ATP-dependent Chromatin Remodeling Complexes Research Articles

Chromatin modification and remodelling: a regulatory landscape for the control of Arabidopsis defence responses upon pathogen attack

Due to their sessile lifestyle, plants have to cope with an ever-changing environment and to defend themselves against a multitude of biotic aggressors that compromise their development and reproduction. Responses to various biotic stresses largely depend on the plant's capacity to modulate rapidly and specifically its transcriptome. In a stress type-dependent manner, external signals are translocated into the nucleus to activate transcription factors, resulting in the increased expression of particular sets of defence-related genes. Among mechanisms of transcriptional regulation, chromatin remodelling accomplished through the activity of histone-modifying enzymes and ATP-dependent chromatin-remodelling complexes is emerging as a key process in the orchestration of plant biotic stress responses. In this review, we summarize and discuss roles that chromatin-remodelling mechanisms may play in regulating Arabidopsis defence responses.

ATP-dependent chromatin remodeling complex SWI/SNF in cardiogenesis and cardiac progenitor cell development

The recent identification of cardiac progenitor cells (CPCs) provides a new paradigm for studying and treating heart disease. To realize the full potential of CPCs for therapeutic purposes, it is essential to understand the genetic and epigenetic mechanisms guiding CPC differentiation into cardiomyocytes, smooth muscle, or endothelial cells. ATP-dependent chromatin remodelers mediate one critical epigenetic mechanism. These large multiprotein complexes open up chromatin to modulate transcription factor access to DNA. SWI/SNF, one of the major types of chromatin remodelers, plays a key role in various aspects of development (de la Serna et al., 2006; Wu et al., 2009), including heart development and disease (Lickert et al., 2004; Wang et al., 2004; Huang et al., 2008; Stankunas et al., 2008; Hang et al., 2010). In this review, we describe the specific function of various SWI/SNF components in cardiogenesis and cardiac progenitor cell (CPC) self-renewal and differentiation. We envision that a detailed understanding of the SWI/SNF in heart development and CPC formation and differentiation will generate novel insights into epigenetic mechanisms that govern CPC differentiation and may have significant implications in understanding and treating heart disease.

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

ATP-dependent chromatin remodeling in the DNA-damage response

The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

Human CCAAT/Enhancer-Binding Protein β Interacts with Chromatin Remodeling Complexes of the Imitation Switch Subfamily

Transcription factor C/EBPβ is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBPβ protein is found as three major isoforms, C/EBPβ1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBPβ1 being the full-length isoform. It has been found that C/EBPβ1 participates in terminal differentiation processes. Conversely, C/EBPβ2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBPβ2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBPβ using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBPβ directly interacts with hACF1. By overexpressing hC/EBPβ, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBPβ target genes, we observed a functional interaction between C/EBPβ and SNF2H/ACF1, characterized mainly by suppression of C/EBPβ transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBPβ, hSNF2H, and hACF1 on the promoter region of this gene.

SRG3/mBAF155 stabilizes the SWI/SNF-like BAF complex by blocking CHFR mediated ubiquitination and degradation of its major components

The murine SWI/SNF-like BAF complex is an ATP-dependent chromatin remodeling complex that functions as a transcriptional regulator in cell proliferation, differentiation and development. The SWI/SNF-like BAF complex consists of several components including core subunits such as BRG1, BAF155/SRG3, BAF47/SNF5/INI1, and BAF170. We have previously shown that the interaction between SRG3/mBAF155 and other components of the complex stabilizes them by attenuating their proteasomal degradation. However, it has not been known how the major components of the SWI/SNF-like BAF complex such as BRG1, SNF5, and BAF60a are targeted for the ubiquitination and degradation, and how SRG3/mBAF155 protects them from the degradation process. Here we report that CHFR interacts with BRG1, SNF5, and BAF60a of the SWI/SNF-like BAF complex and ubiquitinates them to target for degradation through a proteasome-mediated pathway, and that SRG3/mBAF155 stabilizes these components by blocking their interaction with CHFR.

