The reversible epigenetic silencing of BRM: implications for clinical targeted therapy.

The SWI/SNF chromatin-remodeling complex serves as a master switch that directs and limits the execution of specific cellular programs, such as differentiation and growth control. SWI/SNF function requires one of two paralogous ATPase subunits, Brahma (BRM) or BRM-related gene 1 (BRG1), which we previously found are lost together in cancer cell lines and primary lung cancers. Although BRG1 has been found to be mutated in cancer cell lines, the mechanisms underlying BRM silencing are not known. To address this question, we sequenced BRM in 10 BRM/BRG1-deficient cancer cell lines and found that BRM was devoid of abrogating mutations. Moreover, histone deacetylase (HDAC) inhibitors restored BRM expression in each of these BRG1/BRM-deficient cancer cell lines, indicating that epigenetic silencing is a major mechanism underlying the loss of BRM expression. Despite their ability to restore BRM expression, these HDAC inhibitors also blocked BRM function when present. However, after their removal, we observed that BRM expression remained elevated for several days, and during this period, BRM activity was detected. We also found that the suppression of BRM occurs in a broad range of human tumor types and that loss of one or both BRM alleles potentiated tumor development in mice. Thus, BRG1 and BRM are silenced by different mechanisms, and it may be possible to clinically target and reexpress BRM in a number of tumor types, potentially impacting tumor development.

When the SWI/SNF complex remodels...the cell cycle.

Mammalian cells contain several chromatin-remodeling complexes associated with the Brm and Brg1 helicase-like proteins. These complexes likely represent the functional homologs of the SWI/SNF and RSC complexes found in Saccharomyces cerevisiae. The mammalian chromatin-remodeling complexes are involved in both activation and repression of a variety of genes. Several lines of evidence also indicate that they play a specific role in the regulation of cell growth. Brm is down-regulated by ras signaling and its forced re-expression suppresses transformation by this oncogene. Besides, the Brg1 gene is silenced or mutated in several tumors cell lines and a Brg1-associated complex was recently found to co-purify with BRCA1, involved in breast and ovarian cancers. Finally, the gene encoding SNF5/Ini1, a subunit common to all mammalian SWI/SNF complexes, is inactivated in rhabdoid sarcomas, a very aggressive form of pediatric cancer. The current review will address observations made upon inactivation of Brm, Brg1 and SNF5/Ini1 by homologous recombination in the mouse, as well as the possible implication of these factors in the regulation of the Retinoblastoma pRb-mediated repression of the transcription factor E2F.

The human brm protein is cleaved during apoptosis: the role of cathepsin G.

The human brm (hbrm) protein (homologue of the Drosophila melanogaster brahma and Saccharomyces cervisiae SNF-2 proteins) is part of a polypeptide complex believed to regulate chromatin conformation. We have shown that the hbrm protein is cleaved in NB4 leukemic cells after induction of apoptosis by UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is found only in the nucleus, we have investigated the nature of the proteases that may regulate the degradation of this protein during apoptosis. In an in vitro assay, the hbrm protein could not be cleaved by caspase-3, -7, or -6, the "effector" caspases generally believed to carry out the cleavage of nuclear protein substrates. In contrast, we find that cathepsin G, a granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to that observed in vivo during apoptosis. In addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage during apoptosis but does not block activation of caspases or cleavage of the nuclear protein polyADP ribose polymerase (PARP). Although localized in granules and in the Golgi complex in untreated cells, cathepsin G becomes diffusely distributed during apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment containing a bromodomain from the carboxyl terminus of hbrm. This cleavage disrupts the association between hbrm and the nuclear matrix; the 160-kDa hbrm cleavage fragment is less tightly associated with the nuclear matrix than full-length hbrm.

Cloning and characterization of SCHIP-1, a novel protein interacting specifically with spliced isoforms and naturally occurring mutant NF2 proteins.

