with strong lymphocyte infiltration. By contrast, downregulation at the transcript level was observed for BRG1 encoding gene but not for BRCA1. BRG1 and CTCF co-precipitated from cancer cells, indicating the existence of co-interaction between CTCF, BRG1 and BRCA1 proteins. Additionally, overexpression of BRG1 caused increased expression of CTCF in human cells. We also found that BRG1 targets both CTCF and BRCA1 genes. Conclusions: BRCA1, BRG1 and CTCF module is dysregulated in ccRCC cells independently on Fuhrman grade and stage of the disease. This missregulation can have a brought spectrum of changes including 3D chromatin structure, transcription, epigenetic and others. Legal entity responsible for the study: Elzbieta Sarnowska Funding: This work was supported by grant from National Science Center No UMO–2013/11/B/NZ2/00132 Disclosure: All authors have declared no conflicts of interest.

Hypomethylation and decreased expression of BRG1 in the myocardium of patients with congenital heart disease.

BRG1, an ATPase subunit of the SWItch/Sucrose Non-Fermentable complex, is tightly associated with cardiac development. However, little is known about the association between the pathogenesis of CHD and BRG1. The methylation of a BRG1 promoter and a novel CpG island in the second intron was analyzed in the myocardium of congenital heart disease (CHD) patients (n = 24) and normal controls (n = 11) using pyrosequencing and the MassARRAY platform. BRG1 expression was sketched in the normal fetal and postnatal heart using real-time PCR. BRG1 mRNA and protein expression was detected by means of real-time PCR and immunohistochemistry. The expression of GATA4 was analyzed with real-time PCR. The CpG shore in the second intron of BRG1 was hypomethylated in the myocardium of patients (p < 0.05). BRG1 showed a high level of expression in the normal fetal heart in the second trimester (p < 0.01). Compared with that of the normal subjects, BRG1 expression was decreased by 70% in the myocardium of patients (n = 92; p < 0.05). Of note, the expression of GATA4 was significantly correlated with BRG1 expression (r = 0.7475; p = 0.0082) in the myocardium, and it was also decreased by 70% in these patients (n = 92; p < 0.05). These results suggested that the early high expression of BRG1 in fetal hearts maintained normal cardiac development and that the abnormal hypomethylation and decreased expression of BRG1 in human hearts probably affect the expression of GATA4, which affects the pathogenesis of CHD. Birth Defects Research 109:1183-1195, 2017. © 2017 Wiley Periodicals, Inc.

Brg1-mediated Nrf2/HO-1 pathway activation alleviates hepatic ischemia\u2013reperfusion injury

Cytoprotective gene heme oxygenase 1 (HO-1) could be induced by nuclear factor E2-related factor 2 (Nrf2) nuclear translocation. The purpose of this study was to determine the role of Brahma-related gene 1 (Brg1), a catalytic subunit of SWI2/SNF2-like chromatin remodeling complexes, in Nrf2/HO-1 pathway activation during hepatic ischemia–reperfusion (HIR). Our results showed that hepatic Brg1 was inhibited during early HIR while Brg1 overexpression reduced oxidative injury in CMV-Brg1 mice subjected to HIR. Moreover, promoter-driven luciferase assay showed that overexpression of Brg1 by adenovirus transfection in AML12 cells selectively enhanced HO-1 gene expression after hypoxia/reoxygenation (H/R) treatment but did not affect the other Nrf2 target gene NQO1. Furthermore, inhibition of HO-1 by the selective HO-1 inhibitor zinc protoporphyria could partly reverse the hepatic protective effects of Brg1 overexpression while HO-1-Adv attenuated AML12 cells H/R damage. Further, chromatin immunoprecipitation analysis revealed that Brg1 overexpression, which could significantly increase the recruitment of Brg1 protein to HO-1 but not NQO1 promoter, was recruited by Nrf2 to the HO-1 regulatory regions in AML12 hepatocytes subjected to H/R. In conclusion, our results demonstrated that restoration of Brg1 during reperfusion could enhance Nrf2-mediated inducible expression of HO-1 during HIR to effectively increase antioxidant ability to combat against hepatocytes damage.