Function of the INO80 chromatin remodeling complex in DNA replication

ATP-dependent chromatin remodeling complexes are involved in several nuclear processes. In particular the INO80 remodeling complex is an essential factor during transcription and DNA repair. Recently, several labs have described a novel role for INO80 during DNA replication. Moreover, Falbo et al. have presented evidence linking INO80's activities to the DNA damage tolerance pathways during replication (1). In this review we will discuss and integrate the results obtain by these various research groups to describe a novel role for INO80 in DNA replication.

Noncoding RNA-Mediated Chromatin Silencing (RmCS) in Plants

Copyright: © 2013 He Y. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. In a eukaryotic nucleus, DNA strands wrap around histones to form nucleosomes, the basic units of chromatin. Each nucleosome is composed of a histone octomer (two molecules of the highly conserved H2A, H2B, H3 and H4) wrapped by about 147-bp DNA. Both DNA and histones can be covalently modified. The cytosines of DNA can be methylated, whereas histone residues are often reversibly modified by acetylation, methylation, phosphorylation and/or ubiquitination. In addition, canonical nucleosomal histones can be replaced by histone variants, and nucleosomes are often remodeled (e.g. reposition) by ATPdependent chromatin-remodeling complexes. These modifications can regulate chromatin structure and modulate DNA accessibility to control gene expression and other DNA-templated activities.

Diverse functions of ATP-dependent chromatin remodeling complexes in development and cancer

Mammalian SWI/SNF like Brg1/Brm associated factors (BAF) chromatin-remodeling complexes are able to use energy derived from adenosine triphosphate (ATP) hydrolysis to change chromatin structures and regulate nuclear processes such as transcription. BAF complexes contain multiple subunits and the diverse subunit compositions provide functional specificities to BAF complexes. In this review, we summarize the functions of BAF subunits during mammalian development and in progression of various cancers. The mechanisms underlying the functional diversity and specificities of BAF complexes will be discussed.

Chromatin Remodeler Sucrose Nonfermenting 2 Homolog (SNF2H) Is Recruited onto DNA Replication Origins through Interaction with Cdc10 Protein-dependent Transcript 1 (Cdt1) and Promotes Pre-replication Complex Formation

From late mitosis to the G(1) phase of the cell cycle, ORC, CDC6, and Cdt1 form the machinery necessary to load MCM2-7 complexes onto DNA. Here, we show that SNF2H, a member of the ATP-dependent chromatin-remodeling complex, is recruited onto DNA replication origins in human cells in a Cdt1-dependent manner and positively regulates MCM loading. SNF2H physically interacted with Cdt1. ChIP assays indicated that SNF2H associates with replication origins specifically during the G(1) phase. Binding of SNF2H at origins was decreased by Cdt1 silencing and, conversely, enhanced by Cdt1 overexpression. Furthermore, SNF2H silencing prevented MCM loading at origins and moderately inhibited S phase progression. Although neither SNF2H overexpression nor SNF2H silencing appeared to impact rereplication induced by Cdt1 overexpression, Cdt1-induced checkpoint activation was inhibited by SNF2H silencing. Collectively, these data suggest that SNF2H may promote MCM loading at DNA replication origins via interaction with Cdt1 in human cells. Because efficient loading of excess MCM complexes is thought to be required for cells to tolerate replication stress, Cdt1- and SNF2H-mediated promotion of MCM loading may be biologically relevant for the regulation of DNA replication.

