When the SWI/SNF Complex Remodels . . . the Cell Cycle

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

Christian Muchardt*,1 and Moshe Yaniv1

1UniteÂ des Virus OncogeÁnes, URA1644 du CNRS, DeÂpartement des Biotechnologies, Institut Pasteur, Paris, France

Mammalian cells contain several chromatin-remodeling particle. The exact eect of this modi®cation is still complexes associated with the Brm and Brg1 helicase- unclear but it is likely that the loss of positive charges like proteins. These complexes likely represent the modify interactions between the histone tails and the functional homologs of the SWI/SNF and RSC DNA or the neighboring nucleosomes. The reaction complexes found in Saccharomyces cerevisiae. The can be reversed by histone deacetylases (HDACs) that mammalian chromatin-remodeling complexes are in- have a negative eect on transcriptional activity. The volved in both activation and repression of a variety of second category of factors that aect chromatin genes. Several lines of evidence also indicate that they encompasses protein complexes that use the energy of play a speci®c role in the regulation of cell growth. Brm ATP-hydrolysis to weaken the interaction between is down-regulated by ras signaling and its forced re- histone core particles and DNA. These complexes expression suppresses transformation by this oncogene. facilitate the binding of transcription factors to speci®c Besides, the Brg1 gene is silenced or mutated in several DNA sites and may thus mediate both activation and tumors cell lines and a Brg1-associated complex was repression, depending on the activity of the recruited recently found to co-purify with BRCA1, involved in transcriptional regulator. 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 SWI/SNF complexes in Saccharomyces cerevisiae very aggressive form of pediatric cancer. The current review will address observations made upon inactivation The prototype of the chromatin-remodeling complexes of Brm, Brg1 and SNF5/Ini1 by homologous recombina- is the SWI/SNF complex. This complex of 11 subunits tion in the mouse, as well as the possible implication of was initially characterized in S. cerevisiae through these factors in the regulation of the Retinoblastoma genetic and biochemical studies. Its chromatin-remo- pRb-mediated repression of the transcription factor E2F. deling activity relies on the SWI2/SNF2 subunit that Oncogene (2001) 20, 3067 ± 3075. contains a large helicase-like domain functioning as a DNA-dependent ATPase. Mutations in any of the swi/ Keywords: N-CoR; HDAC; Cyclin A; Cyclin E; G0 snf genes give rise to various phenotypes, including poor growth, the inability to use speci®c carbon sources and defects in mating type switching. However, these genes are not required for viability. Whole Introduction genome expression analysis has shown that the SWI/ SNF complex is required for normal expression of only Within the nucleus of an eucaryotic cell, DNA is 3 to 6% of all the S. cerevisiae genes, depending on the wrapped around the histone octamers composed of two study (Holstege et al., 1998; Sudarsanam et al., 2000). copies of each of the histones H2A, H2B, H3 and H4. About half of the genes aected more then threefold in These histone-DNA complexes known as the nucleo- swi/snf mutant strains are upregulated rather than somes form a potent obstacle for biological processes downregulated, indicating that the SWI/SNF complex requiring access to the DNA, like transcription, is also involved in transcriptional repression. It must replication or DNA repair. The cellular machineries also be noted that the genes regulated by SWI/SNF are allowing transcription factors to gain access to their scattered all over the genome, suggesting that the target promoters have been extensively studied in the complex aects chromatin structure locally rather than last decade. These machineries can be divided in two remodeling larger chromosomal domains (Sudarsanam categories: the histone acetylases and the chromatin et al., 2000). remodeling complexes. The histone acetylases or HATs S. cerevisiae expresses several other helicase-like add acetyl groups to the N-terminal tails of the proteins related to the catalytic subunit of the SWI/ histones that protrude out of the nucleosome core SNF complex. Among these proteins, Sth1p has the highest degree of homology with SWI2/SNF2 and it is associated with a large complex containing at least four other proteins homologous to SWI/SNF subunits *Correspondence: C Muchardt, UniteÂ des Virus OncogeÁ nes, DeÂ partement des Biotechnologies, Institut Pasteur, 25, rue du (Table 1). In addition, this complex, known as RSC Docteur Roux, 75724 Paris Cedex 15, France (for Remodel the Structure of Chromatin) contains two Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3068 Table 1 SWI/SNF-related complexes in yeast, Drosophila and human Yeast Drosophila Human SWI/SNF RSC BAP 1 BAF 2 PBAF 3 N-CoR 4

