BRG1 mutations found in human cancer cell lines inactivate Rb-mediated cell-cycle arrest

BRG1 Mutations Found inHuman Cancer Cell LinesInactivate Rb-MediatedCell-Cycle ArrestCHRISTOPHER BARTLETT,1,2 TESS J. ORVIS,2 GARY S. ROSSON,2

AND BERNARD E. WEISSMAN1,2,3*1Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina2Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina3Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina

Eukaryotic organisms package DNA into chromatin for compact storage in the cell nucleus. However, this process promotestranscriptional repression of genes. Toovercome the transcriptional repression, chromatin remodeling complexes have evolved that alterthe configuration of chromatin packaging of DNA into nucleosomes by histones. The SWI/SNF chromatin remodeling complex usesenergy from ATP hydrolysis to reposition nucleosomes and make DNA accessible to transcription factors. Recent studies showingmutations of BRG1, one of twomutually exclusive ATPase subunits, in human tumor cell lines and primary tissue samples have implicated arole for its loss in cancer development. While most of the mutations lead to complete loss of BRG1 protein expression, others result insingle amino acid substitutions. To better understand the role of these BRG1 point mutations in cancer development, we characterizedSWI/SNF function in human tumor cell lines with these mutations in the absence of BRM expression, the other ATPase component. Wefound that the mutant BRG1 proteins still interacted with the core complex members and appeared at the promoters of target genes.However, while these mutations did not affect CD44 and CDH1 expression, known targets of the SWI/SNF complex, they did abrogateRb-mediated cell-cycle arrest. Therefore, our results implicate that thesemutations disrupt the de novo chromatin remodeling activity ofthe complex without affecting the status of existing nucleosome positioning.J. Cell. Physiol. 226: 1989–1997, 2011. � 2010 Wiley-Liss, Inc.

Eukaryotic organisms package DNA into condensed solenoidchromatin for compact storage in the cell nucleus, resulting intranscriptional repression of genes. Therefore, mechanismsof chromatin remodeling have evolved to overcome thisrepressive nature of chromatin and make DNA accessible tosequence-specific transcription factors and transcriptionmachinery (Clapier and Cairns, 2009). Chromatin remodelingresults in an alteration of nucleosomes to allow the binding oftranscription factors and initiation of transcription (Wang et al.,2007a,b; Clapier and Cairns, 2009). Two classes of chromatinremodeling enzymes exist, histone modifying enzymes andATP-dependent chromatin remodeling enzymes.

The SWI/SNF complex is an ATP-dependent chromatinremodeling complex that is conserved from humans to yeast(Clapier and Cairns, 2009). The complex is approximately2-Mda in size and consists of 9–12 members varying in a tissue-specific manner (Wang et al., 1996). To date, six SWI/SNFcomplexes have been isolated with each complex consisting ofone catalytic subunit, BRG1 or BRM, plus 8–11 BAFs (BRG1-associated factors). A minimum catalytic core of BRG1 or BRMand BAF155 or BAF170 is required for disruption ofnucleosome arrays in vitro, but all members of the complex arerequired for proper functioning in yeast (Laurent et al., 1991;Peterson and Herskowitz, 1992).

Themain function of the SWI/SNF complex is transcriptionalregulation. The complex has been shown to be required forexpression of a variety of genes involved in cellular adhesionsuch as CD44 (Strobeck et al., 2001) and CDH1 (Banine et al.,2005), and genes involved in cellular proliferation such as cyclinA (Zhang et al., 2000) and cyclin E (Zhang et al., 2000), CSF1(Liu et al., 2001), and p53-dependent target promoters (Leeet al., 2002). The complex has also been shown to be requiredfor RB-mediated arrest by upregulating p21, which inhibits

cyclin-dependent kinases and leads to RB hypophosphorylation(Hendricks et al., 2004; Kang et al., 2004). Due to the role of thecomplex in cellular adhesion and growth arrest, it is notsurprising that evidence has linked loss of SWI/SNF complexmembers with human disease.

