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SNF chromatin remodeling and cancer
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SWI/SNF chromatin remodeling and cancer Agnès Klochendler-Yeivin*, Christian Muchardt† and Moshe Yaniv† The SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure. Depending on the context, it can be involved in either transcriptional activation or repression. Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in human and mice. Results from biochemical and transfection studies suggest also that SWI/SNF participates either in the inhibition or activation of several oncogenes and tumor suppressor genes and/or control their transcriptional activity. These activities provide molecular insight into the mechanism underlying SWI/SNF function in tumor suppression. Addresses *Department of Cellular Biochemistry and Human Genetics, The Hebrew University–Hadassah Medical School, Ein Kerem, Jerusalem 91120, Israel; e-mail: [email protected] † Oncogenic Viruses Unit/URA1644 of the Centre National de la Recherche Scientifique, Department of Biotechnology, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Correspondence: Agnès Klochendler-Yeivin Current Opinion in Genetics & Development 2002, 12:73–79 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BAF Brg1-associated factor CNS central nervous system dTCF Drosophila T-cell factor HDAC histone deacetylase ICM inner cell mass LOH loss of heterozygosity PBAF Polybromo- and Brg1-associated factors pRB retinoblastoma protein RSC remodels the structure of chromatin SWI/SNF mating type switching/sucrose non-fermenting TSG tumor suppressor gene
Introduction Histones serve a dual role in the nucleus of eukaryotic cells. First, they are assembled with DNA into nucleosomes that can form higher-order structures. Second, they establish a dynamic molecular interface and play an active role in the regulation of transcription. This regulation occurs at least in part by covalent modifications of the tails of core histones. Modifications such as acetylation, phosphorylation and methylation modulate the nucleosome structure and the interaction with activators and repressors. Furthermore, over the past few years, a growing number of studies have led to the identification of additional mechanisms that regulate chromatin function in conjunction with histone covalent modifications. These involve enzymatic complexes that remodel chromatin and serve as transcriptional cofactors (reviewed in [1]). One class of such co-factors is represented by the SWI/SNF remodeling complexes that alter the path of DNA around the nucleosomal histone core in an ATP-dependent manner, resulting in nucleosome mobilization (reviewed in [2]). First identified in the yeast
Saccharomyces cerevisiae, SWI/SNF is a 2MDa multisubunit assembly that is highly conserved in eukaryotes. Mammalian SWI/SNF complexes contain one of the two potential catalytic ATPase subunits, Brm or Brg1. They further diverge biochemically in their subunit composition, suggesting that they might have specialized cellular functions [3]. Whereas chromatin-remodeling complexes are generally thought to promote gene expression, recent genetic and biochemical studies suggest that the SWI/SNF complex may also be involved in transcriptional repression [4–6,7•]. The subunit composition of the different human complexes that belong to this family is listed in Table 1. Several of the subunits, including SNF5/INI1, are common to all complexes and may constitute its core. Genetic alterations or dysregulated expression of genes involved in cell-cycle control, differentiation, cell death or maintenance of genomic integrity may be sufficient to drive malignant transformation. The precise transcriptional response to cellular regulatory circuits involves the core transcription machinery, gene-specific activators or repressors, as well as chromatin-remodeling activities that may either antagonize or enhance the repressive effects of chromatin. It is not difficult to imagine that balanced chromatin remodeling activities are crucial to ensure accurate responses to developmental or environmental cues, and to prevent the transition of normal cells into cancer cells. In this review, we describe recent genetic studies supporting the idea that the SWI/SNF complex is involved in tumor suppression. We also discuss protein interactions and functions focusing on the regulatory pathways of tumor suppressors and oncogenes.
Mutations in human primary tumors and tumor-derived cell lines Accumulating molecular genetic evidence suggests that ATPdependent chromatin remodeling by the SWI/SNF complex plays a crucial role in human tumorigenesis. Bi-allelic deletions or truncating mutations of SNF5/INI1/BAF47 on chromosome 22q11 were shown to be associated with most cases of malignant rhabdoid tumor. This rare but very aggressive pediatric cancer was initially described in the kidney, and subsequently reported as occurring elsewhere, including liver, lung and CNS where it is termed atypical teratoid and rhabdoid tumor [8]. SNF5 mutations were also observed occurring at a high frequency in another early childhood neoplasm, choroid plexus carcinoma, as well as in some cases of medulloblastoma and cPNET (central primitive neuroectodermal tumor) [9]. Deletions of SNF5 have also been reported in chronic phase and blast crisis of chronic myeloid leukemia [10]. In addition to somatic alterations, constitutive mutations of SNF5 predispose to renal and extra-renal malignant
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Oncogenes and cell proliferation
Table 1 Chromatin-remodeling complexes across species. Yeast
Drosophila
Human
SWI/SNF
RSC
BAP
BAF(a)
PBAF(a)
SWI1
–
Osa
BAF250/p270
–
hBrm complex (b)
Brg1 complex I (b)
Bgr1 complex II (b)
BAF250/p270
BAF250/p270
BAF250/p270
p220
p220
–
SWI2/SNF2
Sth1
Brm
Brm or Brg1
Brg1
Brm
Brg1
Brg1
–
Rsc1, Rsc2 and Rsc4
–
–
Polybromo/ BAF180
–
–
–
SWI3
RSC8
BAP155/Moira
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
–
–
–
p66
p66
p66
SWP73
RSC6
BAP60
BAF60a
BAF60a
BAF60a
BAF60a
–
–
–
BAP111
BAF57
BAF57
BAF57
BAF57
–
Arp7
Arp7
BAP55
β-actin
β-actin
–
–
–
Arp9
Arp9
BAP47/ACT1/ACT2
BAF53
BAF53
BAF53
BAF53
BAF53
SNF5
Sfh1
SNR1
SNF5/INI1
SNF5/INI1
SNF5/INI1
SNF5/INI1
SNF5/INI1
–
–
–
–
—
mSin3A
mSin3A
mSin3A
–
–
–
–
–
HDAC1
–
–
–
–
–
–
–
HDAC2
HDAC2
HDAC2
–
–
–
RbAp48
RbAp48
–
–
–
–
RbAp48
Three recent biochemical studies describe the purification of partly divergent human SWI/SNF complexes (a) [20•,21•] (b) [7•]. The BAF complex was described as the true homolog of the yeast SWI/SNF complex whereas PBAF seems more related to the yeast RSC complex. The ‘hBrm complex’ and the ‘Brg1 complex’ are distinguished by the presence of subunits associated with
co-repression (four last lines of the table). Note that SNF5/INI1/BAF47 are three designations for the same protein homologous to the S. cerevisiae SNF5 protein. INI1 was isolated in a two-hybrid screen as a protein interacting with the HIV integrase. BAF47 denotes a subunit of biochemically purified BAF complex and was later identified as being identical to SNF5/INI1.
rhabdoid tumor as well as CNS tumors, further supporting the hypothesis that SNF5/Ini1 is a tumor suppressor gene (TSG) [11,12].
with variable features of rhabdoid tumors [14•–16•]. Loss of the wild-type SNF5 allele in tumor DNA further demonstrated that SNF5 is a TSG. Brg1+/– mice also develop differentiated epithelial tumors [17•] but loss of heterozygosity (LOH) was not detected. In addition SNF5+/– mice develop and reproduce normally whereas 15% of Brg1 heterozygotes exhibit exencephaly, indicating that Brg1 gene dosage might be important for CNS development. Homozygous mutations, in either SNF5 or Brg1 genes are lethal at the peri-implantation stage. In vitro blastocysts outgrowth studies have shown that neither the trophectoderm nor inner cell mass (ICM) cells can survive in the absence of these proteins [14•,17•]. In contrast, the Brm knockout mice survive and do not exhibit a striking phenotype, except for an increase in body weight. At the cellular level, Brm deficiency does alter cell proliferation control, as demonstrated with primary embryonic fibroblasts in vitro and hepatocytes in vivo [18]. Nonetheless, this partial defect does not result in a tumor-prone phenotype.