SWI2/SNF2 Sth1 5 Brahma 6 Brm or Brg1 7 Brg1 Brg1 SNF5 Sfh1 8 SNR1 9 hSNF5/INI1 10 hSNF5/INI1 hSNF5/INI1 SNF6 SNF11 SWI1 Osa 11 BAF250/p270 12 SWI3 RSC8 BAP155/Moira BAF170 and BAF155 BAF170 and BAF155 BAF170 and BAF155 SWP82 SWP73 RSC6 BAP60 BAF60a BAF60a SWP29/TFG-3/TAF30 SAS-5 Arp7 Arp7 BAP55 b-actin 13 b-actin Arp9 Arp9 BAP47/ACT1/ACT2 BAF53 BAF53 BAP111 BAF57 BAF57 Rsc1, Rsc2 and Rsc4 14 Polybromo/BAF180 15 TIF1b/KAP-1 HDAC3

1(Papoulas et al., 1998), 2(Kwon et al., 1994; Nie et al., 2000), 3(Kwon et al., 1994; Xue et al., 2000), 4(Underhill et al., 2000), 5(Du et al., 1998; Tsuchiya et al., 1998), 6(Tamkun et al., 1992), 7(Chiba et al., 1994; Khavari et al., 1993; Muchardt and Yaniv, 1993), 8(Cao et al., 1997), 9(Dingwall et al., 1995), 10(Kalpana et al., 1994; Muchardt et al., 1995), 11(Kal et al., 2000), 12(Nie et al., 2000), 13(Zhao et al., 1998), 14(Cairns et al., 1999), 15(Xue et al., 2000)

actin-related subunits (Arp7p and Arp9p) also present dominant suppressors of Polycomb mutations (Tam- in the SWI/SNF complex. Several dierences exist kun et al., 1992). Subsequent biochemical studies however between the RSC and the SWI/SNF complex. showed that the protein is associated with a complex RSC is essential for growth and about 10-fold more very similar to the yeast SWI/SNF complex, containing abundant than the SWI/SNF complex. RSC function is at least six subunits related to SWI/SNF subunits required for normal cell cycle progression (Cao et al., (Dingwall et al., 1995; Papoulas et al., 1998). Another 1997; Du et al., 1998; Laurent et al., 1992) but the SWI2/SNF2-related protein, known as ISWI, has been involvement of the RSC complex in RNA Polymerase found associated with dierent chromatin remodeling II transcription has been somewhat controversial. complexes (Ito et al., 1997; Tsukiyama and Wu, 1995; Unlike most SWI/SNF proteins, RSC subunits do Varga-Weisz et al., 1995). However, it is clear that only not activate transcription when tethered to DNA the Brahma-associated (or BAP) complex has a through a heterologous DNA-binding domain (Sigrid composition and an activity closely related to yeast Schaper and Moshe Yaniv, unpublished observation). SWI/SNF (Kal et al., 2000). Besides, at non-permissive temperatures, a ts mutation In mammalian cells, the situation is far more in Sth1 induces alterations of centromeric chromatin complicated. These cells contain at least two proteins structure and perturbs chromosome segregation (Tsu- closely related to SWI2/SNF2, known as Brm and chiya et al., 1998). Other observations do however Brg1 (or SNF2a and SNF2b, respectively). In addition, connect the RSC complex with transcription. Some rsc biochemical studies have identi®ed dierent variants of mutations aect the expression of the CHA1 gene as the mammalian SWI/SNF complex that all contain well as certain genes involved in early meiosis (Moreira subunits related to several yeast SWI/SNF proteins and Holmberg, 1999; Yukawa et al., 1999). The RSC (Table 1). The SWI/SNF complexes elute in two complex also appears to cooperate with histone distinct fractions during puri®cation. The ®rst fraction acetylases. Indeed, the rsc1 and rsc2 genes code for contains two dierent complexes associated with either similar proteins and only mutation of both genes is Brm or Brg1, respectively. Because of their similar lethal. However a rsc1 or a rsc2 mutation, in biochemical properties, they are both referred to as the combination with mutations that impair the SAGA BAF or the SWI/SNF-A complex. They are character- histone acetylase complex, either cause lethality or ized by the presence of BAF250, a low speci®city strong mutant phenotypes. This clearly suggests a role DNA-binding subunit with homology to yeast SWI1 for the RSC complex in transcription regulation and Drosophila OSA. A subfraction of the BAF through chromatin remodeling (Cairns et al., 1999). complexes apparently also contains the tumor suppressor protein BRCA1 (Bochar et al., 2000). The second chromatographic fraction contains a complex called SWI/SNF complexes in higher eucaryotes PBAF or SWI/SNF-B. This complex is apparently associated only with Brg1 and not Brm. It also SWI/SNF proteins have been identi®ed in several contains the Polybromo protein that is not present in species other than yeast, including Arabidopsis, Droso- the BAF complex. Polybromo, also called BAF180, phila, C. elegans, Xenopus, chick, mouse and human. In contains six successive Bromodomains and shares some Drosophila, the closest relative of SWI2/SNF2 is homology with three subunits of the yeast RSC Brahma. It was initially isolated in a screen for complex, Rsc1, Rsc2 and Rsc4, both within and