The first association between loss of a SWI/SNF complexmember and human disease was established when loss of SNF5expression was found to lead to development of malignantrhabdoid tumors (Versteege et al., 1998). Increasing evidencehas also indicated a role for inactivation of other members ofthe SWI/SNF complex including BRG1, BRM, BAF155, andBAF57 in cancer development and/or cancer progression(Weissman and Knudsen, 2009). As expected, loss of thecatalytic subunits, BRG1 and BRM, leads to loss of function ofthe complex. In fact, loss of BRG1 and BRM is found in humantumor cell lines and primary tumor of lung, breast, and prostate

Contract grant sponsor: NIH/NCI;Contract grant number: CA138841.Contract grant sponsor: NIH/NIEHS;Contract grant number: T32 ES007126.

Christopher Bartlett’s present address is Scimetrika, LLC, 100Capitola Drive, Suite 104, Durham, NC 27713.

*Correspondence to: Bernard E. Weissman, UNC LinebergerComprehensive Cancer Center, University of North Carolina,CB#7259, Room 32-048, Chapel Hill, NC 27599-7295.E-mail: [email protected]

Received 30 October 2010; Accepted 2 November 2010

Published online in Wiley Online Library(wileyonlinelibrary.com), 6 December 2010.DOI: 10.1002/jcp.22533

ORIGINAL RESEARCH ARTICLE 1989J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

� 2 0 1 0 W I L E Y - L I S S , I N C .

(Weissman and Knudsen, 2009).Wong et al. (2000) screened apanel of tumor cell lines to determine if BRG1 is targeted formutation. They identified 16/180 human tumor cell lines thatpossessed mutations in BRG1. We have found loss ofexpression of BRG1 and BRM in approximately 20% of NSCLCcell lines (Reisman et al., 2003). In most cases, loss of BRG1protein expression arises from gene deletions or truncatingpointmutations (Wong et al., 2000).However, primaryNSCLCtumors and several human tumor cell lines contain pointmutations in BRG1 (Wong et al., 2000; Medina et al., 2004).Several groups, including our own, have found loss of BRG1and/or BRM expression in primary NSCLC (Reisman et al.,2003; Fukuoka et al., 2004). Most importantly, loss of BRG1/BRM is an indicator of poor prognosis. NSCLC BRG1/BRM-negative tumor patients have a shorter survival time thenNSCLC BRG1/BRM-positive tumor patients (Reisman et al.,2003; Fukuoka et al., 2004).

In a previous study, tumor cell lines lacking both BRG1 andBRM expression were analyzed for impaired RB-mediatedgrowth arrest and for expression of CD44 and CDH1(Strobeck et al., 2001, 2002; Reisman et al., 2002; Banine et al.,2005). It was found that either BRM or BRG1 was sufficient torestore RB-mediated growth arrest and CD44 and CDH1expression, indicating that BRM can compensate for BRG1 loss.In contrast, Brm�/�;Brg1wt/wt mice failed to express CD44 in alltissues examined (Reisman et al., 2002). As a furthercharacteristic of tumor suppressor genes, the stablereintroduction of BRG1 into the tumor cell lines resulted inreplicative senescence (Hendricks et al., 2004; Kang et al.,2004).

In this study, we analyzed if point mutations in BRG1 found inhuman tumor cell lines resulted in altered functions. Wesuppressed BRM expression in each cell line by stableexpression of shRNA and assessed them for impaired RB-mediated arrest, CD44 expression, and CDH1 expression.Wefound that two of the BRG1 mutations have lost the ability topromote RB-mediated cell-cycle arrest and CD44 expression.We also showed that these mutant BRG1 proteins stillassociate with the other core members of the complex by co-immunoprecipitation and were present at target promoters bychromatin immunoprecipitation (ChIPs) analysis. Our resultssuggest that the point mutations found in these cell lines impairthe function of BRG1 by altering the ability of the complex toremodel chromatin. The sites of these point mutations shouldprovide insight into the structure and function of this importantcellular regulatory protein.

Materials and MethodsCell lines

MCF7, HeLa, SW13, NIH-OVCAR3, Hs578t, and HCT116 wereobtained from ATCC. All cell lines were grown in RPMI with 10%FBS exceptHCT116whichwas grown inDMEMwith 10%FBS. Celllines were routinely checked for mycoplasma contamination andwere found to be negative.

