Increased DNA Damage Sensitivity and Apoptosis in Cells Lacking ...

MOLECULAR AND CELLULAR BIOLOGY, Apr. 2006, p. 2661–2674 Vol. 26, No. 70270-7306/06/$08.00�0 doi:10.1128/MCB.26.7.2661–2674.2006Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Increased DNA Damage Sensitivity and Apoptosis in Cells Lacking theSnf5/Ini1 Subunit of the SWI/SNF Chromatin Remodeling Complex

Agnes Klochendler-Yeivin,1* Eli Picarsky,2 and Moshe Yaniv3

Department of Animal and Cell Biology, The Institute for Life Sciences, The Hebrew University of Jerusalem, Givat-Ram,Jerusalem 91904, Israel1; Department of Pathology, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel2;

and Gene Expression and Disease Unit, Department of Developmental Biology, Pasteur Institute, Paris, France3

Received 6 October 2005/Returned for modification 4 November 2005/Accepted 5 January 2006

The gene encoding the SNF5/Ini1 core subunit of the SWI/SNF chromatin remodeling complex is a tumorsuppressor in humans and mice, with an essential role in early embryonic development. To investigate furtherthe function of this gene, we have generated a Cre/lox-conditional mouse line. We demonstrate that Snf5deletion in primary fibroblasts impairs cell proliferation and survival without the expected derepression ofmost retinoblastoma protein-controlled, E2F-responsive genes. Furthermore, Snf5-deficient cells are hyper-sensitive to genotoxic stress, display increased aberrant mitotic features, and accumulate phosphorylated p53,leading to elevated expression of a specific subset of p53 target genes, suggesting a role for Snf5 in the DNAdamage response. p53 inactivation does not rescue the proliferation defect caused by Snf5 deficiency butreduces apoptosis and strongly accelerates tumor formation in Snf5-heterozygous mice.

Chromatin-remodeling complexes modulate nucleosomestructure in an ATP-dependent manner (40, 49). The 2-MDamultisubunit SWI/SNF complex was the first such complexidentified in yeast and is highly conserved among eukaryotes(14, 54). Mammalian SWI/SNF complexes are heterogenousbut have several common subunits, including SNF5 (Ini1),BAF155/BAF170, and one of the highly homologous ATPasesubunits (Brm or Brg1) (46, 72). SWI/SNF complexes playimportant roles in transcriptional regulation (both activationand repression) and contribute to the control of diverse cellu-lar processes, such as proliferation and differentiation (21, 39,44, 59, 63). Gene-targeting experiments with mice have shownthat the mammalian SWI/SNF complex is essential for earlyembryonic development, since homozygous inactivation ofgenes encoding different subunits (Brg1, Snf5, or Swi3/Srg3)results in peri-implantation lethality (11, 22, 37, 38, 57).

Accumulating genetic evidence has defined SNF5/Ini1 as atumor suppressor gene in humans and mice. Homozygous in-activating mutations or deletions in the SNF5 gene in humansare associated with malignant rhabdoid and atypical teratoid/rhabdoid tumors (7, 62, 69). These are rare, but very aggres-sive, pediatric tumors that arise primarily in the brain andkidney. Furthermore, we and others have shown that Snf5functions as a tumor suppressor gene in mice. HeterozygousSnf5�/� animals develop tumors at a high incidence via loss ofheterozygosity (LOH) at the Snf5 locus. These tumors sharehistological features with their human counterparts and de-velop at very specific sites, mainly in the nervous system (22,38, 57). Recently, it was shown that mice with a reversibleconditional mutation of Snf5 developed early, and fully pene-trant, T-cell lymphomas (58). Several studies have also re-vealed that Brg1 is mutated or deleted in a variety of human

tumor cell lines (74). Mice heterozygous for a Brg1-null muta-tion are cancer prone and develop tumors of epithelial originwithout loss of heterozygosity (11).