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3069 ¯anking the Bromodomains (Cairns et al., 1999; up-regulation of the Brm gene (CM, unpublished Callebaut et al., 1999). The presence of Polybromo in observation). the PBAF complex suggests that PBAF may in fact Inactivation of the mouse Brm gene by homologous correspond to the yeast RSC complex. On the other recombination has con®rmed that it is dispensable for hand, only the bona ®de yeast SWI/SNF complex viability. Brm nullizygous mice are born at normal contains a SWI1 subunit. Therefore, the BAF250- mendelian ratios. They are viable and fertile. Several associated BAP complex may be the true mammalian observations suggest, however, a moderate deregula- SWI/SNF complex (Nie et al., 2000; Xue et al., 2000). tion in the control of cell growth. First, mutant Recently, subunits of the BAF/PBAF complexes have animals exhibit a 10 to 15% increase in body mass, been found in a fourth complex named N-CoR, for relative to their wild-type littermates. Increased cell `Nuclear Receptor CoRepressor'. Within this complex, proliferation was also observed in the liver, and Brg1, Baf170, Baf155 and SNF5/INI1 are associated embryonic ®broblasts isolated from the mutant animals with the histone deacetylase HDAC3 and the co- failed to show proper growth arrest at con¯uency repressor Kap1/TIF1b (Underhill et al., 2000). In (Reyes et al., 1998). Interestingly, the Brm knockout lymphoid cells, SWI/SNF subunits are also associated mice showed a 2 ± 6-fold increase in the expression of with the transcriptional regulator Ikaros required for Brg1 in several tissues. It is likely that this upregulated normal B and T cell dierentiation (Kim et al., 1999; Brg1 expression compensates for the lack of Brm. O'Neill et al., 2000). Similarly, erythroid cells contain a Inactivation of the Brg1 gene is lethal at a very early SWI/SNF-like complex associated with EKLF, a key stage and embryos die before implantation. Explanted regulator of b-globin gene expression (Armstrong et blastocytes carrying the Brg1 null mutation fail to al., 1998; Kadam et al., 2000). This wealth of SWI/ hatch from their zona pellucidae and no cellular SNF complexes suggests that in mammals, some SWI/ proliferation is observed. Heterozygote Brg1 mutants SNF sub-complexes are dedicated to either activation are under-represented at birth and about 15 to 30% of or repression of speci®c pathways, possibly with a these mutants show signs of exencephaly in utero. After restricted tissue-distribution. birth, heterozygotes are viable and fertile but around 16 months of age, a fraction of these mice display large subcutaneous tumors localized to the neck or ingineal Inactivation of Brm and Brg1 in mammalian cells regions (Bultman et al., 2000). The tumors appearing in the Brg1 heterozygous mice have apparently not lost Early studies have shown that two human cell lines, the second Brg1 allele. Taken together with the C33A and SW13, contain either very low or exencephaly observed on the heterozygous embryos, undetectable levels of both Brm and Brg1 (Muchardt this suggests that the dosage of the Brg1 protein is and Yaniv, 1993). Systematic screening has further important for proper function. identi®ed at least half a dozen human tumor cell Given the high degree of homology between Brg1 lines carrying truncating mutations in the Brg1 gene and Brm (86% similar and 75% identical), the very on both alleles (Wong et al., 2000). All of these lines dierent phenotypes of the respective null mutants are have lost Brg1 expression entirely and in most cases, rather surprising. As mentioned above, the PBAF Brm is either present at reduced levels or absent version of the mammalian SWI/SNF complex may (Table 2). The Brg1 gene can also be inactivated in only contain Brg1 whereas the BAF complex can primary mouse embryo ®broblasts without aecting contain either Brg1 or Brm. If this observation is cell viability (Bultman et al., 2000). In mouse correct, the PBAF complex is inactivated in Brg1 null embryonic ES and F9 cells, the Brm protein is low mutants whereas the BAF complex will still be able to or absent and its expression is only induced upon form with Brm. It has therefore been suggested that the dierentiation. Finally, many murine cell lines lose PBAF complex is more important for cell viability than Brm expression upon transformation (LeGouy et al., the BAF complex and that the absence of this PBAF 1998; Muchardt et al., 1998). From these observa- complex could be responsible for the early lethality tions, it appears clearly that expression of Brm is induced by Brg1 inactivation (Xue et al., 2000). This cell-type and transformation-dependent and that situation would mimic the situation in S. cerevisiae neither Brm nor Brg1 are required at all times. The where the RSC complex is required for viability existence of cell lines apparently missing both Brm whereas the SWI/SNF complex is dispensable. Alter- and Brg1 further suggests that in speci®c cases, SWI/ natively, Brg1 and Brm may potentially be able to SNF activity is dispensable in mammalian cells. This compensate for each other, but may in some cases be may however not be a general rule. Inactivation of prevented from doing so because of restricted expres- one allele of the Brg1 gene by homologous sion in time and in space. In fact, it was shown that recombination in F9 cells severely aects prolifera- levels of Brm increase gradually during mouse tion, and a homozygous deletion of the gene in these development to reach a maximum in adult post-mitotic cells is lethal (Sumi-Ichinose et al., 1997). We have tissues. Brg1, on the other hand, is apparently present also noted that the human ovary carcinoma cell line at similar levels throughout development (LeGouy et OV-1063 that does not express Brg1, contains levels al., 1998; Reyes et al., 1998). Likewise, in tissue culture of Brm 2 ± 3-fold higher than average, suggesting that mouse ®broblasts, Brg1 protein levels appear stable in some cases, loss of Brg1 must be compensated by through the cell cycle, whereas Brm is only present at