Recently, the gene encoding Brg1, the catalytic subunit of the SWI/SNF complex, was found to be mutated in human tumor cell lines from various tissues including prostate, lung and breast [13]. In many cases, alteration of this gene results in loss of the expression of wild-type Brg1. Interestingly, the related Brm protein is also either downregulated or not expressed in several Brg1 mutant cell lines. These results are reminiscent of our early findings that several human tumor-derived cell lines lack Brg1 and Brm. It would be important to extend these studies to primary tumor material.
Mouse models The association of human malignancies with homozygous deletions or inactivating mutations of SNF5 and Brg1 suggested that SWI/SNF loss-of-function may contribute to oncogenesis in different cell types. The development of mouse models has provided further evidence that these critical components of the chromatin-remodeling machinery act as growth suppressors. Both SNF5 and Brg1 heterozygous mice display a cancer-prone phenotype. Several groups have shown recently that heterozygosity at the SNF5 locus predisposes mice to the development of soft-tissue sarcomas
The mild phenotype of Brm mutants compared to Brg1 and SNF5 can be accounted for by the capacity of Brg1 to replace Brm in the SWI/SNF complexes during development. In contrast to Brg1, Brm is expressed at very low levels in preimplantation embryos [19]. This could explain why the lack of Brg1 cannot be compensated by its close homolog, resulting in a strong decrease in the abundance
SWI/SNF chromatin remodeling and cancer Klochendler-Yeivin et al.
of the SWI/SNF complexes and death at this time of development. A recent biochemical study suggests an alternative explanation for the different phenotypic severity of Brm and Brg1-null mice [20•]. This study suggests that, apart from a distinguishable pattern of expression in early embryogenesis, Brg1 and Brm might not be fully functionally redundant, as previously suggested. Two distinct subfamilies of SWI/SNF-related complexes have been purified from human cells, BAF (SWI/SNF A) and PBAF (SWI/SNF B). These complexes share at least 6 common subunits (including SNF5) and display similar nucleosome disruption activity in vitro, yet they diverge structurally. BAF250 and BAF180 (a polybromoprotein) are unique subunits of BAF and PBAF, respectively. Furthermore, whereas BAF can contain either Brm or Brg1, Brg1 is the sole catalytic subunit of PBAF ([20•,21•]; Table 1). PBAF may regulate a distinct subset of genes and/or mediate specific biological functions required for proper growth and development. The severe phenotype of Brg1 knockout mice might therefore reflect the disruption of PBAF-specific functions. In addition, two recent biochemical studies bring evidence for the existence of additional Brg1 and Brm complexes that contain subunits of the mammalian mSin3A complex, including histone deacetylases (HDACs). These Brg1 and Brm complexes diverge in composition and display different chromatin-remodeling activities ([6,7•]; Table 1). We cannot rule out that the severe phenotype of Brg1 knockout mice might therefore reflect the disruption of specific Brg1-containing complexes.
Mechanism of SWI/SNF function in tumor suppression The Rb connection
Early transfection studies (e.g. [22]) demonstrated that Brg1 and Brm can associate with the retinoblastoma protein (pRB) to induce growth arrest. More recently, LKB1, a serine-threonine kinase mutated in patients with Peutz–Jeghers Syndrome was found to interact with Brg1 and its kinase activity is necessary for Brg1/Rb-dependent cell-cycle arrest [23]. The Rb TSG plays a fundamental role in cell-cycle control, apoptosis and development. A major cellular target of pRB is the E2F family of transcription factors that stimulate expression of genes required for G1 → S progression. pRB inhibits gene expression via at least two mechanisms: first it binds to the E2F family of transcription factors, blocking their transactivation potential. In addition, pRB–E2F actively inhibits transcription by recruiting HDACs and/or the SUV39H1 histone H3 methylase and the methyl-binding protein HP1 to generate a repressive chromatin conformation [24••,25]. Several studies have suggested a role for SWI/SNF in pRB inhibition of E2F transactivation. A genetic screen for modifiers of an E2F overexpression phenotype in the Drosophila eye identified enhancer mutations in Osa, Brahma and Moira genes, which encode homologs of SWI1, SWI2 and SWI3, respectively, indicating that the SWI/SNF complex downregulates E2F activity [26]. Results from transfection
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assays in cultured cells also support a role for chromatinremodeling enzymes in active repression by the pRB–E2F complex [27]. Overexpression of Brg1 in cells deficient in Brg1 and Brm induces growth arrest if functional pRB is present [22]. Similarly, expression of a dominant negative Brg1 in proliferating NIH3T3 reverses Rb or p16(INK4a)mediated cell-cycle arrest [28]. Transfection studies indicate that class I HDACs and SWI/SNF are recruited by pRB and may coordinate the sequential expression of E2F target genes to regulate progression through G1 and S phases [29••]. It has been suggested that a trimeric complex pRB–HDAC–SWI/SNF inhibits Cyclin E expression in quiescent and early G1 cells. As cells progress toward S phase, active Cyclin D–Cdk4 phosphorylates pRB, causing the dissociation of HDAC, but leaving the RB–SWI/SNF interaction unaffected. This repressor complex inhibits Cyclin A and Cdc2 but allows accumulation of active Cyclin E–Cdk2, which, in turn, phosphorylates pRB and/or Brg1. These modifications are suggested to disrupt the repressing complex, promoting cyclin A and cdc2 transcription allowing progression into G2/M phase. This model is compatible with a previous study showing that Brg1 and BAF155 (SWI3) associate with Cyclin E and are potential substrates of Cyclin E–Cdk2 kinase activity in vitro [30]. The in vivo relevance of this activity, and its effect on pRB–Brg1 association and function, however, has not been established. Moreover, considering that the expression levels of the different cellcycle regulators must be finely balanced to ensure proper signaling, it will be important to examine the assembly or dissociation of pRB–SWI/SNF complexes and their recruitment to endogenous promoters during the cell cycle in a physiological context. Functional SWI/SNF is not absolutely necessary for cell-cycle arrest to occur. The expression of a dominant negative mutant form of Brm or Brg1 in NIH3T3 cells prevents MyoD-induced muscle cell differentiation [31••]. Experiments to elucidate whether SWI/SNF is required for MyoD-mediated cell-cycle withdrawal and muscle-specific gene activation, revealed that SWI/SNF is crucial to induce muscle-specific genes but dispensable for cell-cycle arrest [32••]. Moreover, SWI/SNF is neither required for MyoDmediated induction of p21, Cyclin D3 and pRb, nor for repression of Cyclin A. Under culture conditions used for differentiation (confluency and serum-starvation) both mockdifferentiated and MyoD-differentiated cells arrested in G1, regardless of whether functional SWI/SNF complexes were present. This cell-cycle withdrawal may be mediated by p27kip1, which is expressed at high levels in all cells. Beyond pRB
Although the studies described above provide support for a model in which SWI/SNF collaborates with pRB to inhibit cell proliferation, other studies show that SWI/SNF may participate in additional growth-signaling pathways. Control of transcriptional activity of growth regulators
Recently, components of the SWI/SNF complex have been shown to associate with BRCA1, a tumor suppressor linked
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Oncogenes and cell proliferation
Figure 1 (a) Transcriptional repression
(b) Transcriptional activation
SWI/SNF SWI/SNF TCF Rpd3
β-cat TCF
Gro Repression of Wg-target genes in Drosophila
Activation of Wg/Wnt target genes in Drosophila and mammals
Growth arrest?