It is still unclear whether the tumor suppressor function ofSnf5 depends on SWI/SNF activity. Although it has beenshown that the expression of several Brg1 target genes is notaffected by the absence of Snf5 and that the SWI/SNF complexremains intact in Snf5-deficient cells (17), the similar pheno-types of Snf5 and Brg1 knockout mice suggest that both genesfunction in partially redundant pathways. The molecular mech-anisms dictating the tumor-suppressing functions of Snf5 andBrg1 are not fully understood. The antiproliferative activity ofBrg1 has been shown to depend on the repression of specificE2F target genes (those for cyclin E and cyclin A and cdc2) viaits association with the retinoblastoma gene product (pRb) (65,80). Reexpression of Brg1 in mutant cell lines can restore pRbfunction via induction of the cyclin-dependent kinase inhibi-tors p15INK4b and p21cip1 (27). Similarly, reexpression ofSNF5 in human rhabdoid cell lines causes G0/G1 arrest. Thismay occur via downregulation of specific cyclin-encoding genesor via direct activation of p16INK4a, implicating the cyclinD/CDK4-pRb-E2F pathway in tumor formation (6, 51, 68, 81).

Studies of mouse models for Snf5 inactivation indicate thatSnf5 has a dual role: it prevents tumorigenesis, and paradoxi-cally, it is required for cell survival, as both the Snf5-nulltrophectoderm and inner-cell-mass-derived cell lineages die ofapoptosis (38). Furthermore, mice with a conditionally inacti-vated Snf5 locus die and display bone marrow failure, indicat-ing that Snf5 is required for the survival of hematopoietic cells(58). These results suggest that malignancy due to loss of Snf5depends on the specific cellular context and/or additionalmutations, whereas most cells will not survive the biallelicinactivation event. To understand better the molecular basisunderlying the survival or lethal phenotype triggered by Snf5inactivation, we used a Cre/lox-conditional targeting approachto disrupt Snf5 in cultured primary murine embryonic fibro-blasts (MEFs) (61). MEFs represent a cell type that has been

* Corresponding author. Mailing address: Department of Animal andCell Biology, The Institute for Life Sciences, The Hebrew University ofJerusalem, Givat-Ram, Jerusalem 91904, Israel. Phone: 00972-2-6585945.Fax: 00972-2-6585417. E-mail: [email protected].

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widely used to identify the consequences of gene ablation incell cycle control. We report that inactivation of Snf5 in MEFsimpairs cell growth and survival. This phenotype includes hy-persensitivity to genotoxic stress and signs of defective mitosisand occurs concomitantly with p53 induction and altered ex-pression of several key players involved in cell cycle regulation.Although p53 inactivation cannot rescue the growth arrestphenotype in cultured Snf5-deficient MEFs, we show that itsignificantly reduces the apoptotic response and considerablyaccelerates the onset of rhabdoid tumor formation in Snf5-heterozygous mice.

MATERIALS AND METHODS

Mice. The targeting vector and the conditional-deletion procedure have beendescribed in detail elsewhere (21). The floxed allele contains two loxP sites, thefirst one located 1 kb upstream of exon 1 and the other one 0.6 kb downstreamof exon 2. Genotyping of mice and embryos was performed by PCR using primersflanking the second loxP site (5�-CTTGCCAGGTGAGCAGTCTG and 5�-GCCACCAGCCAGATGTCATAC). Mice carrying a null allele of Snf5 (designatedSnf5�) have been described previously (38). Snf5-heterozygous mice were bredwith mice carrying a mutated null p53 allele (33). All the animals were on a mixed129/SV � C57BL/6 genetic background.

Survival curves were compiled from animals that died or were sacrificed whenseriously ill or displaying an obvious tumor.

MEF generation, culture, and infection. Primary MEFs were isolated from day13.5 postcoitum embryos by standard methods. Briefly, the brains and livers wereused for genotyping, and the remainder of the embryos were treated with trypsin,washed once in phosphate-buffered saline (PBS), and cultured in Dulbeccomodified Eagle medium (DMEM) supplemented with 10% fetal calf serum(FCS). MEFs at passage 2 or 3 were infected with adenovirus type 5-cytomeg-alovirus Cre (AdCre) (University of Iowa Gene Transfer Vector Core) in 2%FCS-DMEM at a multiplicity of infection of 100 per cell. At 18 to 24 h after thestart of infection, the virus-containing medium was removed and replaced withfresh 10% FCS-DMEM.