RSC Facilitates Rad59-Dependent Homologous Recombination between Sister Chromatids by Promoting Cohesin Loading at DNA Double-Strand Breaks

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

Diverse Roles of RNA Polymerase II-associated Factor 1 Complex in Different Subpathways of Nucleotide Excision Repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two pathways of nucleotide excision repair (NER). In Saccharomyces cerevisiae, Rad26 is important but not absolutely required for TCR. Rpb4, a nonessential RNA polymerase II (Pol II) subunit that forms a subcomplex with Rpb7, and the Spt4-Spt5 complex, a transcription elongation factor, have been shown to suppress Rad26-independent TCR. The Pol II-associated factor 1 complex (Paf1C) has been shown to function in transcription elongation, 3'-processing of mRNAs, and posttranslational modification of histones. Here we show that Paf1C plays a marginal role in facilitating Rad26-dependent TCR but significantly suppresses Rad26-independent TCR. The suppression of Rad26-independent TCR is achieved by cooperating with Spt4-Spt5. We propose a model that, in the absence of Rad26, a lesion is "locked" in the active center of a Pol II elongation complex, which is stabilized by the coordinated interactions of Rpb4-Rpb7, Spt4-Spt5, and Paf1C with each other and with the core Pol II. We also found that Paf1C facilitates GGR, especially in internucleosomal linker regions. The facilitation of GGR is achieved through enabling monoubiquitination of histone H2B lysine 123 by Bre1, which in turn permits di- and trimethylation of histone H3 lysine 79 by Dot1. To our best knowledge, among the NER-modulating factors documented so far, Paf1C appears to have the most diverse functions in different NER pathways or subpathways.

EsBAF safeguards Stat3 binding to maintain pluripotency

How the unique chromatin configuration of embryonic stem cells (ESCs) integrates inputs from exogenous stimuli to maintain pluripotency remains largely unknown. The ESC-specific ATP-dependent chromatin-remodelling (esBAF) complex maintains the accessibility of the target sites of Stat3, a leukaemia inhibitory factor (LIF) signalling effector, by preventing repressive localized polycomb-mediated trimethylation of Lys 27 of histone 3 (H3K27me3).

SWI/SNF Complexes Containing Brahma or Brahma-Related Gene 1 Play Distinct Roles in Smooth Muscle Development

SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1(flox/flox) mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.

Involvement of a Toxoplasma gondii Chromatin Remodeling Complex Ortholog in Developmental Regulation

The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

Abstract LB-18: BRG1 promotes melanoma survival in response to DNA damage

Abstract The objective of this study is to elucidate the mechanisms by which BRG1 promotes melanoma survival in response to DNA damage. Cutaneous malignant melanoma, is an aggressive malignancy that arises from the transformation of epidermal melanocytes and is notoriously chemoresistant. Exposure to ultraviolet radiation (UVR) from sunlight is the most critical environmental agent involved in melanoma genesis. Melanocytes protect surrounding skin cells from UVR damage and because of this protective role, melanocytes have evolved to be particularly resistant to apoptosis when exposed to UVR. The lineage specific mechanisms that prevent cell death of melanocytes exposed to UVR are thought to contribute to melanoma chemoresistance. Microphthalmia-associated transcription factor (MITF), a master regulator of differentiation in melanocytes, is a lineage addiction oncogene in melanoma. We previously demonstrated that MITF interacts with the SWI/SNF complex to activate MITF target gene expression. SWI/SNF enzymes are multi-subunit ATP dependent chromatin remodeling complexes that regulate gene expression and promote DNA repair. Several of the subunits act as tumor suppressors and are mutated or absent in many forms of cancer. Re-expression of SWI/SNF subunits in cancer cell lines deficient in a particular subunit often results in cell cycle arrest and reversion of the tumorigenic phenotype. However, re-expression of the SWI/SNF subunit, BRG1, in BRG1 deficient melanoma cells increases survival to cisplatin and to UVR exposure. Furthermore, we have found that BRG1 is highly expressed in melanoma tumors and its expression increases during late stages of melanoma, potentially contributing to the chemoresistance typical of advanced melanoma. Our hypothesis is that BRG1promotes lineage specific activation of anti-apoptotic pathways through interactions with MITF. Data indicate that BRG1and MITF expression are closely correlated with expression of the melanoma inhibitor of apoptosis (ML-IAP) gene and that BRG1 promotes dramatic chromatin modifications on the ML-IAP regulatory region, strongly activating expression of this MITF target gene. ML-IAP is a potent inhibitor of apoptosis, which is highly expressed in melanomas and contributes to the survival of melanoma cells exposed to DNA damaging agents. Our current studies are focused on the mechanisms by which BRG1 activates ML-IAP expression and on elucidating the role of ML-IAP in mediating BRG1 induced survival of melanoma cells exposed to UVR. We expect that down-regulation of ML-IAP will significantly compromise BRG1 mediated survival of melanoma cells exposed to UVR. The results from these studies should illuminate a lineage specific mechanism by which BRG1 promotes survival of melanoma cells exposed to a DNA damaging agent and implicate BRG1 as a target for epigenetic based therapeutics to treat this highly resistant malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-18. doi:10.1158/1538-7445.AM2011-LB-18