Immunoblotting

Protein levels were determined as previously described (Chai et al.,2005). Briefly, cells were grown in 100mm tissue-culture dishes,

harvested in extraction buffer, and collected with a scraper. Afterclarification by centrifugation, the supernatant was collected andprotein concentration was determined by Bradford protein assay.Equal amounts of protein lysate (30mg) were separated by SDS–polyacrylamide gel electrophoresis (either 7.5% or 4–20%gradient) for 1 h at 100V. Protein was then transferred toImmobilon-P membranes (Millipore, Billerica, MA) for 16–18 h at80mA. Membranes were blocked for 1 h in 5% non-fat dry milk/0.2% Tween-20 in PBS. Membranes were then incubated for 2 h inprimary antibody at room temperature. Primary antibodiesincluded BRG1 (Santa Cruz Biotechnology, G7, Santa Cruz, CA),BRM (Dr. Yaniv Moshe, Pasteur Institut or Abcam, 15597,Cambridge, MA), CDH1 (Transduction Labs 610181), CD44 (H3)(Dr. Larry Sherman, Oregon Health Sciences University), and actin(Sigma, A2066, St. Louis, MO). Membranes were washed andincubated in a 1:2,000 dilution of either anti-mouse or anti-rabbitsecondary antibody for 1 h. Membranes were then washed andprotein bandswere detectedwith ECL chemiluminescence reagent(Amersham, Piscataway, NJ) on Biomax ML film (Kodak, NewYork, NY) or by Licor fluorescent imaging.

Nuclear protein extraction

1� 109 cells were collected, washed, and resuspended inhypotonic buffer (10mM Hepes, pH 7.9/10mM NaCl/1.5mMMgCl2/2mM DTT) for 10min. The pellet was the collected bycentrifugation, resuspended in 2� original pellet volume withhypotonic buffer and extracted with a Dounce homogenizer. Thenucleiwere collected by centrifugation and resuspended inNuclearExtract Buffer (20mM Hepes, pH 7.9/420mM NaCl/1.5mMMgCl2/2.0mMDTT/0.2mM EDTA/25% glycerol). The nuclei werethen sonicated and nuclear proteins extracted with a douncehomogenizer. Nuclear proteins were collected by centrifugationand then dialyzed against three changes of dialysis buffer (20mMHepes, pH 7.9/50mM KCl/1.0mM DTT/0.2mM EDTA/10%glycerol). Protein concentrations were determined by Bradfordassay.

Immunoprecipitation

Nuclear extracts were precleared with Protein A and G sepharosebeads (1:1) (Amersham) inRIPAbuffer for 2 h at 48C.After removalof beads of centrifugation, equal amounts of protein wereincubatedwith each antibody alongwith ProteinA andG sepharosebeads (1:1) (Amersham) saturated with BSA and salmon sperm inRIPA buffer at 48C overnight. Primary antibodies included normalrabbit serum, rabbit anti-BRG1 (J1, Weidong Wang, NationalInstitute on Aging), rabbit anti-BAF155 (H-76, Santa CruzBiotechnology), and rabbit anti-SNF5 (Tony Imbalzano, Universityof Massachusetts). The next day, the beads were collected bycentrifugation and washed once in RIPA buffer, IP wash buffer(100mM Tris–HCl, pH 8.5/500mM LiCl/1% NP40/1%deoxycholate), and in PBS. The proteins were then releaseddirectly from the beads by heating in loading buffer and loaded ontoSDS–polyacrylamide gels for immunoblot analysis. Immunoblottingwas carried out as above using the following antibodies—mouseanti-BRG1 (G7, Santa Cruz Biotechnology), rabbit anti-BAF155(Weidong Wang, National Institute of Aging), and mouse anti-SNF5 (612111, BDTransduction Laboratories, Franklin Lakes, NJ).

Chromatin immunoprecipitation. ChIP was carried outas described by Kuwahara et al. (2010). Immunoprecipitation was

TABLE 1. Sequences of QT-PCR primers used for ChIP

Gene/position Forward primer Reverse primer

CDH1/�354 bp 50-TCG AAC CCA GTG GAA TCA GAA-30 50-GGG TCT AGG TGG GTT ATG GGA C-30CD44/TSS 50-TCA GCC TTT GGC CTC TCC TT-30 50-CTC CAG CCG GAT TCA GAG AA-30p21/�20 bp 50-TAT ATC AGG GCC GCG CTG-30 50-GGC TCC ACA AGG AAC TGA CTT C-30p21/�3 kb 50-CCG GCC AGT ATA TAT TTT TAA TTG AGA-30 50-AGT GGT TAG TAA TTT TCA GTT TGC TCA-30

TSS, transcription start site.