Cell proliferation? Current Opinion in Genetics & Development
to familial breast and ovarian cancers. Moreover, transient transfection assays suggest that the ability of BRCA1 to act as a coactivator of p53-dependent transcription is mediated through Brg1 [33]. Among the human tumor cell lines screened for Brg1-inactivating mutations, 2 out of 22 breast carcinoma cell lines displayed biallelic mutations resulting in loss of wild-type protein expression [13]. Furthermore, re-introduction of Brg1 into one of the cell lines (ALAB) induced G1 arrest. It is worth noting that Brg1 deficiency in ALAB cells correlates with a defective pRB pathway as shown previously with SW13 and C33a cells, whereas alterations in BRCA1 activity have not been examined. The SWI/SNF complexes may also regulate either the synthesis or the stability of growth inhibitors. Confluent cultures of mouse embryonic fibroblasts from Brm–/– mice accumulate less p27kip1 protein than their wild-type counterparts [18]. Ras-transformation of rodent cells was shown to downregulate the synthesis of Brm. And re-expression of ectopic Brm in these cells reversed their transformed phenotype [34]. These studies suggest that some SWI/SNF activities can antagonize Ras transformation, perhaps by controlling the pRB/E2F checkpoint, and that they in turn are downregulated by oncogenic Ras. c-myc, a proto-oncogene involved in cell proliferation, differentiation and apoptosis might also require SWI/SNF for its transcriptional activity. SNF5 has been reported to interact with c-Myc and co-expression of a dominantnegative mutant of BRG1 suppressed reporter gene activation by Myc in a transient transfection assay [35]. Consistent with SNF5 tumor suppressor activity, it has been suggested that c-Myc might recruit SWI/SNF and facilitate transcription of a subset of target genes, specifically those involved in apoptosis. Whether c-Myc associates with the intact SWI/SNF complex and directs chromatin remodeling at distinct endogenous promoters remains to be demonstrated. Genetic data in Drosophila provide evidence that Brm complexes modulate target gene expression by the
Antagonistic functions of SWI/SNF in the transcriptional regulation of the Wingless (Wg)/Wnt pathway. (a) Genetic interactions in Drosophila indicate that in the absence of β-catenin, the Brm complex cooperates with TCF and Groucho (Gro) to repress Wingless target genes. (b) Following Wingless/Wnt signaling, β-catenin accumulates in the nucleus, interacts with Brg1, promoting transcriptional activation of TCF target genes. Of note, the SWI/SNF complex participating in either reaction may have a specific subunit composition that facilitates interaction with co-repressors or co-activators and/or directs opening or closing of the chromatin structure.
Wingless/Armadillo pathway. Wingless signaling triggers the stabilization of Armadillo which translocates to the nucleus and binds to dTCF (Drosophila T-cell factor) to stimulate transcription of Wingless target genes. Vertebrate homologs of all Wingless pathway genes have been identified and their function is known to be highly conserved. Deregulation of β-catenin (the vertebrate homolog of Armadillo) results in increased transcription of TCF target genes such as myc or cyclin D1, and malignant transformation of the mammalian gut epithelium (reviewed in [36]). In the absence of Wingless signaling in Drosophila, dTCF target genes are actively repressed by recruitment of negative regulators such as Groucho and Rpd3 HDAC that associate with dTCF [37]. One report [38••] has demonstrated genetic interaction between the Wingless pathway and components of the Brm complex (Osa, Moira and Brahma), as well as strong genetic interaction between Osa and Groucho, and suggested that the Brm complex is required for the repression of dTCF target genes in the absence of Wingless signaling. If this function is conserved in vertebrates and SWI/SNF is required to ensure effective repression of TCF target gene activity in the absence of β-catenin, it might provide a novel mechanism by which this chromatin-remodeling complex prevents tumor growth. Paradoxically, a second study [39••] has shown that Brg1 interacts with β-catenin and promotes TCF target gene activity in mammalian cells. Genetic studies in Drosophila also support a role for the Brm complex in the stimulation of β-catenin–TCF transcriptional activity [39••]. These two studies imply that, depending on the upstream acting signal, the SWI/SNF complexes might function in both activation and repression of the same set of genes, resulting in either positive or negative control of cell proliferation. A putative oncogenic activity of SWI/SNF is suggested by its association with c-Fos and c-Jun through BAF60a [40]. This association has been shown to potentiate the transactivating activity of the c-Fos/c-JunAP1 heterodimer.
SWI/SNF chromatin remodeling and cancer Klochendler-Yeivin et al.
Transcriptional regulation of factors involved in tumorigenesis
The proto-oncogene c-fos promoter has been shown to be repressed following ectopic expression of Brg1. This repression requires a functional ATP-binding site and depends on Rb but not on E2F activity [41]. CD44, a transmembrane glycoprotein whose deregulation contributes to cancer progression, is downregulated in cell lines lacking Brg1. Furthermore, ectopic expression of functional Brg1 restores CD44 expression [42]. These studies, however, do not demonstrate clearly whether and how the SWI/SNF complex is recruited to the c-fos and CD44 promoters. Moreover, c-fos repression could be an indirect consequence of Brg1/pRb-mediated growth arrest.
Conclusions Genetic studies in human and mouse have shown that inactivating mutations or deletions of genes encoding subunits of the SWI/SNF complex are associated with cancer, qualifying these genes as putative tumor suppressors. Although there has been much focus on the role of the pRB pathway, biochemical and molecular studies have revealed an increasing number of potential targets of the SWI/SNF complex that function as either positive or negative regulators of cell growth. Thus, the SWI/SNF complex can be considered as an integrator of multiple signaling networks. Taken together, these studies support a role for SWI/SNF in transcriptional regulation, via its interaction with either activator or repressor proteins, some of which may function within the same pathway (Figure 1). In the future, it will be crucial to investigate whether the SWI/SNF complex participates in other molecular processes, such as DNA repair, recombination and replication or chromosome segregation in which chromatin structure plays an important role. Disruption of such functions could contribute to malignant transformation. In this context, the SNF5 subunit has been shown to stimulate replication of the human papillomavirus, and the SWI/SNF complex to increase the efficiency of DNA replication in yeast [43,44]. Moreover, SWI/SNF has been observed to stimulate RAG1 and RAG2 mediated V(D)J recombination in vitro [45•]. Furthermore the initial isolation of SNF5/INI1 as a protein interacting with and stimulating the HIV integrase [46] and the recent report on the association of SNF5 with the incoming viral particle [47] support a role for at least SNF5 in recombination. The association of BRCA1 with SWI/SNF could imply a role for the chromatin-remodeling complex in DNA repair. The S. cerevisiae RSC complex (the second SWI/SNF-like complex in yeast), or its putative human homolog, may also be important for chromosome segregation. It was suggested that RSC is involved in centromere remodeling during G2/M [48] and some rsc3 mutant alleles trigger G2/M arrest and cell death [49]. Such a role is further supported by the association of the PBAF complex with mammalian kinetochores [20•]. There are several additional questions about SWI/SNF function that are yet to be resolved. Different cells appear
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to have significantly divergent responses to SWI/SNF inactivation. Neither ICM nor trophectoderm cells derived from Brg1 or SNF5-null blastocysts survive in culture. Likewise, a Brg1-null mutation in F9 embryonic carcinoma cells resulted in death [50]. Yet, some cells in mice and humans become transformed upon SNF5 inactivation, and several human tumor cell lines lacking Brg1 survive and proliferate normally. As SWI/SNF has a role in Rb inhibition of E2F-dependent transactivation, it is possible that loss of SWI/SNF leads to release of free E2F that can trigger apoptosis by activating the p53-inhibitor p19ARF in many cell types [51]. It will be of great interest to analyze the status of the ARF–p53 pathway in SWI/SNF-deficient tumor cells. Another issue concerns the difference between human and mice. Children with a constitutive germline mutation are afflicted by a cancer predisposition syndrome with high penetrance before the age of 3. Furthermore, the low number of other chromosomal rearrangements outside of the SNF5 locus suggests a major role for this alteration. In contrast, only 30–40% of SNF5+/– mice develop tumors by the age of 12–15 months, suggesting that this pathway may be less crucial in rodents and that tumor formation in this case requires additional mutations.