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3070 Table 2 Cell lines aected in the expression of at least one SWI/SNF subunit. Unless otherwise indicated, all data are based on detection of the proteins by Western blotting. When nothing is indicated, the expression of the protein has not been tested Cell line Origin Brm Brg1 SNF5/INI1 BAF250 Polybromo BAF53 BAF155 Reference

Human OV-1063 Ovary High Absent Present 6 NIH-OVCAR3 Ovary Present Monoallelic 5,6 mutation T47D Breast Absent Present 1 ALAB Breast Absent Absent Present Present 5 Hs578T Breast Absent 5 PANC-1 Pancreas Very low to 4 absent Su86.86 Pancreas Monoallelic 5 mutation Hs700T Pancreas Present Absent Present Present 5 TSU-Pr1 Prostate Absent Absent Present Present 5 DU145 Prostate Low Absent Present Low 5 NCI-H1299 Lung Low Absent Present Present 5 A-427 Lung Absent Absent Present Absent 5 Calu-1 Lung Low Present 6 HCT-15 Colon Monoallelic 5 mutation WiDr Colon Monoallelic 5 mutation EJ-35 Bladder Absent Very low and 6 shorter T24 Bladder Low Low 6 C33A Cervix Very low to absent Absent Present Present 5,7,8 SW13 Adrenal Absent Absent Present Present 4,5 SNU-C2B Cecum Monoallelic 5 mutation Hs683 Brain Monoallelic 5 mutation 5 A204 Rhabdomyosarcoma Present Absent Present 3 G401 Rhabdoid Absent Present Absent Present Present 2,3,6 TM87-16 Rhabdoid Present Absent Present 2 TTC549 Rhabdoid Present Absent Present 3 TTC642 Rhabdoid Present Absent Present 3 TTC709 Rhabdoid Present Absent Present 3 TTC1240 Rhabdoid Present Absent Present 3 DL Rhabdoid Absent Present Absent Present 2,6 LP Rhabdoid Absent Present Absent Present 2,6 WT Rhabdoid Biallelic 2 mutation MON Rhabdoid Absent 2 TM Rhabdoid Absent 2 2004 Rhabdoid Biallelic 2 mutation KD Rhabdoid Absent Present Biallelic Present 2,6 mutation LM Rhabdoid Absent 2 Wa2 Rhabdoid Absent Present Biallelic Present 2,6 mutation AS Rhabdoid Biallelic 2 mutation Mouse ES Mouse embryonic stem Low or absent 9±11 depending on the source F9 Teratocarcinoma Low or absent 9,12 depending on the source DT Mouse fibroblast Absent Present Present 9 transformed by ras EHneo Fibroblasts Absent Present 9 transformed by raf MCO-1 Fibrosarcoma Absent Present 9 Rat PYF3.2 Fibroblasts transformed Absent Present 9 by Polyoma middle T