Abstract 4028: Identification of novel binding targets of hSNF5 in malignant rhabdoid tumors

Abstract Loss of SNF5, a core subunit of the SWI/SNF ATP-dependent chromatin-remodeling complex, has been linked to development of malignant rhabdoid tumors (MRT). MRTs, a deadly form of pediatric cancer, can occur in almost any soft tissue of the body, including the brain. We have previously shown that introduction of SNF5, a known tumor suppressor gene, into deficient cell lines results in a G1 growth arrest through p21(CIP1/WAF1) mRNA induction. However, additional gene expression changes that may contribute to MRT development or may be involved in SNF5 cell cycle arrest remain uncharacterized. To discover novel binding targets of SNF5, we used a Biosciences Human Cell Cycle RT2Profiler PCR expression array to identify genes whose expression significantly changed upon reexpression of SNF5-GFP in the MRT cell line A204.1. The genes identified include p16(INK4A), p21(CIP1/WAF1), CCNG2, CCNH, CDK8, and HERC5. p16(INK4A) and p21(CIP1/WAF1) have been previously validated as direct binding targets of SNF5. Cyclin G2 (CCNG2) is a noncanonical cyclin that appears to negatively control cell cycle progression. HERC5 is involved in interferon signaling and ISGylation of proteins. CDK8 is a member of the mediator complex that plays a role in transcription. Cyclin H (CCNH) has been linked to development of some neural cancers. We developed an adenoviral vector system containing a novel HA tagged SNF5 construct. We used RT-PCR to validate the gene expression changes seen in the array in A204.1 and other MRT cell lines using this novel construct. An empty adenoviral vector was used as a control. While all genes showed increases in A204.1 after SNF5 reexpression, CCNG2 and CCNH mRNA levels increased in only a few other MRT cell lines. CDK8 expression levels were unchanged or decreased in all other MRT cell lines examined. HERC5 mRNA expression was greatly increased in all MRT cell lines at significantly higher levels than any of the other genes identified in this study. Chromatin immunoprecipitation (ChIP) analysis of the HERC5 and CCNG2 promoter regions in A204.1 confirmed direct SNF5 binding. Maximal enrichment was observed upstream of the transcriptional start site of both promoters. RNA Polymerase II also increased across the promoter regions in a pattern similar to SNF5 binding. Western blotting was also performed, confirming the upregulation of HERC5. CCNG2 protein levels did not follow mRNA expression, possibly because of tight regulation due to involvement in the cell cycle. Overall, we have identified the HERC5 and CCNG2 promoters as direct binding targets of SNF5 that may play roles in the development of this cancer. Interestingly, previous studies have shown that interferon signaling is activated upon SNF5 introduction into MRTs, indicating the potential biological relevance of HERC5. Future studies are needed to fully characterize their relationship to MRT oncogenesis and the mechanism of SWI/SNF regulation at their proximal promoter region. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2011-4028

Abstract LB-130: Abnormal expression of SWI/SNF chromatin remodeling complex genes in lung cancer