Brg1 chromatin remodeling ATPase balances germ layer patterning by amplifying the transcriptional burst at midblastula transition.

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning.

Chromatin-remodelling factor Brg1 regulates myocardial proliferation and regeneration in zebrafish.

The zebrafish possesses a remarkable capacity of adult heart regeneration, but the underlying mechanisms are not well understood. Here we report that chromatin remodelling factor Brg1 is essential for adult heart regeneration. Brg1 mRNA and protein are induced during heart regeneration. Transgenic over-expression of dominant-negative Xenopus Brg1 inhibits the formation of BrdU+/Mef2C+ and Tg(gata4:EGFP) cardiomyocytes, leading to severe cardiac fibrosis and compromised myocardial regeneration. RNA-seq and RNAscope analyses reveal that inhibition of Brg1 increases the expression of cyclin-dependent kinase inhibitors such as cdkn1a and cdkn1c in the myocardium after ventricular resection; and accordingly, myocardial-specific expression of dn-xBrg1 blunts myocardial proliferation and regeneration. Mechanistically, injury-induced Brg1, via its interaction with Dnmt3ab, suppresses the expression of cdkn1c by increasing the methylation level of CpG sites at the cdkn1c promoter. Taken together, our results suggest that Brg1 promotes heart regeneration by repressing cyclin-dependent kinase inhibitors partly through Dnmt3ab-dependent DNA methylation.

Abstract PR467

Background & Objectives: Isoflurane postconditioning (IsoPostC) against myocardial ischemia reperfusion injury (IRI) is significantly decreased in diabetes. The underlying mechanism is unclear. Signal transducer and activator of transcription 3 (STAT3) is critical in ischemic postconditioning cardioprotection which is reduced in diabetic hearts as well as decreased expressions of cardiac Brahma-related gene 1 (Brg1) and nuclear factor-erythroid 2-related factor 2 (Nrf2). We hypothesized that Brg1/Nrf2 and STAT3 activation may play a critical role in IsoPostC-mediated cardioprotection in normal and diabetic hearts. Materials & Methods: Non-diabetic (C) and 8-week streptozotocin-induced type-1 diabetic (D) rat hearts were perfused ex vivo using Langendorff-perfused model. Rat hearts were subjected to 30 minutes global ischemia and 120 minutes of reperfusion with or without IsoPostC. IsoPostC was achieved by administrating emulsified isoflurane(2.0%, v/v) at the onset of reperfusion for 10 min. Left ventricular (LV) function was assessed throughout the experiment. Myocardial infarct size (IS) in relation to the area at risk (AAR) was used to measure the extent of myocardial injury. Cell injury was assessed by quantification of lactate dehydrogenase (LDH). Cardiac Nrf2, STAT3 and Brg1 protein expressions were measured using western blot. Results: (1)IsoPostC reduced myocardial IS in normal but not diabetic rats. (2) IsoPostC attenuated myocardial dysfunction in normal but not diabetic rats. (3) Brg1, Nrf2 and STAT3 protein expressions were enhanced in normal but decreased in diabetic rats. (4) The beneficial effects of IsoPostC were abolished by Brg1, Nrf2 or STAT3 gene knockdown.Conclusion: Our results indicate that IsoPostC confers cardioprotection through Brg1/Nrf2/STAT3 signaling, and impairment of this pathway may be responsible for the loss of sensitivity to IsoPostC cardioprotectio in type 1 diabetes.

Diabetes blocks the cardioprotective effects of sevoflurane postconditioning by impairing Nrf2/Brg1/HO-1 signaling