Acknowledgements We apologize to those whose work was not cited directly because of space constraints. We thank Jonathan Weitzman and Olivier Delattre for critical reading and advice on the manuscript. We thank the AICR Foundation, ARC and LNFCC for financial support.
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10. Grand F, Kulkarni S, Chase A, Goldman JM, Gordon M, Cross NC: Frequent deletion of hSNF5/INI1, a component of the SWI/SNF complex, in chronic myeloid leukemia. Cancer Res 1999, 59:3870-3874. 11. Sevenet N, Sheridan E, Amram D, Schneider P, Handgretinger R, Delattre O: Constitutional mutations of the hSNF5/INI1 gene predispose to a variety of cancers. Am J Hum Genet 1999, 65:1342-1348. 12. Taylor MD, Gokgoz N, Andrulis IL, Mainprize TG, Drake JM, Rutka JT: Familial posterior fossa brain tumors of infancy secondary to germline mutation of the hSNF5 gene. Am J Hum Genet 2000, 66:1403-1406. 13. Wong AK, Shanahan F, Chen Y, Lian L, Ha P, Hendricks K, Ghaffari S, Iliev D, Penn B, Woodland AM et al.: BRG1, a component of the SWI-SNF complex, is mutated in multiple human tumor cell lines. Cancer Res 2000, 60:6171-6177. 14. Klochendler-Yeivin A, Fiette L, Barra J, Muchardt C, Babinet C, • Yaniv M: The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression. EMBO Rep 2000, 1:500-506. See annotation [16•]. 15. Roberts CW, Galusha SA, McMenamin ME, Fletcher CD, Orkin SH: • Haploinsufficiency of Snf5 (integrase interactor 1) predisposes to malignant rhabdoid tumors in mice. Proc Natl Acad Sci USA 2000, 97:13796-13800. See annotation [16•]. 16. Guidi CJ, Sands AT, Zambrowicz BP, Turner TK, Demers DA, • Webster W, Smith TW, Imbalzano AN, Jones SN: Disruption of Ini1 leads to peri-implantation lethality and tumorigenesis in mice. Mol Cell Biol 2001, 21:3598-3603. Using different targeting strategies, these studies [14•–16•] reveal that the SNF5 gene is essential for murine early development and tumor suppression. SNF5-null mice die at peri-implantation and neither ICM nor trophectoderm cells derived from SNF5–/– blastocysts survives in vitro. Consistent with human genetic data indicating that hSNF5 is a TSG, heterozygous mice are predisposed to tumors that show LOH at the SNF5 locus. 17. •
Bultman S, Gebuhr T, Yee D, La Mantia C, Nicholson J, Gilliam A, Randazzo F, Metzger D, Chambon P, Crabtree G et al.: A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell 2000, 6:1287-1295. This study reveals that Brg1 loss-of-function mutation in mice results in early lethality similar to SNF5 targeted mutation, suggesting that either SNF5 or Brg1 disruption inactivates the SWI/SNF complex and demonstrating that despite the multiplicity of chromatin-remodeling complexes in mammals, the Brg1 complex is irreplaceable. Brg1+/– mice are predisposed to exencephaly and tumors that fail to show LOH. This suggests that Brg1 might be haploinsufficient for tumorigenesis and CNS development in the mouse. 18. Reyes JC, Barra J, Muchardt C, Camus A, Babinet C, Yaniv M: Altered control of cellular proliferation in the absence of α). EMBO J 1998, 17:6979-6991. mammalian brahma (SNF2α 19. LeGouy E, Thompson EM, Muchardt C, Renard JP: Differential preimplantation regulation of two mouse homologues of the yeast SWI2 protein. Dev Dyn 1998, 212:38-48. 20. Xue Y, Canman JC, Lee CS, Nie Z, Yang D, Moreno GT, Young MK, • Salmon ED, Wang W: The human SWI/SNF-B chromatinremodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes. Proc Natl Acad Sci USA 2000, 97:13015-13020. See annotation [21•]. 21. Nie Z, Xue Y, Yang D, Zhou S, Deroo BJ, Archer TK, Wang W: • A specificity and targeting subunit of a human SWI/SNF family-related chromatin-remodeling complex. Mol Cell Biol 2000, 20:8879-8888. The authors of [20•,21•] have immunopurified two highly similar SWI/SNFrelated complexes: BAF and PBAF. These complexes display two main differences. First, whereas PBAF contains BAF180 — a homolog of chicken PB1 and a combined homolog of yeast Rsc1, Rsc2 and Rsc4 — that localizes at mitotic kinetochores and spindle poles, BAF250 (a homolog of
SWI1) is a specific marker of BAF. Second, PBAF contains only Brg1 in contrast to BAF that can contain either hBrm or Brg1. On the basis of these findings, the authors suggest that PBAF represents the human homolog of the RSC complex, whereas BAF is more related to yeast SWI/SNF. 22. Dunaief JL, Strober BE, Guha S, Khavari PA, Alin K, Luban J, Begemann M, Crabtree GR, Goff SP: The retinoblastoma protein and BRG1 form a complex and cooperate to induce cell cycle arrest. Cell 1994, 79:119-130. 23. Marignani PA, Kanai F, Carpenter CL: Lkb1 associates with brg1 and is necessary for brg1-induced growth arrest. J Biol Chem 2001, 276:32415-32418. 24. Nielsen SJ, Schneider R, Bauer UM, Bannister AJ, Morrison A, •• O’Carroll D, Firestein R, Cleary M, Jenuwein T, Herrera RE et al.: Rb targets histone H3 methylation and HP1 to promoters. Nature 2001, 412:561-565. This paper reveals that Rb interacts with and targets the histone methylase SUV39H1 and HP1 to the cyclin E promoter. Furthermore, histone methylase activity is shown to be required for Rb-mediated repression of the cyclin E and cyclin A2 genes. These findings suggest a role for histone methylation and HP1 in transcriptional repression of euchromatic genes and in the regulation of cell cycle progression. 25. Harbour JW, Dean DC: The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev 2000, 14:2393-2409. 26. Staehling-Hampton K, Ciampa PJ, Brook A, Dyson N: A genetic screen for modifiers of E2F in Drosophila melanogaster. Genetics 1999, 153:275-287. 27.
Trouche D, Le Chalony C, Muchardt C, Yaniv M, Kouzarides T: RB and hbrm cooperate to repress the activation functions of E2F1. Proc Natl Acad Sci USA 1997, 94:11268-11273.