JOURNAL OF CELLULAR PHYSIOLOGY

1990 B A R T L E T T E T A L .

performed with an antibody specific to SNF5 (Dr. Tony Imbalzano,University of Massachusetts School of Medicine), BRG-1 (J1; Dr.Weidong Wang, NIA), BAF155 (H76, Santa Cruz Biotechnology), ornormal rabbit IgG (sc-2027; Santa Cruz Biotechnology). DNA presentin each IP was quantified by QT-PCR using gene-specific primers on anABI 7000 sequence detection system (Table 1). All expression valueswere normalized against input DNA.

Generation of stable RNAi clones

The generation and characterization of the BRM and BRG1 shRNAexpression vectors were previously reported (Link et al., 2005;Rosson et al., 2005). Cell lines were transfected with these shRNAivectors using Lipofectamine 2000 (Invitrogen, San Diego, CA)following the manufacturers instructions. Clones were selectedusing puromycin selectionmedia based on death curves establishedfor each cell line; 0.4mg/ml puromycin in RPMI for Hs578t cells,1mg/ml puromycin in RPMI for OVCAR3, and 2mg/ml puromycinin DMEM for HCT116 cells. Messenger RNA and protein levels foreach clone were screened by QT-PCR and Western blotting aspreviously described (Kuwahara et al., 2010; Livak and Schmittgen,2001).

Brdu incorporation assay

The assay was carried out as previously described (Betz et al.,2002). Briefly, cells were plated in eight well-cover slips andtransfectedwith 1mg of pcDNA3 or p16/PSMRB and 0.2mg of GFPusing lipofectamine 2000 (Invitrogen), following manufacturer’sinstructions. After 36 h, 1mg/ml Brdu was added for an additional12 h. The cells were then fixed in 10% formalin for 15–20min. Brduincorporation was detected using mouse anti-Brdu (Amersham)followed by alexa fluor 546 anti-mouse IgG (Molecular Probes, SanDiego, CA). Fifty to 100 GFP-positive cells were counted for Brduincorporation by fluorescent microscopy. The average of threeexperiments per sample was normalized to the parent cell linetransfected with pcDNA3.

Transient transfection

Cell lines were transfected with shRNAi vectors usingLipofectamine 2000 (Invitrogen), following the manufacturersinstructions. Transfected cells were harvested for protein 48 hafter transfection with detergent-based extraction buffer (Banineet al., 2005).

ResultsCharacterization of BRG1 mutant cell lines

To determine whether point mutations in BRG1 affected itsnormal functions, we chose three human tumor cell lines withknownmutations (Fig. 1). These cell lines were chosen becausethe mutations appear in three different domains of BRG1. The

Fig. 1. LocationofBRG1mutations.The locationofthethreepointmutations inhumancancercell linesHs578t,NIH-OVCAR3,andHCT116areindicated along with known functional domains of BRG1.

Fig. 2. Western blot analysis of BRG1 mutant cell lines. Proteinfrom BRG1 mutant cell lines was separated by SDS–PAGE andimmunoblotted for BRG1, BRM, CD44, CDH1, and b-actin. MCF7cells served as a positive control forCDH1 expression.b-Actin servedas a loading control.


F U N C T I O N A L S I G N I F I C A N C E O F B R G 1 M U T A T I O N S 1991

Hs578t mutation contains a P196S mutation the proline richdomain I, near an area shown to be required for B-cateninsignaling (Barker et al., 2001). The OVCAR3 E402G mutationis hemizygous, within the highly charged domain II, whosefunction remains unknown (Wong et al., 2000). The HCT116L1149P mutation maps to the ATPase domain, which isresponsible for hydrolyzing ATP (Wong et al., 2000).Previously, our lab determined a functional copy of either BRG1or BRM was enough for CDH1 or CD44 expression and RB-mediated arrest (Strobeck et al., 2000, 2001, 2002; Reismanet al., 2002; Banine et al., 2005). These cell lines have previouslybeen described for their CD44 expression and RB-sensitivity(Strobeck et al., 2002). Interestingly, a Western blot screenof our BRG1 mutant cell lines shows OVCAR3 lacks CD44expression and Hs578t lacks CDH1 expression, even thoughthey presumably contain at least a functional copy of BRM(Fig. 2).