Sevofluane postconditioning (SPostC) protects heart against ischemia/reperfusion injury. However, SPostC cardioprotection is lost in diabetes whose cardiac heme oxygenase-1 (HO-1) is reduced. Brahma-related gene 1 (Brg1) facilitates nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. However, cardiac Brg1 is reduced in diabetes. We hypothesized that SPostC confers cardioprotection by activating HO-1 through Nrf2/Brg1 and that impaired Nrf2/Brg1/HO-1 in diabetes is responsible for the loss of SPostC. Control and streptozotocin-induced diabetic mice were subjected to 45min coronary artery occlusion followed by 2h reperfusion with or without SPostC achieved by exposing the mice to 2% sevoflurane for 15min at the onset of reperfusion. In invitro study, H9c2 cells were exposed to normal or high glucose and subjected to 3h hypoxia followed by 6h reoxygenation. Diabetic mice displayed larger post-ischemic infarct size, severer cardiomyocytes apoptosis, and increased oxidative stress concomitant with reduced HO-1, nuclear Nrf2 and Brg1 protein expression. These changes were prevented/reversed by SPostC in control but not in diabetic mice, and these beneficial effects of SPostC were abolished by HO-1 inhibition. In H9c2 cells exposed to normal glucose but not high glucose, SPostC significantly attenuated hypoxia/reoxygenation-induced cellular injury and oxidative stress with increased HO-1 and nuclear Nrf2. These SPostC beneficial effects were canceled by HO-1 inhibition. In conclusion, SPostC protects against myocardial ischemia/reperfusion injury through activation of Nrf2/Brg1/HO-1 signaling and impairment of this signaling may be responsible for the loss of SPostC cardioprotection in diabetes.

Abstract IA02: Dissection of the BRG1 and MYC/MAX biological connection and its use in lung cancer therapeutics

Abstract Lung cancer is the first cause of death due to cancer in most western countries, in part because of the low efficacy of most current therapies. In this regard, understanding the genetic architecture of lung malignancies has proven to constitute a reliable strategy to develop novel anticancer agents and foretell response to therapies. Previously, we reported that one third of the lung tumors of the non-small cell lung cancer type (NSCLC) endure inactivation of BRG1, which constitutes the fourth most commonly altered gene in this type of cancer. Interestingly, we also observed that BRG1 inactivation and MYC amplification are mutually exclusive events in lung cancer, supporting previous observations of a functional relationship between the MYC and BRG1 proteins. The BRG1 gene codes for a member of the SWI/SNF chromatin remodeling complex, and its role in cancer development is still poorly understood, which hinders its potential use in clinical settings. Here, I report our discovery on how the expression of wild type BRG1 in lung cancer cells up-regulates lung-specific transcripts, significantly restoring the gene expression signature of normal lung. Using cell lines from several cancer types we found that those lacking BRG1 do not respond to retinoic acid (RA) or glucocorticoids (GC), while restoration of BRG1 restitutes sensitivity, as determined by changes in cell morphology and gene expression. Conversely, in SH-SY5Y cells, a paradigm of RA-dependent differentiation, abrogation of BRG1 prevented the response to RA. We also demonstrated an antagonistic functional connection between BRG1 and MYC, whereby refractoriness to RA and GC by BRG1 inactivation involves deregulation of MYC activity. Mechanistically, these effects are mediated by BRG1 binding to MYC and MYC-target promoters to regulate their expression. Finally, BRG1 restoration significantly dampened invasion and progression, decreased MYC and restored responsiveness to RA in lung cancer cells orthotopically implanted in nude mice. This supports that BRG1 inactivation enables the cancer cell to sustain undifferentiated gene expression programs and prevent its response to environmental stimuli. On the other hand, small cell lung cancer (SCLC) accounts for one-fifth of lung cancer diagnoses and is a highly aggressive malignancy. However, our knowledge of small cell lung cancer (SCLC) genetics is still very limited, amplification of L-MYC, N-MYC and C-MYC being some of the well-established gene alterations. In this presentation I will also report our discovery of biallelic and tumor-specific inactivation of the MYC-associated factor X gene, MAX, in SCLC. Alterations in MAX were mutually exclusive with alterations at MYC and BRG1, the latter coding for an ATPase of the SWI/SNF complex. Taking advantage of three lung cancer cell lines lacking MAX we studied the effects of restoring MAX activity and depleting BRG1 in these cells. The current work demonstrates that BRG1 regulates the expression of MAX through direct recruitment to the MAX promoter, and that depletion of BRG1 strongly hinders cell growth, specifically in MAX-deficient cells. This heralds a synthetic lethal interaction between MAX and BRG1, and raise the possibility of developing a therapeutic strategy for patients with MAX-deficient tumors. Furthermore, it was observed that MAX requires BRG1 to activate neuroendocrine transcriptional programs and to up-regulate MYC-targets, such as glycolytic-related genes (e.g., LDHA, HK2, PDK1, PKM2, and PGK1). Finally, we also report genetic inactivation of the MAX dimerization protein, MGA, in lung cancers with wild type components of the SWI/SNF or MYC pathways. Taken together, these observations demonstrate that an aberrant SWI/SNF-MYC network is essential for lung cancer development, and opens novel therapeutic possibilities for the treatment of SCLC patients with MAX-deficient tumors. Citation Format: Montse Sanchez-Cespedes. Dissection of the BRG1 and MYC/MAX biological connection and its use in lung cancer therapeutics. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA02.