1(Nie et al., 2000), 2(Versteege et al., 1998), 3(DeCristofaro et al., 1999), 4(Strobeck et al., 2000a), 5(Wong et al., 2000), 6(B Bourachot and C Muchardt, unpublished observations), 7(Muchardt and Yaniv, 1993), 8(Dunaief et al., 1994), 9(Muchardt et al., 1998), 10(LeGouy et al., 1998), 11(Bultman et al., 2000), 12(Sumi-Ichinose et al., 1997)

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3071 high levels in growth arrested cells (Muchardt et al., Many of these observations were con®rmed by a 1998). It is therefore possible that Brg1 plays the second study (Roberts et al., 2000). In this case, it principal role in SWI/SNF activity during development was noted that a majority of the tumors were found in and cannot be replaced by Brm because of its structures derived from the ®rst bronchial arch. In the insucient expression. Studies of the early mouse two studies, a large fraction of the tumors showed embryo also suggest that timing of the Brg1 and Brm similarities with human rhabdoid sarcomas although expression could explain the early lethality induced by the onset of the tumorigenesis was clearly delayed in the Brg1 null mutation. Indeed, the oocyte contains mice compared to human. Besides, in humans, the both Brg1 and Brm of maternal origin. However, at `rhabdoid predisposition syndrome' caused by consti- the 4-cell stage, when zygotic transcription starts, the tutional SNF5/INI1 mutations has a very high Brg1 gene is clearly expressed whereas Brm mRNA is penetrance (SeÂ venet et al., 1999b), whereas only one- almost undetectable. Some Brm mRNA is detected at third of the heterozygous mice develop tumors. The the 8-cell stage but abundant transcription only starts molecular events causing these dierences are still at the blastocyst stage (Bultman et al., 2000; LeGouy et unknown. al., 1998). It therefore appears that the Brg1 nullizygote mice go through a short window of time when they are missing both Brg1 and Brm. The Why does the SWI/SNF complex aect cell growth? absence of a functional SWI/SNF complex at the The pRb connection onset of zygotic transcription may lead to defective reprogramming of the embryonic chromatin, ultimately As described above, many observations made in both leading to death before implantation. cell lines and knockout animals suggest that the mammalian SWI/SNF complex is linked to the control of cell proliferation. As we will see, there are several Inactivation of the SNF5/INI1 gene possible explanations for this connection, most likely re¯ecting the large number of genes transcriptionally One subunit of the mammalian SWI/SNF complexes regulated by the SWI/SNF complex. The best has been clearly demonstrated to function as a tumor documented link between members of the SWI/SNF suppressor. This protein known as SNF5/INI1 or complex and regulators of the cell cycle, concerns the BAF47 is a homolog of S. cerevisiae SNF5 (Kalpana interaction between Brm or Brg1 and the Retinoblas- et al., 1994; Muchardt et al., 1995; Wang et al., 1996). toma protein pRb. This interaction was initially It is present in all versions of the mammalian SWI/ discovered in yeast two-hybrid screen using pRb as a SNF complex puri®ed to date but has also been shown bait (Dunaief et al., 1994). Association of pRb with to be involved in HIV integration (Kalpana et al., Brm has also been described, as well as interaction of 1994; Morozov et al., 1998; Parissi et al., 2000). other Rb family members with both Brm and Brg1 Recently, biallelic mutations in the SNF5/INI1 gene (Fryer and Archer, 1998; Singh et al., 1995; Strober et were shown to be the causative event leading to al., 1996; Trouche et al., 1997). The fragment of Brg1 malignant rhabdoid sarcomas, a very aggressive form encoded by the partial cDNA isolated in the initial of pediatric cancer (Versteege et al., 1998). Mutations two-hybrid screen, contained an LXCXE motif. This in the SNF5/INI1 gene are also found in choroid motif is known to mediate interaction of the `pocket' plexus carcinomas and in a subset of central primitive domain of pRb with Adenovirus E1a, SV40 large T neuroectodermal tumors, meduloblastomas and pri- and Papilloma E7 as well as several cellular proteins, mary rhabdomyosarcomas (DeCristofaro et al., 1999; such as the histone deacetylases HDAC1 and HDAC2. SeÂ venet et al., 1999a,b). In all cases tested, mutations However, later studies using Brg1 derivatives carrying in the SNF5/INI1 gene compromise expression of the point mutations in the LXCXE motif have shown the SNF5/INI1 protein and re-introduction of SNF5/INI1 integrity of this motif to be dispensable for interaction protein into defective cell lines causes growth arrest (O with Brm or Brg1 (Dahiya et al., 2000; Zhang et al., Delattre, personal communication). 2000). Regions neighboring the LXCXE motif also Inactivation of the mouse SNF5/INI1 gene by show homology to Adenovirus E1a and may be the homologous recombination is lethal at a very early real site of pRb interaction (Trouche et al., 1997). stage. No homozygous mutants are detected at 6.5 The pRb protein is one of the major cell cycle days p.c and explanted blastocysts either fail to hatch regulators that controls the G1/S transition as well as from their zona pellucida or do not proliferate properly progression through S phase. In early G1, when the after hatching. These observations suggest that like protein is hypophosphorylated, it associates with the Brg1 null mutants, SNF5/INI1 nullizygotes die at a transcription factor E2F and recruits histone deacety- peri-implantation stage. Heterozygous SNF5/INI1 lases. These events cause the repression of several E2F- mutants show, like in human, increased susceptibility dependent genes involved in cell cycle progression, like to an early onset of cancer. In our study, 32% of the Cyclin A and Cyclin E. In late G1, accumulation of heterozygous animals developed tumors by the age of Cyclin D/Cdk4/6 complexes results in phosphorylation 15 months. These tumors were detected at dierent of the pRb pocket. These phosphorylation events lead locations but were most frequent in the nervous system to the displacement of the HDAC activities and and in soft tissues (Klochendler-Yeivin et al., 2000). ultimately to the release and the activation of E2F