Mutant BRG1 proteins still interact with corecomplex members

Point mutations in proteins can affect their folding or theirbinding properties. To determine if the BRG1 mutations inHCT116, Hs578t, and OVCAR3 inhibited its interaction withother members of the SWI/SNF complex, we performedimmunoprecipitation of nuclear extracts with antibodiesagainst BRG1 and BAF155. Immunoprecipitated proteinwas analyzed by Western blot analysis (Fig. 3). Both of theother core complex members, BAF155 and SNF5,

co-immunoprecipitated from HeLa, our positive control forwild-type BRG1 (Fig. 3). However, the stoichiometry of thecomplex appears to vary depending upon which complexmember we used for immunoprecipitation. In contrast, onlySNF5 co-immunoprecipitated with BAF155 in the BRG1-deficient SW13 (Fig. 3).We did observe a small amount of bothBAF155 and SNF5 protein co-precipitating with BRG1 due to asmall subpopulation of BRG1-positive cells within this cell line(Yamamichi-Nishina et al., 2003). In general, the results withthe HCT116 cell line appear similar to those observed with theHeLa cell line indicating that the BRG1 mutation did notapparently affect the ability of the protein to interact with thesecore proteins (Fig. 3). However, the immunoprecipitationresults with the Hs578t suggest a reduced interaction with theBAF155 subunit (Fig. 3). The resultswith theNIH-OVCAR3 cellline also indicate altered protein interactions. These cellsexpress both wtBRG1 and mutant BRG1 proteins. Therefore,the absence of BAF155 protein in the BRG1 pull-down lane butnot the BAF155 pull-down lane may indicate a reduced bindingof BAF155 by the BRG1 mutant protein.

Analysis of BRM-deficient HCT116 and Hs578t cell lines

Because the BRG1mutant protein appeared to be in a complexwith at least one other core member of the SWI/SNF complex,we wanted to determine if the BRG1 mutant complexesremained functional. To test this possibility, we decided toabrogate BRM function to negate its ability to compensate forBRG1 loss. To create HCT116, NIH-OVCAR3, and Hs578t

Fig. 3. Immunoprecipitation of SWI/SNFcomplex core components.Nuclear proteins fromBRG1mutant cell lineswere immunoprecipitatedfor BRG1 (J1 antibody) of BAF155 (H76, Santa Cruz Biotechnology) and then analyzed by immunoblot for expression of the other core complexmembers of the SWI/SNF complex. Input samples were run as positive controls. PBS precipitated protein served as a negative control. Proteinprecipitated with normal rabbit serum (NRS) indicated any background protein precipitated by IgG. HeLa cells were precipitated as a positivecontrol for an intact complex while SW13 serves as a negative control for complex formation because only aminor population expresses BRG1.



stable knockdowns of BRM, we transfected each cell line withshRNAi vectors against BRM and selected stable clones. Wealso isolated cell lines with stable expression of a BRG1 shRNAor vector alone as controls.

Western blot analysis confirmed that stable expression ofthe RNAi vectors in the HCT116 and Hs578t cell lines resultedin downregulation of BRG1 by the BRG1 RNAi (Fig. 4a,c) anddownregulation of BRM by the BRM RNAi vector (Fig. 4b,d).Attempts to generate NIH-OVCAR3 stable clones expressinga BRM shRNAi vector were unsuccessful. Although we wereable to obtain colonies for vector alone and BRG1 shRNAi-transfected cells, no colonies were able to grow out in BRMshRNAi-transfected cells. Transient transfection with BRMshRNAi confirmed initial reduction of BRM expression in NIH-OVCAR-3 cells but no stable colonies with reduced expressionproliferated (data not shown). Therefore, we chose the celllines indicated by a star in Figure 4 and tested each one forthree phenotypes associated with SWI/SNF complex function:RB-mediated cell-cycle arrest and expression of CD44 andCDH1 mRNA (Table 2).