Southern blot analysis. Genomic DNA was extracted from mock- or AdCre-infected MEFs and from tumor samples to monitor Cre-mediated deletion andLOH, respectively. Tumor DNA was digested with PstI, separated by electro-phoresis on a 1% agarose gel, and transferred to a Hybond N� membrane(Amersham) using standard Southern blotting techniques. Blots were probedwith a radiolabeled genomic 5� external probe as described previously (38).

Cell proliferation assays. For growth curves, 300,000 mock- or AdCre-infectedMEFs were seeded in duplicate 2 days after infection. At the indicated timeperiods, cell cultures were trypsinized and washed, and live-cell numbers weredetermined by trypan blue exclusion and plated at the same initial density.

To monitor cells in S phase, we performed BrdU incorporation assays. Cellsplated on coverslips were labeled with 10 �M BrdU for 30 min. Cells were thenfixed in ice-cold methanol for 10 min, incubated in 2 N HCl for 10 min, andwashed with 0.1 M sodium borate (pH 8.5) and then with PBS. Incorporation ofBrdU was monitored by immunostaining with anti-BrdU antibodies (Ab-3; Neo-markers) and with fluorescein isothiocyanate-conjugated goat anti-mouse immu-noglobulin Gs (IgGs). The percentage of BrdU-labeled nuclei was quantified bycounting at least 300 DAPI (4�,6�-diamidino-2-phenylindole)-counterstained nu-clei for each MEF preparation.

Genotoxin sensitivity. Three days after infection, mock- and AdCre-infectedMEFs plated at 5 � 104 to 10 � 104 cells/well in six-well dishes were exposed toUV light (254 nm) or doxorubicin at the doses indicated in Fig. 5. UV exposurewas achieved using StrataLinker source (Stratagene Corp.) after gentle aspira-tion of the culture medium. Cell survival was determined 24 h (doxorubicin) or3 days (UV) later by trypan blue exclusion and expressed as a percentagecompared to that of untreated, nonconfluent cells. All experiments were con-ducted in triplicate, and the cell survival rate for each replicate was determinedtwice at each experimental point. Thus, each data point represents the mean ofsix observations.

Flow cytometry. Cells (1 � 106 to 2 � 106) were harvested at various timepoints after infection, pelleted, and washed in PBS. Cells were fixed in ice-cold70% ethanol. After fixation, cells were washed in PBS and stained in PBScontaining 50 �g/ml propidium iodide (Sigma) and 100 �g/ml RNase A. Flowcytometry analysis was carried out (BD Biosciences) and analyzed usingCellQuest software.

Protein analysis. Whole-cell extracts were prepared in lysis buffer (20 mMphosphate buffer [pH 7], 250 mM NaCl, 30 mM Na4P2O7, 0.1% NP-40, 5 mMEDTA) containing the complete cocktail of protease inhibitors (Roche). Proteinlevels were determined by Western blot analyses by following established pro-tocols. The rabbit polyclonal anti-SNF5 was described previously (48). Antibod-ies against p21 (F-5), cyclin E (M-20), and cyclin A (C-19) were obtained fromSanta Cruz. Antibodies against �-tubulin were from Sigma. The phospho-p53(Ser15) antibodies were obtained from Cell Signaling Technology, and p19ARF

antibodies were from Novus Biologicals. The mouse monoclonal anti-p53 (248and 421) and anti-mdm2 (2A10 and 4B2) were a kind gift from Y. Haupt(Hebrew University, Jerusalem, Israel). Secondary antibodies coupled to horse-radish peroxidase were purchased from Jackson Immunoresearch Laboratories.Detection was performed by chemiluminescence.