The neurofibromatosis type 2 (NF2) protein, known as schwannomin or merlin, is a tumor suppressor involved in NF2-associated and sporadic schwannomas and meningiomas. It is closely related to the ezrin-radixin-moesin family members, implicated in linking membrane proteins to the cytoskeleton. The molecular mechanism allowing schwannomin to function as a tumor suppressor is unknown. In attempt to shed light on schwannomin function, we have identified a novel coiled-coil protein, SCHIP-1, that specifically associates with schwannomin in vitro and in vivo. Within its coiled-coil region, this protein is homologous to human FEZ proteins and the related Caenorhabditis elegans gene product UNC-76. Immunofluorescent staining of transiently transfected cells shows a partial colocalization of SCHIP-1 and schwannomin, beneath the cytoplasmic membrane. Surprisingly, immunoprecipitation assays reveal that in a cellular context, association with SCHIP-1 can be observed only with some naturally occurring mutants of schwannomin, or a schwannomin spliced isoform lacking exons 2 and 3, but not with the schwannomin isoform exhibiting growth-suppressive activity. Our observations suggest that SCHIP-1 interaction with schwannomin is regulated by conformational changes in schwannomin, possibly induced by posttranslational modifications, alternative splicing, or mutations.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

ATP-dependent chromatin remodelling: SWI/SNF and Co. are on the job.

SWI/SNF, RSC, NURF, CHRAC, ACF, RSF and NuRD are highly conserved multiprotein complexes that use the energy of ATP-hydrolysis to remodel chromatin. These complexes that have different subunit composition, all rely on helicase-like enzymes for ATPase activity and affect chromatin structure in similar ways. The specific function of the different complexes remains unclear, but many of them seem to be involved in transcriptional regulation. Although all cellular genes may not depend on chromatin remodelling for normal expression, recent data has shown that the complexes are required for both positive and negative control of a variety of cellular pathways.

The mammalian SWI/SNF complex and the control of cell growth.

The mammalian SWI/SNF complex is a chromatin remodelling complex that uses the energy of ATP hydrolysis to facilitate access of transcription factors to regulatory DNA sequences. This complex, that was initially described as a co-factor for nuclear receptors, has recently been associated with the control of cell growth. Two of the subunits known as BRG-1 and brm can associate with the Retinoblastoma tumour suppressor gene product and co-operate with this protein for repression of E2F activity. In addition, expression of brm is frequently down-regulated upon cellular transformation and re-introduction of this protein into fibroblasts transformed by activated ras induces partial reversion of the transformed phenotype. Finally, the hSNF5/INI1 gene, encoding another subunit of the SWI/SNF complex, is subject to bi-allelic mutations in rhabdoid tumours, a very aggressive form of paediatric cancers. These observations provide a novel link between malignant transformation and chromatin remodelling machineries.

The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain.

The mammalian SWI-SNF complex is a chromatin-remodelling machinery involved in the modulation of gene expression. Its activity relies on two closely related ATPases known as brm/SNF2alpha and BRG-1/SNF2beta. These two proteins can cooperate with nuclear receptors for transcriptional activation. In addition, they are involved in the control of cell proliferation, most probably by facilitating p105(Rb) repression of E2F transcriptional activity. In the present study, we have examined the ability of various brm/SNF2alpha deletion mutants to reverse the transformed phenotype of ras-transformed fibroblasts. Deletions within the p105(Rb) LXCXE binding motif or the conserved bromodomain had only a moderate effect. On the other hand, a 49-amino-acid segment, rich in lysines and arginines and located immediately downstream of the p105(Rb) interaction domain, appeared to be essential in this assay. This region was also required for cooperation of brm/SNF2alpha with the glucocorticoid receptor in transfection experiments, but only in the context of a reporter construct integrated in the cellular genome. The region has homology to the AT hooks present in high-mobility-group protein I/Y DNA binding domains and is required for the tethering of brm/SNF2alpha to chromatin.

Altered control of cellular proliferation in the absence of mammalian brahma (SNF2alpha).