Loss of BRM expression abrogates Rb sensitivity

Normally, cells transfected with p16INK4A or a constitutivelyactive form of Rb, PSM-RB, undergo growth arrest (Strobecket al., 2002). However, in previous studies, we and others haveshown that cell lines without a functional BRM and BRG1 wereinsensitive to RB-mediated cell-cycle arrest (Strobeck et al.,2002). Previously, Hs578t was found to be sensitive to RB-mediated arrest due to the presence of functional BRM. In asimilar fashion, the p16-deficient HCT116 cell line undergoescell-cycle arrest when transfected with PSM-RB. To determineif the BRG1 mutations affected RB sensitivity, we analyzed theRNAi stable clones ofHCT116 andHs578t for incorporation ofBrdu during the S phase of the cell cycle. In parent cell lines,stable vector clones, and BRG1 RNAi stable clones,transfection of Hs578t with p16, and transfection of HCT116cells with PSM-RB caused reduction of Brdu incorporation dueto growth arrest of the cells (Fig. 5). In contrast, the BRM RNAistable clones became resistant to Rb-mediated growth arrestand continued to incorporate Brdu (Fig. 5). These data indicatethat the mutations in BRG1 abrogated its ability to regulate thecell cycle.

Loss of BRM expression does not alter CD44 orCDH1 expression

We also assessed if the BRM and BRG1 knockdown cell linesshowed altered expression of cell adhesion genes previouslyassociated with BRG1 and BRM regulation. Using Westernblotting, we examined CD44 and CDH1 expression in a subsetof these cell lines compared to BRG1 and BRM expression. Asshown in Figure 6, we did not observe a correlation betweenBRG1 mutations and reduced CD44 or CDH1 expression.Therefore, in contrast to responsiveness to RB-mediatedgrowth arrest, transcription of these genes does not seemaffected by either of these mutations.

BRG1 mutant proteins still bind to the CD44 andCDH1 promoters

Although the mutant BRG1 proteins could still associate withthe core complex members (Fig. 3), they may still compromisethe ability of the complex to bind to chromatin. To assess thispossibility, we examined the binding of BRG1, BAF155, andSNF5 at the CD44, CDH1, and p21 promoters by ChIPs. We

Fig. 4. Analysis of RNAi stable clones by protein expression. Hs578tandHCT116 cells were transfectedwith shRNAi vectors and selectedin growth medium supplemented with puromycin. Total cellularprotein was extracted from colonies using urea, separated bySDS–PAGE and immunoblotted for BRG1 (G-7, Santa CruzBiotechnology) in Hs578t cells (A) and HCT116 cells (C) and BRM(Dr. Yaniv, Pasteur Institut) in Hs578t cells (B) andHCT116 cells (D).b-Actin served as a loading control. TABLE 2. Characteristics of the BRG1/BRM-deficient cell lines

Cell line RB sensitivity CD44 expression CDH1 expression

HCT116 S þ þHs578t S þ �NIH-OVCAR3 R � þ

S, sensitive; R, resistant.



and others had previously shown the presence of the complexat these three promoters (Hendricks et al., 2004; Kang et al.,2004; Banine et al., 2005). We looked at binding in the parentalcell lines and representative vector control and BRMrnai celllines. As shown in Figure 7A,B, we observed similar levelsof BRG1 and SNF5 binding at all three promoters in all Hs578tcell lines. In contrast, an increase in BRG1 and SNF5 bindingoccurred at all promoters in the HCT116BRMrnai cell linecompared to the parent and vector control (Fig. 7C,D).However, this appears to result from a non-specific increase inbinding because it occurs at both the low binding (�3 kb) andhigh binding (�20 bp) p21 sites (Kuwahara et al., 2010).

Discussion

Research focused on the SWI/SNF complex has increasedrecently due to expanding evidence that aberrations in thecomplex lead to cancer development (Weissman and Knudsen,2009). Several lines of evidence implicate loss of catalyticsubunits in cancer development. Loss of BRG1 is found inhuman tumor cell lines, and primary tumors (Reisman et al.,2003; Medina et al., 2004; Weissman and Knudsen, 2009).