Abstract 2965: Identification of SMARCA4/BRG1 mutation types in NSCLC cell lines and tumors

Abstract Purpose: Identification of SMARCA4/BRG1 mutation types in NSCLC cell lines and tumors. Background: The SMARCA4 gene encodes the major catalytic subunit of the SWI/SNF chromatin remodeling complex and is necessary for the transcriptional regulation of multiple cellular pathways. SMARCA4 is frequently mutated in lung cancer cell lines and in lung cancers. Methods: Sixty four NSCLC cell lines were analyzed. The Cancer Genome Atlas (TCGA) data were examined. SMARCA4 mutations were identified by NextGen and/or Sanger sequencing using genomic DNA or cDNA. Gene expression profiles and global methylation was determined by Illumina BeadChips. BRG1 protein was detected by immunoblotting. Results: SMARCA4 abnormalities were divided into Type1 (loss of full length protein, usually due to deletions, non-sense mutations or splicing site mutations) and Type 2 (homozygous or heterozygous missense mutations with protein present). Sequencing often failed to detect large deletions. Loss of SMARCA4/BRG1 resulted in profound downstream effects on the transcriptome and methylome, while existence of BRG1 with mutations has much less effects. Analysis using most significantly regulated transcripts can distinguish SMARCA4 type 1 abnormality from WT group, and indicate that type 2 abnormality effects were heterogeneous. SMARCA4 regulated transcripts are involved in many important cellular functions such as cell movement, death and proliferation and participate in many cytokines and hormone/nuclear receptors-mediated pathways. The expressions of transcription factors FOXA2 and NKX2-1 were decreased, while two groups of cancer testis antigens (CTAs), PRAME and MAGEA family were elevated in NSCLC cell lines and tumors with SMARCA4 mutations/low expression. Furthermore, FOXA2 and NKX2-1 were downregulated in an inducible SMARCA4 knock- down immortalized human lung epithelial cell line. Conclusions: SMARCA4 mutations can be divided into two types having varying downstream effects. Citation Format: Wei Zhang, Yibing Yao, Yunyun Zhou, Xiaotu Ma, Jingsheng Yan, Tao Wang, Luc Girard, John Minna, Guanghua Xiao, Yang Xie, Adi Gazdar. Identification of SMARCA4/BRG1 mutation types in NSCLC cell lines and tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2965. doi:10.1158/1538-7445.AM2015-2965

Central nervous system-specific knockout of Brg1 causes growth retardation and neuronal degeneration

Changes in chromatin structure (chromatin remodeling) are essential regulatory processes for neuronal development, but the molecular mechanisms are unclear. The aim of the present study was to assess the effects of conditional knockout (Ko) of the Brahma-related gene-1 (Brg1) in the mouse central nervous system (CNS) on postnatal development. Brg1 was deleted in the CNS by crossing mice carrying the Brg1 conditional allele with a transgenic line expressing Cre under the control of the Nex 1 promoter. Brg1, PSD-95, NR2A and NR2B protein expressions were assessed using western blotting. Immunofluorescence, Nissl and TUNEL staining were used to assess cortical neuron viability. Hippocampal neurons were extracted from mouse embryos to observe the effects of neuronal degeneration associated with oxidative stress using Paraquat dichloride x-hydrate or 80% oxygen. Brg1fx/fx;NEX-Cre mice were significantly smaller in both body size and brain size after P35 conditional Ko of Brg1 in mouse cortical progenitors. The amount of neurons and their dendritic branches were significantly reduced in Brg1 Ko cortexes during early postnatal development. Absence of Brg1 may result in increased number of astrocytes. Loss of Brg1 increased damaged and dying neurons associated with oxidative stress. Furthermore, loss of NR2A in the Brg1 Ko cortex during early postnatal development, and delayed the NR2B–NR2A switch. Therefore, Brg1 may play a critical role in neuronal growth by regulating the NR2B–NR2A switch. Our findings provide an insight in chromatin remodeling regulation in postnatal neuronal development.