Abstract Objective: To study deregulation of the SWI/SNF chromatin remodeling complex in lung cancer tumors and cell lines with the emphasis on SMARCA2, SMARCA4 and ARID1A. Background: The DNA in each mammalian cell is compacted about 5000 fold into chromatin, and in the compacted state is unavailable for transcription. This compaction is controlled by three mechanisms: including the ATP-dependent chromatin remodeling complex SWI/SNF, consisting of 20 genes that code for about 12 protein subunits (some of which can substitute for each other). There are over 280 possible subunit permutations, influencing transcription, chromatin binding and remodeling, and tissue and gene specificity. Several subunit genes function as tumor suppressor genes (TSGs) including the interchangeable ATP catalytic components SMARCA2 (protein = BRM) and SMARCA4 (protein = BRG1) and the ARID1A (protein = BAF250a) accessory gene. The catalytic components are known to be inactivated in several cancers including lung, and ARID1A in ovarian cancer. Materials and Methods: Up to 58 cell lines and 60 non small cell lung cancer tumors arising in smokers and never smokers were studied. NextGen and Sanger sequencing of the SWI/SNF complex genes were performed on the cell lines. Western blots of nuclear extracts, methylation and qPCR were also performed on cell lines. Genome wide gene copy number (by SNP analyses) and microarray expression studies were performed on the tumors and cell lines. Immunostaining (for BRG1 and BAF250a) were performed on lung cancer microarrays. Results: Mutations rates in NSCLC lines were: SMARCA4 28% (mainly homozygous deletions), SMARCA2 0%, ARID1A 11% (mainly heterozygous point mutations). However, a marked decrease in nuclear protein expression was frequently present in NSCLC lines: BRG1 28%, BRM 28%, BAF250a 31%. Loss of BRG1, BRM, or BAF250a was present in 41% of the NSCLC lines, and 16% had loss of two or three. Combined analyses with microarray expression, immunostaining and qPCR studies of these three genes indicated frequent low expression in both NSCLC tumors and cell lines. In addition, DNA copy number by SNP analyses of NSCLC tumors indicated widespread loss of alleles of multiple members of the SWI/SNF complex. Evidence for epigenetic inactivation of one or more complex genes was present. Conclusions: Our data indicate frequent inactivation of one or more members of the SWI/SNF complex by a variety of mechanisms including deletions, mutations, epigenetic, transcriptional and translational control. The predicted downstream effects on transcription and histone regulation are likely to be widespread and possibly demonstrating cell type and gene specific effects. Although highly complicated, elucidation of the precise chromatin complex abnormalities, mechanisms and downstream effects indicate the possibility of multiple new therapeutic targets for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-130. doi:10.1158/1538-7445.AM2011-LB-130

Identification and characterization of novel potentially oncogenic mutations in the human BAF57 gene in a breast cancer patient

BAF57 is a core subunit present in all mammalian SWI/SNF ATP-dependent chromatin remodeling complexes, which regulates important biological processes including gene transcription, DNA recombination, DNA repair, and DNA replication. Among other functions, BAF57 mediates the recruitment of SWI/SNF to sequence-specific transcription factors. Thus, BAF57 plays a crucial role in regulating estrogen-dependent gene expression and proliferation in human cell lines derived from breast tumors. Increasing genetic and biochemical evidences suggest that mutations in BAF57 or alterations in its expression could play an oncogenic role in the mammary gland. Here, we describe two novel mutations in the BAF57 gene found in a breast cancer patient. Both mutations originate premature stop codons, leading to truncated proteins, structurally similar to another BAF57 mutant previously found in a human cell line derived from a breast tumor (BT-549). The expression of these novel BAF57 mutants has abnormally high estrogen receptor alpha (ERα) coactivating potential, suggesting that they might be involved in the aberrant estrogen-dependent proliferation that occur in the majority of breast tumors that retain ERα expression. In addition, the mutations in BAF57 affect its functional interaction with the androgen receptor and ETS2, two transcription factors that play an important role in breast cell biology. Therefore, mutations in BAF57 could impinge on several oncogenic signaling pathways contributing to the origin and/or development of breast cancer.

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and β-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC)(n) repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.