Similarly, mutations in BRG1 are found in human tumor celllines and primary tumors (Wong et al., 2000; Medina et al.,2004). Interestingly, the presence of point mutations andinternal deletions shows that BRG1 loss is not due to thepresence of a larger genomic deletion. Loss of BRG1 or BRMmost likely contributes to cancer development by altering geneexpression on a global scale and/or disrupting RB-mediatedgrowth arrest. Importantly, our results show that mutationsthat alter BRG1 function do not a priori affect expression ofknown target genes, that is, CD44 and CDH1. Presumably,nucleosome positioning remained in place in the absence ofSWI/SNF complex function. Rather, the effects of themutationsappeared upon the requirement for active nucleosomerepositioning. In this study, the mutant BRG1 proteins lost theability to respond to Rb-mediated growth arrest, presumablythrough the absence of chromatin remodeling activity atthe necessary promoter regions. Further studies examiningnucleosome positioning at promoters such as p21WAF1/CIP1 willaddress this possibility.

Previous examination of human tumor cell lines revealed thatcell lines deficient for both BRG1/BRM were impaired for theirability to express CD44 protein and were unable to growth

Fig. 5. RBsensitivityofHCT116andHs578tRNAistableclones.HCT116andHs578tRNAistableclonesweretransfectedwitheitherpcDNA3orp16forHs578tcells,orPSM-RBforHCT116cells.Brdu incorporationwasassessedforHs578tandHCT116parentcell linesandvectoralonestableclones (A), BRG1 RNAi stable clones (B), and BRM RNAi stable clones (C) as described in the Materials and Methods Section.



arrest through RB indicating compensatory function betweenthe two (Strobeck et al., 2002). Expression of BRG1or BRMhasbeen shown to rescue these defects. HCT116 cells contain amutation in the ATPase domain in Motif V (Wong et al., 2000;Medina et al., 2004; Smith and Peterson, 2005). A screen of20 NSCLC found two mutations in BRG1, both of which are inMotif V of the ATPase domain (Medina et al., 2004). Thesefindings indicate, although rare, BRG1 mutations maycontribute to tumor development. Additional research hasindicated how this mutation may impair BRG1 function.Mutations in Motif V do not impair nucleosomal binding but doappear to reduce ATPase activity (Smith and Peterson, 2005),so mutations in Motif V are believed to alter the coupling ofATPase activity to the specific function of BRG1 (Smith andPeterson, 2005). The BRG1 mutation in breast cancer cellHs578t is in domain I (Wong et al., 2000). Part of domain I hasbeen shown to interact with B-catenin (Barker et al., 2001).Previous studies have shown that these cell lines are capable ofCD44 protein expression and undergo RB-mediated arrest dueto presence of functional BRM (Strobeck et al., 2002). In thisstudy we wanted to determine if the function of BRG1 mutantsin human tumor cell lines was impaired in the absence of BRMusing BRM shRNAi.We found that both the mutations found inHCT116 and Hs578t were impaired in terms of RB sensitivity.Neither mutation showed consistent effects on proteinexpression.

CDH1 is not expressed in BRG1/BRM-deficient cells but canbe upregulated by transfection of BRG1 or BRM into deficientcells. In aWestern blot screenofHs578t andHCT116we foundHs578t does not express CDH1. Apparently, BRM is unable tocompensate for the BRG1 mutation in this cell line and/orCDH1 remains silent due to other mechanisms. The BRG1mutation in breast cancer cell Hs578t is in domain I, a domainthat is partially required to interact with B-catenin and isrequired for transcriptional activation of Tcf-dependentpromoters (Barker et al., 2001). CDH1 is regulated by thispathway, and aberrations in B-catenin signaling and loss ofCDH1 commonly occurs in breast cancer (Graff et al., 1995).The Hs578t CDH1 promoter is methylated (Graff et al., 1995),but treatment with 5-azacytidine does not restore CDH1expression, indicating the requirement of other transcriptionalfactors (Hajra et al., 1999). We found transfection of Hs578tcells with BRG1 does not upreguate CDH1 expression(data not shown). Other labs have demonstrated inducibleexpression of K789R, a DNBRG1 with no ATPase activity,reduces expression of Tcf target genes (Barker et al., 2001). TheBRG1 mutation in Hs578t may also be acting as a dominantnegative and inhibiting BRM from compensating in the absenceof functional BRG1. Reduction of BRM in HCT116 stable RNAiclones did not result in consistent reduction of CDH1expression. BRG1 and BRM are approximately 76%homologous but they lack homology in theN-terminal region oftheir proteins including the area of the Hs578t mutation. Soalternatively, Hs578t may not express CDH1 with functionalBRM because it may be functionally unique to BRG1. Itsinduction by BRM may result from the high non-physiologicallevels used in previous experiments.