Adiponectin ameliorates hyperglycemia-induced cardiac hypertrophy and dysfunction by concomitantly activating Nrf2 and Brg1

Hyperglycemia-induced oxidative stress is implicated in the development of cardiomyopathy in diabetes that is associated with reduced adiponectin (APN) and heme oxygenase-1 (HO-1). Brahma-related gene 1 (Brg1) assists nuclear factor-erythroid-2-related factor-2 (Nrf2) to activate HO-1 to increase myocardial antioxidant capacity in response to oxidative stress. We hypothesized that reduced adiponectin (APN) impairs HO-1 induction which contributes to the development of diabetic cardiomyopathy, and that supplementation of APN may ameliorate diabetic cardiomyopathy by activating HO-1 through Nrf2 and Brg1 in diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were untreated or treated with APN adenovirus (1×109 pfu) 3 weeks after diabetes induction and examined and terminated 1 week afterward. Rat left ventricular functions were assessed by a pressure–volume conductance system, before the rat hearts were removed to perform histological and biochemical assays. Four weeks after diabetes induction, D rats developed cardiac hypertrophy evidenced as increased ratio of heart weight to body weight, elevated myocardial collagen I content, and larger cardiomyocyte cross-sectional area (all P<0.05 vs C). Diabetes elevated cardiac oxidative stress (increased 15-F2t-isoprostane, 4-hydroxynonenal generation, 8-hydroxy-2′-deoxyguanosine, and superoxide anion generation), increased myocardial apoptosis, and impaired cardiac function (all P<0.05 vs C). In D rats, myocardial HO-1 mRNA and protein expression were reduced which was associated with reduced Brg1 and nuclear Nrf2 protein expression. All these changes were either attenuated or prevented by APN. In primarily cultured cardiomyocytes (CMs) isolated from D rats or in the embryonic rat cardiomyocytes cell line H9C2 cells incubated with high glucose (HG, 25 mM), supplementation of recombined globular APN (gAd, 2μg/mL) reversed HG-induced reductions of HO-1, Brg1, and nuclear Nrf2 protein expression and attenuated cellular oxidative stress, myocyte size, and apoptotic cells. Inhibition of HO-1 by ZnPP (10μM) or small interfering RNA (siRNA) canceled all the above gAd beneficial effects. Moreover, inhibition of Nrf2 (either by the Nrf2 inhibitor luteolin or siRNA) or Brg1 (by siRNA) canceled gAd-induced HO-1 induction and cellular protection in CMs and in H9C2 cells incubated with HG. In summary, our present study demonstrated that APN reduced cardiac oxidative stress, ameliorated cardiomyocyte hypertrophy, and prevented left ventricular dysfunction in diabetes by concomitantly activating Nrf2 and Brg1 to facilitate HO-1 induction.

BRG1 Expression in Diffuse Large B Cell Lymphoma (DLBCL) Is Associated with Advanced Disease Stage