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3072 (Harbour et al., 1999). Several functional assays have What is the function of the SWI/SNF complex when demonstrated an implication of Brg1 and Brm in the associated with pRb and E2F? control exerted by pRb on the G1/S transition. Transfection experiments have shown a cooperation Like down-regulation of Drosophila Cyclin E, over- between Brm and pRb in the repression of E2F- expression of dE2F and the associated dDP disrupt dependent transcriptional activation (Trouche et al., the patterning of the Drosophila eye. In ¯ies over- 1997). Furthermore, several studies have shown a expressing these two proteins, the `rough eye' cooperation between pRb and Brm or Brg1 for phenotype can be compensated by overexpression of induction of growth arrest. In SW13 cells that express the Drosophila pRb homolog RBF. Interestingly, the neither Brm nor Brg1, but contain a wild type pRb, phenotype can also be enhanced by `loss of function' overexpression of either of the two SWI/SNF proteins mutations in three genes encoding members of the leads to the formation of ¯at growth arrested cells. Drosophila SWI/SNF complex, namely Brahma, This eect is inhibited by co-transfection of Adeno- Moira and Osa (Staehling-Hampton et al., 1999). virus E1a (Dunaief et al., 1994; Strober et al., 1996). This observation indicates that the SWI/SNF complex Cell lines lacking Brm and Brg1 are also resistant to is required for E2F activation and not just for growth arrest induced by a constitutively active pRb regulation of pRb mediated repression. It is therefore that cannot be phosphorylated. This resistance can be possible that in early G1, the SWI/SNF complex overcome by re-introducing Brg1 into the cells facilitates the binding of E2F to its target promoters (Strobeck et al., 2000a,b). One study based on pRb by increasing their accessibility. The simultaneous overexpression in tumor cell lines also suggests that the recruitment of pRb and the associated histone mammalian SWI/SNF complex is necessary for the deacetylase activities may then prevent the binding orderly expression of Cyclin E and Cyclin A during of other transcription factors to the promoters. It has late G1 and S (Zhang et al., 2000). As mentioned been demonstrated in vitro that the SWI/SNF complex above, HDACs interact with the pRb pocket via their can catalyze the opening of the nucleosome structure LXCXE motif whereas Brg1 apparently associates with but also the reverse reaction (Schnitzler et al., 1998). the Retinoblastoma protein through a neighboring It is therefore possible that after facilitating the domain. The formation of a ternary complex contain- recruitment of E2F, the SWI/SNF complex also plays ing HDAC, pRb and Brg1 is therefore possible. an active role in preventing this transcription factor According to Zhang et al. (2000), this complex from mediating activation. What determines the represses both the Cyclin E and the Cyclin A activating or the repressing eect of the SWI/SNF promoters. Phosphorylation of the pocket region of complex remains unclear, but the eect of the complex pRb in late G1 by Cyclin D/Cdk4 seemingly displaces seems to be dependent on the presence of the histone HDAC from the ternary complex without aecting the tails and on their state of acetylation (Agalioti et al., Brg1-pRb interaction. The resulting binary complex 2000; Logie et al., 1999; Syntichaki et al., 2000). still represses the Cyclin A, but not the Cyclin E Furthermore, both Brg1 and Brm contain a Bromo- promoter. This residual complex is only disrupted in S domain. This conserved motif is known from other phase upon accumulation of CyclinE/Cdk2. These proteins to mediate interaction with acetylated observation are compatible with the ®nding that cells histones (Dhalluin et al., 1999; Hudson et al., 2000; growth-arrested by Brg1 and pRb accumulate in S Jacobson et al., 2000; Owen et al., 2000). Thus, the phase and not in G1. Interestingly, Brg1-pRb-induced equilibrium between histone acetylating and deacety- repression of the Cyclin A promoter was relieved by lating activities may be the main determinant of E2F- Cyclin E/Cdk2 even when wild type pRb was replaced induced promoter activation and this may be what by a mutant RbDcdk, which cannot be inactivated Cdk pRb is in fact regulating. In parallel, it must be noted phosphorylation. This suggests that pRb phosphoryla- that embryonic ®broblasts isolated from Brm7/7 tion by Cyclin E/Cdk2 is not the causative event mice fail to up-regulate p27kip1 at con¯uency. leading to activation of the Cyclin A promoter. An p27kip1 is a member of the p21 family that inhibits earlier study has shown that Brg1 associates with Cyclin D/Cdk4, Cyclin E/Cdk2 and Cyclin A/Cdk2. Cyclin E and can be inactivated by Cyclin E/Cdk2 Brm and Brg1 expression may therefore cause down- phosphorylation (Shanahan et al., 1999). Furthermore, regulation of the kinases inactivating pRb and thereby in Drosophila, reduced Cyclin E function in the eye provide indirect stimulation of pRb-mediated repres- imaginal disk leads to disruption of the regular sion (Reyes et al., 1998). patterning of the eye, due to defective S phase entry The respective roles of Brm and Brg1 in pRb of cells posterior to the morphogenetic furrow (de mediated growth arrest is still debated. It is clear that Nooij and Hariharan, 1995). Mutations in the both proteins can cooperate with pRb upon over- Drosophila Brahma gene can compensate for this expression (Strober et al., 1996). However, in tissue `rough eye' phenotype, demonstrating a genetic link culture cells, the level of Brm expression is low during between the SWI/SNF complex and Cyclin E (H exponential growth. For example in NIH3T3 cells, Richardson, personal communication). It is therefore Brm accumulates in G0 but becomes almost undetect- possible that Brg1 phosphorylation by Cyclin E/Cdk2 able after stimulation of the cells with serum. On the is a major event leading to progression through S other hand, Brg1 is abundant throughout the cell cycle phase. (Muchardt et al., 1998). Under physiological condi-