The mammalian SWI-SNF complex is an evolutionarily conserved, multi-subunit machine, involved in chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2alpha) and BRG1 (SNF2beta) proteins are mutually exclusive subunits that are believed to affect nucleosomal structures using the energy of ATP hydrolysis. In order to characterize possible differences in the function of BRM and BRG1, and to gain further insights into the role of BRM-containing SWI-SNF complexes, the mouse BRM gene was inactivated by homologous recombination. BRM-/- mice develop normally, suggesting that an observed up-regulation of the BRG1 protein can functionally replace BRM in the SWI-SNF complexes of mutant cells. Nonetheless, adult mutant mice were approximately 15% heavier than control littermates. This may be caused by increased cell proliferation, as demonstrated by a higher mitotic index detected in mutant livers. This is supported further by the observation that mutant embryonic fibroblasts were significantly deficient in their ability to arrest in the G0/G1 phase of the cell cycle in response to cell confluency or DNA damage. These studies suggest that BRM participates in the regulation of cell proliferation in adult mice.

RB and c-Myc activate expression of the E-cadherin gene in epithelial cells through interaction with transcription factor AP-2.

E-cadherin plays a pivotal role in the biogenesis of the first epithelium during development, and its down-regulation is associated with metastasis of carcinomas. We recently reported that inactivation of RB family proteins by simian virus 40 large T antigen (LT) in MDCK epithelial cells results in a mesenchymal conversion associated with invasiveness and a down-regulation of c-Myc. Reexpression of RB or c-Myc in such cells allows the reexpression of epithelial markers including E-cadherin. Here we show that both RB and c-Myc specifically activate transcription of the E-cadherin promoter in epithelial cells but not in NIH 3T3 mesenchymal cells. This transcriptional activity is mediated in both cases by the transcription factor AP-2. In vitro AP-2 and RB interaction involves the N-terminal domain of AP-2 and the oncoprotein binding domain and C-terminal domain of RB. In vivo physical interaction between RB and AP-2 was demonstrated in MDCK and HaCat cells. In LT-transformed MDCK cells, LT, RB, and AP-2 were all coimmunoprecipitated by each of the corresponding antibodies, and a mutation of the RB binding domain of the oncoprotein inhibited its binding to both RB and AP-2. Taken together, our results suggest that there is a tripartite complex between LT, RB, and AP-2 and that the physical and functional interactions between LT and AP-2 are mediated by RB. Moreover, they define RB and c-Myc as coactivators of AP-2 in epithelial cells and shed new light on the significance of the LT-RB complex, linking it to the dedifferentiation processes occurring during tumor progression. These data confirm the important role for RB and c-Myc in the maintenance of the epithelial phenotype and reveal a novel mechanism of gene activation by c-Myc.

Differential preimplantation regulation of two mouse homologues of the yeast SWI2 protein.

Epigenetic regulation of gene expression through modification of chromatin organization is an important mechanism in the development of eucaryotic organisms. We investigated the developmentally regulated expression of the mouse mBRG1 and mbrm genes, which are homologous to the yeast SWI2 gene. Both proteins are involved in chromatin remodeling as components of the mammalian SWI/SNF complex. The analysis was performed at a time in mouse development when the formation of a functional zygotic nucleus is closely linked to extensive chromatin modifications. Reverse transcription-polymerase chain reaction (RT-PCR) analysis in mature oocytes and through the first cleavage stages showed that both genes were highly expressed as maternal products but that they subsequently exhibited considerable differences in their level of expression when the transition to zygotic transcription occurred. Immunodetection of the two proteins with specific antibodies paralleled the RT-PCR analysis. The mBRG1 protein was present throughout preimplantation development, whereas zygotic mbrm was clearly detectable only when differentiation first occurs at the blastocyst stage. At this stage, mbrm was restricted to the inner cell mass. Cell type-specific expression of mbrm was also observed after in vitro differentiation of embryonic stem cells. These results indicate that the two murine homologues of SWI2 have substantially different roles in chromatin organization during the onset of embryonic development.

ras transformation is associated with decreased expression of the brm/SNF2alpha ATPase from the mammalian SWI-SNF complex.