Background: BRG1 is a component of the highly conserved multisubunit, 2.0-MDa mammalian transcriptional coactivator complex SWI/SNF. This chromatin-remodeling complex is critical for the regulation of gene transcription, thereby controlling diverse cellular processes including differentiation, proliferation, tumorigenesis and DNA repair. BRG1 gene is frequently down regulated or conversely, in some cases, up regulated in cancer cell lines and tumors, implicating that BRG1 may regulate cancer proliferation and progression. BRG1 is currently considered a potential selective therapeutic target. BRG1 expression level has been related to cancer cell migration and invasion and studies have suggested that BRG1 may serve as a prognostic marker in certain cancers. Few studies exist on expression of BRG1 in lymphoproliferative disorders and diffuse large B cell lymphoma (DLBCL) in particular. Our study was aimed at determining the immunohistochemical expression of the BRG1 protein in DLBCL and its potential association with clinicopathologic variables.Design: Formalin-fixed, paraffin embedded sections from 49 Diffuse Large B-cell Lymphomas (DLBCL), were immunostained by a manual method (DAKO LSAB+ System-HRP) using rabbit monoclonal BRG1 (Abcam, Cambridge, MA) antibody. Nuclear and cytoplasmic immunoreactivity was semiquantitatively assessed in all cases. Scoring was based on staining intensity (weak, moderate, intense) and percentage of positive tumor cells (focal <= 10%, regional 11-50%, diffuse >50%). Results were correlated with clinicopathologic variables.Results: BRG1 immunoreactivity was predominately nuclear. Intense diffuse nuclear BRG1 overexpression was observed in 22/49 (45%) cases and correlated with tumor stage [73% stage 4 vs 63% stage 3 vs 40% stage 2 vs 13% stage 1, (p=0.005)]. There were no other correlations that reached statistical significance.Conclusion: BRG1 nuclear expression is increased in DLBCL and is associated with advanced tumor stage. This association with an important adverse prognostic indicator warrants further study of BRG1 expression and its potential as a selective therapeutic target in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

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

Abstract LB-89: Germ-line and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type

Abstract Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is an aggressive tumor and the most common type of undifferentiated ovarian malignancy presenting at less than 40 years of age. Its cause and histogenesis remain unknown. We sequenced the exomes of individuals from three familial cases of SCCOHT. Subsequently, we used whole exome sequencing, Sanger sequencing and immunohistochemistry to analyze germline and tumor DNA from three additional familial cases, 35 non-familial cases and one SCCOHT cell line (BIN-67). DNA sequencing revealed likely deleterious germ-line mutations in the chromatin remodeling gene SMARCA4 in all four familial cases of SCCOHT where DNA was available. This was accompanied by either a somatic mutation or loss of the wild-type allele in the tumor. BIN-67 contained bi-allelic deleterious mutations in SMARCA4. Sequencing of 24 non-familial pathologically-confirmed SCCOHT cases revealed at least one germ-line or somatic likely deleterious SMARCA4 mutation in 22 cases. Immunohistochemical (IHC) analyses of these and an additional 11 tumors showed loss of BRG1 protein in 38/40 cases. Our findings identify alterations in SMARCA4 as a major cause of SCCOHT, which could pave the way for genetic counseling and new treatment approaches. Citation Format: Leora Witkowski, Jian Carrot-Zhang, Steffen Albrecht, Nancy Hamel, Eva Tomiak, David Grynspan, Emmanouil Saloustros, Catherine Gilpin, Rachel Silva-Smith, François Plourde, Barbara Rivera, Ester Castellsagué, Mona Wu, Somayyeh Fahiminiya, Javad Nadaf, Avi Saskin, Madeleine Arseneault, Rouzan G. Karabakhtsian, Elizabeth A. Reilly, Frederick R. Ueland, Anna Margiolaki, Kitty Pavlakis, Sharon M. Castellino, Janez Lamovec, Lawrence M. Roth, Thomas M. Ulbright, Tracey Bender, Michel Longy, Andrew Berchuck, Marc Tischkowitz, Reiner Siebert, Inga Nagel, Vassilis Georgoulias, Colin J.r. Stewart, Glenn McCluggage, Jocelyne Arseneau, Blaise A. Clarke, Yasser Riazalhosseini, Martin Hasselblatt, Jacek Majewski, William D. Foulkes. Germ-line and somatic SMARCA4 mutations characterize small cell carcinoma of the ovary, hypercalcemic type. [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 LB-89. doi:10.1158/1538-7445.AM2014-LB-89