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3073 tions, it is therefore likely that in late G1 and S phase, What about SNF5/INI1? the major catalytic subunit of the mammalian SWI/ SNF complex is Brg1 and not Brm. Nevertheless, Brm Although SNF5/INI1 is the only con®rmed tumor may also play an important role in the control of cell suppressor protein present in the SWI/SNF complex, cycle. First, as mentioned above, Brm expression is connections linking this protein to the cell cycle can induced upon dierentiation of F9 and ES cells. only be inferred. The SNF5/INI1 protein is present in Second, it is frequently down-regulated or absent in all mammalian SWI/SNF complex puri®ed to date and transformed mouse cells. Re-introduction of Brm in it is possible that, unlike Brm and Brg1 that are at least ras-transformed mouse ®broblasts leads to reversion of partially redundant, inactivation of the SNF5/INI1 the transformed phenotype (Muchardt et al., 1998). In subunit leads to overall inactivation of SWI/SNF this same assay, Brg1 showed little eect, possibly activity. If a complete SWI/SNF complex is required because ectopic expression of Brg1 leads to down- for pRb mediated repression, inactivation of SNF5/ regulation of the endogenous Brg1 gene, keeping the INI1 may jeopardize this growth control pathway and total levels of Brg1 protein essentially constant (B lead to cancer. It must however be noted that mouse Bourachot and C Muchardt, unpublished observation). Rb 7/7 embryos die much later SNF5/INI1 null It is therefore likely that Brm and Brg1 have the same mutants (Jacks et al., 1992; Klochendler-Yeivin et al., potential eect on the cell cycle but that they are 2000). Furthermore, the spectrum of tumors is dierent regulated in very dierent ways. Brg1 may be the in Rb and SNF5/INI1 heterozygous mice. This is also important protein during interphase where it is present true in human patients. It is possible that SNF5/INI1 in well de®ned amounts and regulated by phosphoryla- cooperates with other Rb family members and that tion. On the other hand, Brm appears to be an inactivation of this gene leads to complete disruption inducible protein that may allow to increase the total of Rb function. However, SNF5/INI1 may also aect amount of active SWI/SNF complex present in the the cell cycle through other pathways. The protein has cells. Augmented levels of SWI/SNF complex may been found to interact with the product of the HRX permit regulation of targets speci®c for growth arrest gene, frequently disrupted in human acute leukemia and dierentiation. (Alder et al., 1999) and interaction between SNF5/INI1 and E1 stimulates replication of the papillomavirus DNA (Lee et al., 1999). Finally, SNF5/INI1 interacts The BRCA1 connection directly with the proto-oncogene c-MYC. This protein is a transcription factor that plays a key role in cell As mentioned above, the BRCA1 tumor suppressor proliferation, dierentiation and apoptosis (see Grand- protein has also been found associated with what ori et al., 2000, for review). When dimerized with appears to be a version of the BAF mammalian SWI/ MAX, c-MYC binds DNA and activates transcription. SNF complex (Bochar et al., 2000). The BRCA1 gene This activation is at least in part due to the recruitment is mutated in 50% of inherited breast cancers and of the