The brm and BRG-1 proteins are mutually exclusive subunits of the mammalian SWI-SNF complex. Within this complex, they provide the ATPase activity necessary for transcriptional regulation by nucleosome disruption. Both proteins were recently found to interact with the p105Rb tumor suppressor gene product, suggesting a role for the mammalian SWI-SNF complex in the control of cell growth. We show here that the expression of brm, but not BRG-1, is negatively regulated by mitogenic stimulation, and that growth arrest of mouse fibroblasts leads to increased accumulation of the brm protein. The expression of this protein is also down-regulated upon transformation by the ras oncogene. Re-introduction of brm into ras transformed cells leads to partial reversion of the transformed phenotype by a mechanism that depends on the ATPase domain of the protein. Our data suggest that increased levels of brm protein favour the withdrawal of the cell from the cycle whereas decreased expression of the brm gene may facilitate cellular transformation by various oncogenes.

RB and hbrm cooperate to repress the activation functions of E2F1.

Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G1 arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. We show here that in transfected cells RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex in vivo. Furthermore, RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, our results point out an unexpected role of corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G1 arrest. Finally, we demonstrate that the domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity.

Components of the human SWI/SNF complex are enriched in active chromatin and are associated with the nuclear matrix.

Biochemical and genetic evidence suggest that the SWI/SNF complex is involved in the remodeling of chromatin during gene activation. We have used antibodies specific against three human subunits of this complex to study its subnuclear localization, as well as its potential association with active chromatin and the nuclear skeleton. Immunofluorescence studies revealed a punctate nuclear labeling pattern that was excluded from the nucleoli and from regions of condensed chromatin. Dual labeling failed to reveal significant colocalization of BRG1 or hBRM proteins with RNA polymerase II or with nuclear speckles involved in splicing. Chromatin fractionation experiments showed that both soluble and insoluble active chromatin are enriched in the hSWI/SNF proteins as compared with bulk chromatin. hSWI/SNF proteins were also found to be associated with the nuclear matrix or nuclear scaffold, suggesting that a fraction of the hSWI/SNF complex could be involved in the chromatin organization properties associated with matrix attachment regions.

Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type.

The hbrm and BRG-1 proteins, components of the human SNF/SWI complex, are phosphorylated and excluded from the condensed chromosomes during mitosis.

In yeast, the SNF/SWI complex is believed to regulate transcription by locally altering the chromatin structure. At the present time, three human homologues of yeast SNF/SWI proteins have been characterized: hbrm and BRG-1, homologues of SNF2/SWI2, and hSNF5, a homologue of SNF5. We show here that, during mitosis, hbrm and BRG-1 are phosphorylated and excluded from the condensed chromosomes. In this phase of the cell cycle, the level of hbrm protein is also strongly reduced, whereas the level of BRG-1 remains constant. The mitotic phosphorylation of hbrm and BRG-1 is found not to disrupt the association of these proteins with hSNF5 but correlates with a decreased affinity for the nuclear structure in early M phase. We suggest that chromosomal exclusion of the human SNF/SWI complex at the G2-M transition could be part of the mechanism leading to transcriptional arrest during mitosis.

A human protein with homology to Saccharomyces cerevisiae SNF5 interacts with the potential helicase hbrm.

In yeast, the SNF/SWI complex is involved in transcriptional activation of several inducible promoters, possibly by causing a local modification of the chromatin structure. Recently, two human homologues of the SNF2/SWI2 protein have been isolated, hbrm and BRG-1. In addition, a complex containing one of the SNF2/SWI2 homologues and having an in vitro activity similar to the yeast complex has been partially purified from HeLa cells. Here we describe the characterization of a cDNA encoding a human nuclear protein containing a large domain of homology with SNF5, another member of the yeast SNF/SWI complex. This protein can be co-immunoprecipitated with hbrm and the interaction between the two proteins is dependent on the region conserved between the human and the yeast SNF5. These findings suggest that the cDNA we have cloned encodes one of the members of the human SNF/SWI complex.

Preparative scale culture of Escherichia coli cells expressing the human immunodeficiency virus type 1 Tat protein.

A procedure leading to a 100-liter fermentor culture of Escherichia coli cells expressing the human immunodeficiency virus type 1 (HIV-1) trans-activator (Tat) protein is described. The effects of growth temperature and of cell density at the time of induction on the yield of Tat were investigated. Tat was identified by SDS-gel electrophoresis and Western blot. Tat represents approximately 10% of the soluble protein in the cell lysate.