Redundant functions of BRG1/BRM suggest that cells mustlose expression of both genes for tumorigenic effects. Althoughredundant functions have been shown for the catalytic subunitsin in vitro experiments, some assays have shown differentialfunctions for the two proteins. Staining of MEFs from BRMknockout mice reveals that CD44 expression is lost even in thepresence of BRG1 (Reisman et al., 2002). This is surprising sincetransfection of BRG1 into deficient cells upregulates CD44protein expression. Again overexpression of BRG1, at non-physiological levels, may be able to regulate CD44 in cellsdeficient for BRG1 and BRM. Kadam and Emerson have shownBRG1 and BRM exhibit transcriptional specificity. They foundBRG1 binds zinc finger protein transcription factors while BRMbinds ankyrin repeat transcription factors involved in notchsignaling (Kadam and Emerson, 2003). Examination of normalhuman tissues shows differential expression of the proteins,implicating distinct roles for the catalytic subunits indifferentiated tissue (Reisman et al., 2005). In fact, homozygousknockout mice indicate differential functions of the twocatalytic subunits. Homozygous knockout mice for BRG1 areembryonic lethal and BRG1 heterozygous mice are prone totumor development by haploinsufficiency (Bultman et al., 2000,2008) while knockout mice for BRM are viable with nosignificant phenotype (Reyes et al., 1998). Due to evidenceindicating differential functions of BRG1 and BRM, inactivatingmutations of BRG1 could lead to cancer development orprogression.

Further characterization of BRG1 and BRM mutations willprovide important insights into the normal functions of theseproteins, their unique properties and how their loss leads tocancer progression or development. Investigating thedifferential expression of the catalytic subunits may also lead toinformation about the role of the complex in different cellularenvironments. It will also be important to study the temporalexpression of the catalytic subunits during development anddifferentiation to see if alterations in BRG1 during these timesresult in tumor development. Loss of the catalytic subunits mayprove more catastrophic during these developmental and

Fig. 6. CD44 and CDH1 protein expression in Hs578t and HCT116RNAi stable clones. Total cellular protein was extracted from theHs578t andHCT116 parental cell lines and representative vector andRNAi stable clones usingeitherurea (CD44)orhigh saltRIPA(CDH1)buffers, separated by SDS–PAGEand immunoblotted forCD44 (A,B)or CDH1 (C). b-Actin served as a loading control.



differentiation windows leading to tumor initiation while loss ofthe catalytic subunits in differentiated tissues may contributemore to cancer progression. Our studies also emphasize theneed for careful mutational analysis of BRG1 in primary tumormaterial because single amino acid changes can lead tosubstantial alterations in protein function.

Acknowledgments

We wish to thank Mr. Nisarg Desai for excellent technicalassistance.

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Fig. 7. ChIP analysis of BRG1binding at cellular promoters. The parent aswell as representative cell lines from the vector andBRMRNAi stablecloneswereharvestedandproteinwasextracted forChIPsassaysasdescribed intheMaterialsandMethodsSection.ChIPsassayswereperformedusingantibodiesdirectedagainst threecoremembersof theSWI/SNFcomplex includinghSNF5,BAF155,andBRG-1.ChIPanalyseswerecarriedoutatthreepromoterswheretheSWI/SNFcomplexhadpreviouslyshownbinding.Normalrabbit IgGservedasacontrol fornon-specificantibodybinding. Values are the mean of triplicates.



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BRG1 mutations found in human cancer cell lines inactivate Rb-mediated cell-cycle arrest

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