TRRAP-GCN5 histone acetylase complex 90% of all familial breast and ovarian cancers. The (McMahon et al., 2000). Activation apparently also BRCA1 protein is involved in DNA damage repair requires the presence of SNF5/INI1 and possibly the and its inactivation leads to genetic instability (see entire SWI/SNF complex (Cheng et al., 1999; Takaya- Deng and Scott, 2000, for review). However, the ma et al., 2000). This observation suggests that loss of protein has also been directly implicated in transcrip- an intact SNF5/INI1 protein will cause down-regula- tional activation. It was found associated with RNA tion of c-MYC activation. It is unclear how this event polymerase II and the co-activators CBP and p300. could lead to cell transformation but it has been BRCA1 was also shown to function as a co-activator suggested that SNF5/INI1 may primarily participate in of p53-mediated transcription (see Monteiro, 2000, for c-MYC-mediated activation of genes involved in review). The p53 protein is responsible for G1 and G2 apoptosis (Cheng et al., 1999). arrest upon DNA damage. It is a transcription factor that upon induction up-regulates expression of the p21WAF1 Cdk-inhibitor and thereby prevent the Pespectives inactivating phosphorylation of pRb (see Vousden, 2000, for review). The eect of BRCA1 on p53- Many questions remain open on the way mammalian mediated transcription is inhibited by a trans- SWI/SNF subunits may regulate the cell cycle. For dominant Brg1 mutated in its ATP-binding site example, although many groups have now demon- (Bochar et al., 2000). This observation raises the strated an interaction between pRb and Brg1 or Brm, possibility that the mammalian SWI/SNF complex pRb was never found to co-elute with the puri®ed also controls cell cycle through the p53 pathway. It SWI/SNF complex (Wang et al., 1996). It can therefore must however be noted that overexpression of BRCA1 not be ruled out that pRb repression is mediated by a can lead to growth arrest in a Rb-dependent manner speci®c SWI/SNF sub-complex, which has yet to be and that an interaction between BRCA1 and pRb has identi®ed. It is even possible that Brg1 or Brm alone been described (Aprelikova et al., 1999). It is therefore are the only subunits required for this process. possible that BRCA1 is in fact part of the Brg1-pRb Similarly, the striking dierences between pRb and cell-cycle control pathway described above. SNF5/INI1 knockout mice clearly tells that we still

Oncogene Mammalian SWI/SNF and the cell cycle C Muchardt and M Yaniv 3074 have a poor understanding on how SNF5/INI1 much needed information on their role in the control mutations can lead to tumorigenesis. Again, it can of cell growth. not be ruled out that this protein has a function beyond the mammalian SWI/SNF complexes as they are known today. Mouse models allowing conditional Acknowledgments inactivation of Brg1 and SNF5/INI1 are on the way. We thank Brigitte Bourachot, Sigrid Schaper, Olivier Unlike the simple knockouts, these mice should give us Delattre, Helena Richardson and Terry Magnuson for some insights on the eect of the SWI/SNF proteins at communicating results prior to publication, and Jacob later stages of development and will hopefully provide Seeler for critical reading of the manuscript.

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Oncogene