28. Strobeck MW, Knudsen KE, Fribourg AF, DeCristofaro MF, Weissman BE, Imbalzano AN, Knudsen ES: BRG-1 is required for RB-mediated cell cycle arrest. Proc Natl Acad Sci USA 2000, 97:7748-7753. 29. Zhang HS, Gavin M, Dahiya A, Postigo AA, Ma D, Luo RX, Harbour JW, •• Dean DC: Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-hSWI/SNF. Cell 2000, 101:79-89. This manuscript strengthens previous findings that pRb cooperates with HDAC and SWI/SNF in the repression of E2F-target genes. The authors demonstrate that distinct repressor complexes regulate the expression of different cell-cycle genes and ensuing progression of cells through G1 and S. 30. Shanahan F, Seghezzi W, Parry D, Mahony D, Lees E: Cyclin E associates with BAF155 and BRG1, components of the mammalian SWI-SNF complex, and alters the ability of BRG1 to induce growth arrest. Mol Cell Biol 1999, 19:1460-1469. 31. de la Serna IL, Carlson KA, Imbalzano AN: Mammalian SWI/SNF •• complexes promote MyoD-mediated muscle differentiation. Nat Genet 2001, 27:187-190. See annotation [32•]. 32. de La Serna IL, Roy K, Carlson KA, Imbalzano AN: MyoD can induce •• cell cycle arrest but not muscle differentiation in the presence of dominant negative SWI/SNF chromatin remodeling enzymes. J Biol Chem 2001, 24:41486-41491. Using NIH3T3 cells expressing an inducible dominant-negative mutant of Brg1 or hBrm, the authors show that SWI/SNF is required for MyoD-mediated transcriptional activation of muscle-specific genes, such as myosin heavy chain, myosin light chain or myogenin. By contrast, the dominant-negative complexes do not affect the expression levels of cell-cycle regulators associated with cell-cycle arrest. 33. Bochar DA, Wang L, Beniya H, Kinev A, Xue Y, Lane WS, Wang W, Kashanchi F, Shiekhattar R: BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer. Cell 2000, 102:257-265. 34. Muchardt C, Bourachot B, Reyes JC, Yaniv M: ras transformation is α ATPase associated with decreased expression of the brm/SNF2α from the mammalian SWI-SNF complex. EMBO J 1998, 17:223-231. 35. Cheng SW, Davies KP, Yung E, Beltran RJ, Yu J, Kalpana GV: c-MYC interacts with INI1/hSNF5 and requires the SWI/SNF complex for transactivation function. Nat Genet 1999, 22:102-105. 36. Bienz M, Clevers H: Linking colorectal cancer to Wnt signaling. Cell 2000, 103:311-320.
SWI/SNF chromatin remodeling and cancer Klochendler-Yeivin et al.
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Cavallo RA, Cox RT, Moline MM, Roose J, Polevoy GA, Clevers H, Peifer M, Bejsovec A: Drosophila Tcf and Groucho interact to repress Wingless signalling activity. Nature 1998, 395:604-608.
38. Collins RT, Treisman JE: Osa-containing Brahma chromatin •• remodeling complexes are required for the repression of wingless target genes. Genes Dev 2000, 14:3140-3152. This genetic study in Drosophila shows that the repression of Wingless target genes requires chromatin remodeling mediated by Osa and Moiracontaining Brm complexes. Loss of function or overexpression of these components of the Brm complex induces ectopic expression or repression of endogenous Wingless target genes respectively. Furthermore, Osa interacts genetically with the corepressor Groucho and the HDAC Rpd3 which are required for the active repression of Wingless target genes in the absence of the Wingless signal. 39. Barker N, Hurlstone A, Musisi H, Miles A, Bienz M, Clevers H: •• The chromatin remodelling factor Brg-1 interacts with β-catenin to promote target gene activation. EMBO J 2001, 20:4935-4943. This paper demonstrates that Brg1 can associate with β-catenin and stimulates TCF-target genes activation in transfected mammalian cells. Genetic experiments in Drosophila also indicate functional interactions between Brahma complex genes and Armadillo. Heterozygosity for Brahma and Moira suppresses the Rough eye phenotype caused by the overexpression of a constitutively active form of Armadillo in the larval eye disc and enhances the mutant wing phenotype caused by Armadillo depletion in the wing disc. 40. Ito T, Yamauchi M, Nishina M, Yamamichi N, Mizutani T, Ui M, Murakami M, Iba H: Identification of SWI.SNF complex subunit BAF60a as a determinant of the transactivation potential of Fos/Jun dimers. J Biol Chem 2001, 276:2852-2857.
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44. Flanagan JF, Peterson CL: A role for the yeast SWI/SNF complex in DNA replication. Nucleic Acids Res 1999, 27:2022-2028. 45. Kwon J, Morshead KB, Guyon JR, Kingston RE, Oettinger MA: • Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA. Mol Cell 2000, 6:1037-1048. This study provides the first evidence supporting a role for mammalian SWI/SNF in processes other than transcriptional regulation. The authors show that histone tails constitute a barrier to RAG binding and V(D)J cleavage in vitro. They further demonstrate that histone acetylation or SWI/SNF-mediated chromatin remodeling can increase the accessibility of mononucleosome substrates to the recombinase machinery. When both chromatin-modifying activities act together, the level of V(D)J cleavage is further enhanced. 46. Kalpana GV, Marmon S, Wang W, Crabtree GR, Goff SP: Binding and stimulation of HIV-1 integrase by a human homolog of yeast transcription factor SNF5. Science 1994, 266:2002-2006. 47.
Turelli P, Doucas V, Craig E, Mangeat B, Klages N, Evans R, Kalpana G, Trono D: Cytoplasmic recruitment of INI1 and PML on incoming HIV preintegration complexes: interference with early steps of viral replication. Mol Cell 2001, 7:1245-1254.
48. Tsuchiya E, Hosotani T, Miyakawa T: A mutation in NPS1/STH1, an essential gene encoding a component of a novel chromatinremodeling complex RSC, alters the chromatin structure of Saccharomyces cerevisiae centromeres. Nucleic Acids Res 1998, 26:3286-3292.
41. Murphy DJ, Hardy S, Engel DA: Human SWI-SNF component BRG1 represses transcription of the c-fos gene. Mol Cell Biol 1999, 19:2724-2733.
49. Angus-Hill ML, Schlichter A, Roberts D, Erdjument-Bromage H, Tempst P, Cairns BR: A Rsc3/Rsc30 zinc cluster dimer reveals novel roles for the chromatin remodeler RSC in gene expression and cell cycle control. Mol Cell 2001, 7:741-751.
42. Strobeck MW, DeCristofaro MF, Banine F, Weissman BE, Sherman LS, Knudsen ES: The BRG-1 subunit of the SWI/SNF complex regulates CD44 expression. J Biol Chem 2001, 276:9273-9278.
β-BRG1 50. Sumi-Ichinose C, Ichinose H, Metzger D, Chambon P: SNF2β is essential for the viability of F9 murine embryonal carcinoma cells. Mol Cell Biol 1997, 17:5976-5986.
43. Lee D, Sohn H, Kalpana GV, Choe J: Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA. Nature 1999, 399:487-491.
51. Bates S, Phillips AC, Clark PA, Stott F, Peters G, Ludwig RL, Vousden KH: p14ARF links the tumour suppressors RB and p53. Nature 1998, 395:124-125.