Semiquantitative RT-PCR. We carried out reverse transcription (RT)-PCR ontotal RNA prepared with the RNeasy mini kit (QIAGEN). RNA (2 �g) wasreverse transcribed into cDNA using random primers and Moloney murineleukemia virus reverse transcriptase (Promega). cDNA aliquots (0.8 ng and 2.4ng) were amplified by PCR and labeled with [�-32P]dCTP, using the minimumnumber of cycles to obtain a clear signal in the linear range. The following are thenumber of cycles and the sequences of 5� and 3� primers used for each of thetested genes encoding the indicated proteins, as follows: GAPDH (glyceralde-hyde-3-phosphate dehydrogenase), 22 cycles, 5�-GCCTGGAGAAACCTGCCAAG and 5�-CTCCTTGGAGGCCATGTAGG; SNF5, 27 cycles, 5�-TCCGGGATCAAGATAGGAACAC and 5�-TGGAATGTGTGCTGAAGGGAG; p16,26 cycles, 5�-CAGACAGACTGGCCAGGGC and 5�-GAGAAGGTAGGGTCCTC; p19ARF, 29 cycles, 5�-GAGGGTTTTCTTGGTGGAGTTC and 5�-GAGAAGGTAGGGTCCTC; mdm2, 24 cycles, 5�-TCACAGTCTATCAGACAGGAG and 5�-TCCCCTTATCGTGAAGC; p21, 24 cycles, 5�-CACGTGGCCTTGTCGCTGTC and 5�-CACACAGAGTGAGGGCTAAGG; Puma, 29 cycles,5�-AGCACTTAGAGTCGCTG and 5�-AGGGTGAGGGTCGGTGTCG; Bax,28 cycles, 5�-CCCTGTGCAACTAAAGTGCCC and 5�-ACCCCTCCCAATAATTACAAAAG; thymidine kinase (TK), 26 cycles, 5�-GCAGCATCTTGAACCTGGTG and 5�-CTCAGTTGGCAGAGTTGTATTG; dihydrofolate reductase(DHFR), 29 cycles, 5�-TTGTGACAAGGATCATGCAGG and 5�-ACTAGGGTTGGGGTGGCTC; cyclin E1, 26 cycles, 5�-CTGGACAAAGCCCAAGCAAAG and5�-AGGCCAGCAACCGCCATGG; E2F1, 26 cycles, 5�-GGAGAAGTCACGCTATGAAACC and 5�-CTATGACCATCTGTTCTGAGG; p73 with an N-terminaltransactivation domain (TA-p73), 38 cycles, 5�-CAGACAGCACCTACTTTGACCand 5�-GGTATTGGAAGGGATGACAGG; p73 without the N-terminal transac-tivation domain (�N-p73), 38 cycles, 5�-CACGAGCCTACCATGCTTTAC and5�-GGTATTGGAAGGGATGACAGG.

Each RT-PCR was performed at least twice with RNAs extracted from twoinfection experiments using different sets of MEF preparations.

Immunofluorescence and histology. Cells plated on coverslips were fixed with4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 inPBS for 10 min. Coverslips were washed with PBS, incubated for 1 h withblocking buffer (1% bovine serum albumin, 0.05% Tween 20 in PBS), andincubated with anti-�-tubulin (dilution, 1:1,000) or anti-�-tubulin (dilution,1:300) primary antibodies (Sigma) for 2 h. Coverslips were rinsed three timeswith PBS and incubated for 1 h with fluorescein isothiocyanate-conjugated sec-ondary anti-mouse antibodies (Calbiochem). After counterstaining with DAPIand mounting using Vectashield (Vector Laboratories), immunofluorescencewas monitored and images were digitally recorded.

Tumors were fixed in phosphate-buffered 4% formaldehyde (pH 7.4), embed-ded into paraffin, and cut in 5-�m sections. Sections were stained with hema-toxylin and eosin.