BRG1 Is Required to Maintain Pluripotency of Murine Embryonic Stem Cells

BAF chromatin remodeling complexes containing the BRG1 protein have been shown to be not only essential for early embryonic development, but also paramount in enhancing the efficiency of reprogramming somatic cells to pluripotency mediated by four transcription factors. To investigate the role of BRG1 in regulating pluripotency, we found that Oct4 and Nanog levels were increased immediately after BRG1 knockdown. While Nanog levels remained elevated over the investigated time period, Oct4 levels decreased at later time points. Additionally, OCT4 target genes were also found to be upregulated upon Brg1 knockdown. SiRNA-mediated BRG1 knockdown in embryonic stem (ES) cells led to Oct4 and Nanog upregulation, whereas F9 cells showed primarily Oct4 upregulation. BRG1 knockdown upregulated the expression of differentiation markers in mouse ES cells as well as differentiated morphology under reduced leukemia inhibitory factor conditions. Our results show that BRG1 plays an important role in maintaining pluripotency by fine-tuning the expression level of Oct4 and other pluripotency-associated genes.

Characterization of a Brg1 hypomorphic allele demonstrates that genetic and biochemical activity are tightly correlated

Mammalian SWI/SNF-related complexes are recruited to the promoters of numerous target genes, and the BRG1 catalytic subunit confers ATPase activity necessary to slide or evict nucleosomes and to regulate transcription. Based on gene-targeting experiments in mice, BRG1 is essential for early embryonic development. However, Brg1 null mutants have provided limited insight into gene-dosage considerations and structure-function relationships. To extend our knowledge of BRG1 function, we describe the genetic and biochemical characteristics of an ENU-induced hypomorphic mutation that encodes a protein with a single amino-acid substitution (E1083G) within the bilobal ATPase/chromatin-remodeling domain. Brg1ENU1/ENU1 mice have ~50% genetic activity and survive embryogenesis but exhibit a postnatal developmental phenotype associated with runting and incompletely penetrant lethality. The E1083G mutant protein is stable, and experiments with recombinant FLAG-tagged BRG1 proteins demonstrated that it retains full ATPase activity. Yet the biochemical activity of the mutant protein is diminished to ~50% of normal in chromatin-remodeling assays. Consistent with these findings, the E1083G substitution is predicted to disrupt a structurally conserved α-helix within the lobe that participates in DNA translocation but does not contain the ATPase catalytic site. We propose that this α-helix participates in the DNA translocation cycle by mechanistically linking DNA interaction surfaces at the DNA entry/anchor point to those within the Helicase C domain of lobe 2 of the bilobal ATPase motor. Taken together, these results demonstrate that BRG1 genetic and biochemical activities are tightly correlated. They also indicate that BRG1 ATPase activity is necessary but not sufficient for chromatin remodeling.

Antioxidant N‐Acetylcysteine attenuates the reduction of cardiac Brg1 protein expression in Type1 Diabetic Rats

Hyperglycemiainduced oxidative stress contributes to the development of diabetic cardiac complications. Brahma‐related gene 1(BRG1) is a key gene in regulating the expression of important endogenous antioxidant enzymes, including heme oxygenase‐1(HO‐1) which is central to cardioprotection, while cardiac HO‐1 is reduced in diabetes. We hypothesized that cardiac Brg1 expression is reduced in diabetes which can be restored by antioxidant treatment with N‐acetylcysteine (NAC). Control and streptozotocin‐induced diabetic rats were treated with NAC in drinking water or placebo for 4 weeks. At termination, cardiac function was assessed by echocardiography, and plasma and heart tissue free 15‐F2t‐isoprostane as well as myocardial Brg1 and HO‐1 protein expression were assessed. Plasma and cardiac free15‐F2t‐isoprostane in diabetes were increased, while cardiac Brg1 and HO‐1 protein expression were significantly decreased. Diabetes decreased the ratio of left ventricular peak velocity of early and late diastolic filling (E/A) and reduced isovolumetric relaxation time, indicative of diastolic dysfunction. NAC significantly increased cardiac Brg1 and HO‐1 protein expression and improved cardiac function. It is concluded that cardiac Brg1 is reduced in diabetes and enhancement of cardiac Brg1 may represent a novel mechanism of NAC cardioprotecion.