SWI/SNF chromatin remodeling and cancer Agnès Klochendler-Yeivin*, Christian Muchardt† and Moshe Yaniv† The SWI/SNF complex contributes to the regulation of gene expression by altering the chromatin structure. Depending on the context, it can be involved in either transcriptional activation or repression. Growing genetic and molecular evidence indicate that subunits of the SWI/SNF complex act as tumor suppressors in human and mice. Results from biochemical and transfection studies suggest also that SWI/SNF participates either in the inhibition or activation of several oncogenes and tumor suppressor genes and/or control their transcriptional activity. These activities provide molecular insight into the mechanism underlying SWI/SNF function in tumor suppression. Addresses *Department of Cellular Biochemistry and Human Genetics, The Hebrew University–Hadassah Medical School, Ein Kerem, Jerusalem 91120, Israel; e-mail: [email protected] † Oncogenic Viruses Unit/URA1644 of the Centre National de la Recherche Scientifique, Department of Biotechnology, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Correspondence: Agnès Klochendler-Yeivin Current Opinion in Genetics & Development 2002, 12:73–79 0959-437X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations BAF Brg1-associated factor CNS central nervous system dTCF Drosophila T-cell factor HDAC histone deacetylase ICM inner cell mass LOH loss of heterozygosity PBAF Polybromo- and Brg1-associated factors pRB retinoblastoma protein RSC remodels the structure of chromatin SWI/SNF mating type switching/sucrose non-fermenting TSG tumor suppressor gene
Introduction Histones serve a dual role in the nucleus of eukaryotic cells. First, they are assembled with DNA into nucleosomes that can form higher-order structures. Second, they establish a dynamic molecular interface and play an active role in the regulation of transcription. This regulation occurs at least in part by covalent modifications of the tails of core histones. Modifications such as acetylation, phosphorylation and methylation modulate the nucleosome structure and the interaction with activators and repressors. Furthermore, over the past few years, a growing number of studies have led to the identification of additional mechanisms that regulate chromatin function in conjunction with histone covalent modifications. These involve enzymatic complexes that remodel chromatin and serve as transcriptional cofactors (reviewed in [1]). One class of such co-factors is represented by the SWI/SNF remodeling complexes that alter the path of DNA around the nucleosomal histone core in an ATP-dependent manner, resulting in nucleosome mobilization (reviewed in [2]). First identified in the yeast
Saccharomyces cerevisiae, SWI/SNF is a 2MDa multisubunit assembly that is highly conserved in eukaryotes. Mammalian SWI/SNF complexes contain one of the two potential catalytic ATPase subunits, Brm or Brg1. They further diverge biochemically in their subunit composition, suggesting that they might have specialized cellular functions [3]. Whereas chromatin-remodeling complexes are generally thought to promote gene expression, recent genetic and biochemical studies suggest that the SWI/SNF complex may also be involved in transcriptional repression [4–6,7•]. The subunit composition of the different human complexes that belong to this family is listed in Table 1. Several of the subunits, including SNF5/INI1, are common to all complexes and may constitute its core. Genetic alterations or dysregulated expression of genes involved in cell-cycle control, differentiation, cell death or maintenance of genomic integrity may be sufficient to drive malignant transformation. The precise transcriptional response to cellular regulatory circuits involves the core transcription machinery, gene-specific activators or repressors, as well as chromatin-remodeling activities that may either antagonize or enhance the repressive effects of chromatin. It is not difficult to imagine that balanced chromatin remodeling activities are crucial to ensure accurate responses to developmental or environmental cues, and to prevent the transition of normal cells into cancer cells. In this review, we describe recent genetic studies supporting the idea that the SWI/SNF complex is involved in tumor suppression. We also discuss protein interactions and functions focusing on the regulatory pathways of tumor suppressors and oncogenes.
Mutations in human primary tumors and tumor-derived cell lines Accumulating molecular genetic evidence suggests that ATPdependent chromatin remodeling by the SWI/SNF complex plays a crucial role in human tumorigenesis. Bi-allelic deletions or truncating mutations of SNF5/INI1/BAF47 on chromosome 22q11 were shown to be associated with most cases of malignant rhabdoid tumor. This rare but very aggressive pediatric cancer was initially described in the kidney, and subsequently reported as occurring elsewhere, including liver, lung and CNS where it is termed atypical teratoid and rhabdoid tumor [8]. SNF5 mutations were also observed occurring at a high frequency in another early childhood neoplasm, choroid plexus carcinoma, as well as in some cases of medulloblastoma and cPNET (central primitive neuroectodermal tumor) [9]. Deletions of SNF5 have also been reported in chronic phase and blast crisis of chronic myeloid leukemia [10]. In addition to somatic alterations, constitutive mutations of SNF5 predispose to renal and extra-renal malignant
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Oncogenes and cell proliferation
Table 1 Chromatin-remodeling complexes across species. Yeast
Drosophila
Human
SWI/SNF
RSC
BAP
BAF(a)
PBAF(a)
SWI1
–
Osa
BAF250/p270
–
hBrm complex (b)
Brg1 complex I (b)
Bgr1 complex II (b)
BAF250/p270
BAF250/p270
BAF250/p270
p220
p220
–
SWI2/SNF2
Sth1
Brm
Brm or Brg1
Brg1
Brm
Brg1
Brg1
–
Rsc1, Rsc2 and Rsc4
–
–
Polybromo/ BAF180
–
–
–
SWI3
RSC8
BAP155/Moira
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
BAF170 and BAF 155
–
–
–
p66
p66
p66
SWP73
RSC6
BAP60
BAF60a
BAF60a
BAF60a
BAF60a
–
–
–
BAP111
BAF57
BAF57
BAF57
BAF57
–
Arp7
Arp7
BAP55
β-actin
β-actin
–
–
–
Arp9
Arp9
BAP47/ACT1/ACT2
BAF53
BAF53
BAF53
BAF53
BAF53
SNF5
Sfh1
SNR1
SNF5/INI1
SNF5/INI1
SNF5/INI1
SNF5/INI1
SNF5/INI1
–
–
–
–
—
mSin3A
mSin3A
mSin3A
–
–
–
–
–
HDAC1
–
–
–
–
–
–
–
HDAC2
HDAC2
HDAC2
–
–
–
RbAp48
RbAp48
–
–
–
–
RbAp48
Three recent biochemical studies describe the purification of partly divergent human SWI/SNF complexes (a) [20•,21•] (b) [7•]. The BAF complex was described as the true homolog of the yeast SWI/SNF complex whereas PBAF seems more related to the yeast RSC complex. The ‘hBrm complex’ and the ‘Brg1 complex’ are distinguished by the presence of subunits associated with
co-repression (four last lines of the table). Note that SNF5/INI1/BAF47 are three designations for the same protein homologous to the S. cerevisiae SNF5 protein. INI1 was isolated in a two-hybrid screen as a protein interacting with the HIV integrase. BAF47 denotes a subunit of biochemically purified BAF complex and was later identified as being identical to SNF5/INI1.
rhabdoid tumor as well as CNS tumors, further supporting the hypothesis that SNF5/Ini1 is a tumor suppressor gene (TSG) [11,12].
with variable features of rhabdoid tumors [14•–16•]. Loss of the wild-type SNF5 allele in tumor DNA further demonstrated that SNF5 is a TSG. Brg1+/– mice also develop differentiated epithelial tumors [17•] but loss of heterozygosity (LOH) was not detected. In addition SNF5+/– mice develop and reproduce normally whereas 15% of Brg1 heterozygotes exhibit exencephaly, indicating that Brg1 gene dosage might be important for CNS development. Homozygous mutations, in either SNF5 or Brg1 genes are lethal at the peri-implantation stage. In vitro blastocysts outgrowth studies have shown that neither the trophectoderm nor inner cell mass (ICM) cells can survive in the absence of these proteins [14•,17•]. In contrast, the Brm knockout mice survive and do not exhibit a striking phenotype, except for an increase in body weight. At the cellular level, Brm deficiency does alter cell proliferation control, as demonstrated with primary embryonic fibroblasts in vitro and hepatocytes in vivo [18]. Nonetheless, this partial defect does not result in a tumor-prone phenotype.
Recently, the gene encoding Brg1, the catalytic subunit of the SWI/SNF complex, was found to be mutated in human tumor cell lines from various tissues including prostate, lung and breast [13]. In many cases, alteration of this gene results in loss of the expression of wild-type Brg1. Interestingly, the related Brm protein is also either downregulated or not expressed in several Brg1 mutant cell lines. These results are reminiscent of our early findings that several human tumor-derived cell lines lack Brg1 and Brm. It would be important to extend these studies to primary tumor material.
Mouse models The association of human malignancies with homozygous deletions or inactivating mutations of SNF5 and Brg1 suggested that SWI/SNF loss-of-function may contribute to oncogenesis in different cell types. The development of mouse models has provided further evidence that these critical components of the chromatin-remodeling machinery act as growth suppressors. Both SNF5 and Brg1 heterozygous mice display a cancer-prone phenotype. Several groups have shown recently that heterozygosity at the SNF5 locus predisposes mice to the development of soft-tissue sarcomas
The mild phenotype of Brm mutants compared to Brg1 and SNF5 can be accounted for by the capacity of Brg1 to replace Brm in the SWI/SNF complexes during development. In contrast to Brg1, Brm is expressed at very low levels in preimplantation embryos [19]. This could explain why the lack of Brg1 cannot be compensated by its close homolog, resulting in a strong decrease in the abundance
SWI/SNF chromatin remodeling and cancer Klochendler-Yeivin et al.
of the SWI/SNF complexes and death at this time of development. A recent biochemical study suggests an alternative explanation for the different phenotypic severity of Brm and Brg1-null mice [20•]. This study suggests that, apart from a distinguishable pattern of expression in early embryogenesis, Brg1 and Brm might not be fully functionally redundant, as previously suggested. Two distinct subfamilies of SWI/SNF-related complexes have been purified from human cells, BAF (SWI/SNF A) and PBAF (SWI/SNF B). These complexes share at least 6 common subunits (including SNF5) and display similar nucleosome disruption activity in vitro, yet they diverge structurally. BAF250 and BAF180 (a polybromoprotein) are unique subunits of BAF and PBAF, respectively. Furthermore, whereas BAF can contain either Brm or Brg1, Brg1 is the sole catalytic subunit of PBAF ([20•,21•]; Table 1). PBAF may regulate a distinct subset of genes and/or mediate specific biological functions required for proper growth and development. The severe phenotype of Brg1 knockout mice might therefore reflect the disruption of PBAF-specific functions. In addition, two recent biochemical studies bring evidence for the existence of additional Brg1 and Brm complexes that contain subunits of the mammalian mSin3A complex, including histone deacetylases (HDACs). These Brg1 and Brm complexes diverge in composition and display different chromatin-remodeling activities ([6,7•]; Table 1). We cannot rule out that the severe phenotype of Brg1 knockout mice might therefore reflect the disruption of specific Brg1-containing complexes.
Mechanism of SWI/SNF function in tumor suppression The Rb connection
Early transfection studies (e.g. [22]) demonstrated that Brg1 and Brm can associate with the retinoblastoma protein (pRB) to induce growth arrest. More recently, LKB1, a serine-threonine kinase mutated in patients with Peutz–Jeghers Syndrome was found to interact with Brg1 and its kinase activity is necessary for Brg1/Rb-dependent cell-cycle arrest [23]. The Rb TSG plays a fundamental role in cell-cycle control, apoptosis and development. A major cellular target of pRB is the E2F family of transcription factors that stimulate expression of genes required for G1 → S progression. pRB inhibits gene expression via at least two mechanisms: first it binds to the E2F family of transcription factors, blocking their transactivation potential. In addition, pRB–E2F actively inhibits transcription by recruiting HDACs and/or the SUV39H1 histone H3 methylase and the methyl-binding protein HP1 to generate a repressive chromatin conformation [24••,25]. Several studies have suggested a role for SWI/SNF in pRB inhibition of E2F transactivation. A genetic screen for modifiers of an E2F overexpression phenotype in the Drosophila eye identified enhancer mutations in Osa, Brahma and Moira genes, which encode homologs of SWI1, SWI2 and SWI3, respectively, indicating that the SWI/SNF complex downregulates E2F activity [26]. Results from transfection
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assays in cultured cells also support a role for chromatinremodeling enzymes in active repression by the pRB–E2F complex [27]. Overexpression of Brg1 in cells deficient in Brg1 and Brm induces growth arrest if functional pRB is present [22]. Similarly, expression of a dominant negative Brg1 in proliferating NIH3T3 reverses Rb or p16(INK4a)mediated cell-cycle arrest [28]. Transfection studies indicate that class I HDACs and SWI/SNF are recruited by pRB and may coordinate the sequential expression of E2F target genes to regulate progression through G1 and S phases [29••]. It has been suggested that a trimeric complex pRB–HDAC–SWI/SNF inhibits Cyclin E expression in quiescent and early G1 cells. As cells progress toward S phase, active Cyclin D–Cdk4 phosphorylates pRB, causing the dissociation of HDAC, but leaving the RB–SWI/SNF interaction unaffected. This repressor complex inhibits Cyclin A and Cdc2 but allows accumulation of active Cyclin E–Cdk2, which, in turn, phosphorylates pRB and/or Brg1. These modifications are suggested to disrupt the repressing complex, promoting cyclin A and cdc2 transcription allowing progression into G2/M phase. This model is compatible with a previous study showing that Brg1 and BAF155 (SWI3) associate with Cyclin E and are potential substrates of Cyclin E–Cdk2 kinase activity in vitro [30]. The in vivo relevance of this activity, and its effect on pRB–Brg1 association and function, however, has not been established. Moreover, considering that the expression levels of the different cellcycle regulators must be finely balanced to ensure proper signaling, it will be important to examine the assembly or dissociation of pRB–SWI/SNF complexes and their recruitment to endogenous promoters during the cell cycle in a physiological context. Functional SWI/SNF is not absolutely necessary for cell-cycle arrest to occur. The expression of a dominant negative mutant form of Brm or Brg1 in NIH3T3 cells prevents MyoD-induced muscle cell differentiation [31••]. Experiments to elucidate whether SWI/SNF is required for MyoD-mediated cell-cycle withdrawal and muscle-specific gene activation, revealed that SWI/SNF is crucial to induce muscle-specific genes but dispensable for cell-cycle arrest [32••]. Moreover, SWI/SNF is neither required for MyoDmediated induction of p21, Cyclin D3 and pRb, nor for repression of Cyclin A. Under culture conditions used for differentiation (confluency and serum-starvation) both mockdifferentiated and MyoD-differentiated cells arrested in G1, regardless of whether functional SWI/SNF complexes were present. This cell-cycle withdrawal may be mediated by p27kip1, which is expressed at high levels in all cells. Beyond pRB
Although the studies described above provide support for a model in which SWI/SNF collaborates with pRB to inhibit cell proliferation, other studies show that SWI/SNF may participate in additional growth-signaling pathways. Control of transcriptional activity of growth regulators
Recently, components of the SWI/SNF complex have been shown to associate with BRCA1, a tumor suppressor linked
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Oncogenes and cell proliferation
Figure 1 (a) Transcriptional repression
(b) Transcriptional activation
SWI/SNF SWI/SNF TCF Rpd3
β-cat TCF
Gro Repression of Wg-target genes in Drosophila
Activation of Wg/Wnt target genes in Drosophila and mammals
Growth arrest?
Cell proliferation? Current Opinion in Genetics & Development
to familial breast and ovarian cancers. Moreover, transient transfection assays suggest that the ability of BRCA1 to act as a coactivator of p53-dependent transcription is mediated through Brg1 [33]. Among the human tumor cell lines screened for Brg1-inactivating mutations, 2 out of 22 breast carcinoma cell lines displayed biallelic mutations resulting in loss of wild-type protein expression [13]. Furthermore, re-introduction of Brg1 into one of the cell lines (ALAB) induced G1 arrest. It is worth noting that Brg1 deficiency in ALAB cells correlates with a defective pRB pathway as shown previously with SW13 and C33a cells, whereas alterations in BRCA1 activity have not been examined. The SWI/SNF complexes may also regulate either the synthesis or the stability of growth inhibitors. Confluent cultures of mouse embryonic fibroblasts from Brm–/– mice accumulate less p27kip1 protein than their wild-type counterparts [18]. Ras-transformation of rodent cells was shown to downregulate the synthesis of Brm. And re-expression of ectopic Brm in these cells reversed their transformed phenotype [34]. These studies suggest that some SWI/SNF activities can antagonize Ras transformation, perhaps by controlling the pRB/E2F checkpoint, and that they in turn are downregulated by oncogenic Ras. c-myc, a proto-oncogene involved in cell proliferation, differentiation and apoptosis might also require SWI/SNF for its transcriptional activity. SNF5 has been reported to interact with c-Myc and co-expression of a dominantnegative mutant of BRG1 suppressed reporter gene activation by Myc in a transient transfection assay [35]. Consistent with SNF5 tumor suppressor activity, it has been suggested that c-Myc might recruit SWI/SNF and facilitate transcription of a subset of target genes, specifically those involved in apoptosis. Whether c-Myc associates with the intact SWI/SNF complex and directs chromatin remodeling at distinct endogenous promoters remains to be demonstrated. Genetic data in Drosophila provide evidence that Brm complexes modulate target gene expression by the
Antagonistic functions of SWI/SNF in the transcriptional regulation of the Wingless (Wg)/Wnt pathway. (a) Genetic interactions in Drosophila indicate that in the absence of β-catenin, the Brm complex cooperates with TCF and Groucho (Gro) to repress Wingless target genes. (b) Following Wingless/Wnt signaling, β-catenin accumulates in the nucleus, interacts with Brg1, promoting transcriptional activation of TCF target genes. Of note, the SWI/SNF complex participating in either reaction may have a specific subunit composition that facilitates interaction with co-repressors or co-activators and/or directs opening or closing of the chromatin structure.
Wingless/Armadillo pathway. Wingless signaling triggers the stabilization of Armadillo which translocates to the nucleus and binds to dTCF (Drosophila T-cell factor) to stimulate transcription of Wingless target genes. Vertebrate homologs of all Wingless pathway genes have been identified and their function is known to be highly conserved. Deregulation of β-catenin (the vertebrate homolog of Armadillo) results in increased transcription of TCF target genes such as myc or cyclin D1, and malignant transformation of the mammalian gut epithelium (reviewed in [36]). In the absence of Wingless signaling in Drosophila, dTCF target genes are actively repressed by recruitment of negative regulators such as Groucho and Rpd3 HDAC that associate with dTCF [37]. One report [38••] has demonstrated genetic interaction between the Wingless pathway and components of the Brm complex (Osa, Moira and Brahma), as well as strong genetic interaction between Osa and Groucho, and suggested that the Brm complex is required for the repression of dTCF target genes in the absence of Wingless signaling. If this function is conserved in vertebrates and SWI/SNF is required to ensure effective repression of TCF target gene activity in the absence of β-catenin, it might provide a novel mechanism by which this chromatin-remodeling complex prevents tumor growth. Paradoxically, a second study [39••] has shown that Brg1 interacts with β-catenin and promotes TCF target gene activity in mammalian cells. Genetic studies in Drosophila also support a role for the Brm complex in the stimulation of β-catenin–TCF transcriptional activity [39••]. These two studies imply that, depending on the upstream acting signal, the SWI/SNF complexes might function in both activation and repression of the same set of genes, resulting in either positive or negative control of cell proliferation. A putative oncogenic activity of SWI/SNF is suggested by its association with c-Fos and c-Jun through BAF60a [40]. This association has been shown to potentiate the transactivating activity of the c-Fos/c-JunAP1 heterodimer.
SWI/SNF chromatin remodeling and cancer Klochendler-Yeivin et al.
Transcriptional regulation of factors involved in tumorigenesis
The proto-oncogene c-fos promoter has been shown to be repressed following ectopic expression of Brg1. This repression requires a functional ATP-binding site and depends on Rb but not on E2F activity [41]. CD44, a transmembrane glycoprotein whose deregulation contributes to cancer progression, is downregulated in cell lines lacking Brg1. Furthermore, ectopic expression of functional Brg1 restores CD44 expression [42]. These studies, however, do not demonstrate clearly whether and how the SWI/SNF complex is recruited to the c-fos and CD44 promoters. Moreover, c-fos repression could be an indirect consequence of Brg1/pRb-mediated growth arrest.
Conclusions Genetic studies in human and mouse have shown that inactivating mutations or deletions of genes encoding subunits of the SWI/SNF complex are associated with cancer, qualifying these genes as putative tumor suppressors. Although there has been much focus on the role of the pRB pathway, biochemical and molecular studies have revealed an increasing number of potential targets of the SWI/SNF complex that function as either positive or negative regulators of cell growth. Thus, the SWI/SNF complex can be considered as an integrator of multiple signaling networks. Taken together, these studies support a role for SWI/SNF in transcriptional regulation, via its interaction with either activator or repressor proteins, some of which may function within the same pathway (Figure 1). In the future, it will be crucial to investigate whether the SWI/SNF complex participates in other molecular processes, such as DNA repair, recombination and replication or chromosome segregation in which chromatin structure plays an important role. Disruption of such functions could contribute to malignant transformation. In this context, the SNF5 subunit has been shown to stimulate replication of the human papillomavirus, and the SWI/SNF complex to increase the efficiency of DNA replication in yeast [43,44]. Moreover, SWI/SNF has been observed to stimulate RAG1 and RAG2 mediated V(D)J recombination in vitro [45•]. Furthermore the initial isolation of SNF5/INI1 as a protein interacting with and stimulating the HIV integrase [46] and the recent report on the association of SNF5 with the incoming viral particle [47] support a role for at least SNF5 in recombination. The association of BRCA1 with SWI/SNF could imply a role for the chromatin-remodeling complex in DNA repair. The S. cerevisiae RSC complex (the second SWI/SNF-like complex in yeast), or its putative human homolog, may also be important for chromosome segregation. It was suggested that RSC is involved in centromere remodeling during G2/M [48] and some rsc3 mutant alleles trigger G2/M arrest and cell death [49]. Such a role is further supported by the association of the PBAF complex with mammalian kinetochores [20•]. There are several additional questions about SWI/SNF function that are yet to be resolved. Different cells appear
77
to have significantly divergent responses to SWI/SNF inactivation. Neither ICM nor trophectoderm cells derived from Brg1 or SNF5-null blastocysts survive in culture. Likewise, a Brg1-null mutation in F9 embryonic carcinoma cells resulted in death [50]. Yet, some cells in mice and humans become transformed upon SNF5 inactivation, and several human tumor cell lines lacking Brg1 survive and proliferate normally. As SWI/SNF has a role in Rb inhibition of E2F-dependent transactivation, it is possible that loss of SWI/SNF leads to release of free E2F that can trigger apoptosis by activating the p53-inhibitor p19ARF in many cell types [51]. It will be of great interest to analyze the status of the ARF–p53 pathway in SWI/SNF-deficient tumor cells. Another issue concerns the difference between human and mice. Children with a constitutive germline mutation are afflicted by a cancer predisposition syndrome with high penetrance before the age of 3. Furthermore, the low number of other chromosomal rearrangements outside of the SNF5 locus suggests a major role for this alteration. In contrast, only 30–40% of SNF5+/– mice develop tumors by the age of 12–15 months, suggesting that this pathway may be less crucial in rodents and that tumor formation in this case requires additional mutations.
Acknowledgements We apologize to those whose work was not cited directly because of space constraints. We thank Jonathan Weitzman and Olivier Delattre for critical reading and advice on the manuscript. We thank the AICR Foundation, ARC and LNFCC for financial support.
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