RESULTS

Conditional inactivation of Snf5 in MEFs causes cell cyclearrest and cell death. To circumvent the embryonic lethalityinherent in homozygous-Snf5 deletion, we used the Cre/loxPrecombination system of bacteriophage P1 to generate condi-tional-Snf5-knockout mice. The Snf5lox allele carries two loxPsites abutting the 5� regulatory sequences and the first two exonsof the murine Snf5 gene (Fig. 1A). We obtained Snf5lox/lox miceat the normal frequency, confirming that the floxed Snf5allele did not have an adverse effect on the development ofanimals. By contrast, homozygosity for the deleted allelewas lethal at peri-implantation, as observed with the Snf5

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null allele, confirming that the Cre-mediated deletion inac-tivates Snf5 (data not shown). To study the effect of Snf5loss on primary cell proliferation, day 13.5 postcoitum em-bryos were isolated from parental crosses of Snf5lox/� mice or

crosses between Snf5lox/lox and Snf5�/� mice (38) to generateSnf5lox/�, Snf5lox/lox, and Snf5lox/� MEFs.

The cells were infected with an AdCre, and we confirmed bySouthern blotting that the infection resulted in a virtually com-

FIG. 1. Proliferative defects and cell death in Snf5-depleted MEFs. (A) Schematic representation of the wild-type (Snf5�), floxed (Snf5L), and deleted(Snf5D) alleles of Snf5. Exons 1 to 3 are indicated by boxes and loxP sites by filled triangles. Relevant restriction sites (EcoRI [E], EcoRV [EV]) and theposition of the probe are indicated. Cre-mediated recombination eliminates exons 1 and 2. Southern blot (B) and Western blot (C) analyses of DNA andlysates prepared from mock- and AdCre-infected MEFs with the indicated genotypes, 4 and 6 dpi. For Southern blot analysis, the DNA was digested withEcoRI and EcoRV and the Snf5 locus was identified by hybridization with the probe indicated in panel A. (D) Exit of Snf5-deficient MEFs from the cellcycle. Cells of indicated genotypes are shown 6 days after infection (magnification, �100). (E) Representative growth curves of mock- and AdCre-infected MEFswith the indicated genotypes. The cells were placed in culture at equal densities, infected, counted, and passaged every 2 days at the same density. Experimentswere performed on two different MEF cultures of each genotype, and duplicate plates were counted. The curves represent the means of these values.

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plete deletion of the floxed sequence of the gene (Fig. 1B).Western blot analysis revealed that 4 days after infection, thelevel of Snf5 protein in infected Snf5lox/lox MEFs droppedsignificantly (Fig. 1C). The protein level remained unchangedin mock- or Cre-infected Snf5lox/� cells, indicating the exis-tence of a compensation mechanism that adjusts the SNF5protein level in heterozygotes. Transcriptional compensationof Snf5 in heterozygous mouse has been described previously(23). We measured the proliferation rate of wild-type, het-erozygous, and Snf5-deficient cells. The difference in thegrowth rates between control- and Cre-treated Snf5lox/� MEFswas modest, suggesting that ectopic Cre expression from ouradenoviral vector did not affect cell growth to any appreciableextent. In contrast, Cre-mediated excision of Snf5 from Snf5lox/

lox and Snf5lox/� MEFs severely impaired their growth (Fig.1E). Moreover, 6 days postinfection (dpi), morphologicalchanges were observed in AdCre-infected Snf5lox/lox MEFs,which did not occur in infected Snf5lox/� MEFs. These changesincluded a significant number of detached cells, with the re-maining adherent cells displaying elongated and flattened orrounded morphology (Fig. 1D). In situ detection of -galacto-sidase activity at pH 6.0 was performed on AdCre-infectedSnf5lox/lox and Snf5lox/� MEFs to assess for senescence (16).

Loss of Snf5 did not affect the percentage of senescence-asso-ciated, -galactosidase-positive cells (data not shown).

To investigate whether Snf5-depleted cells were arrested ata specific stage of the cell cycle, we analyzed the cells for theirDNA content by flow cytometry (Fig. 2A). At 4 days postin-fection, unsynchronized knockout cells had a profile of DNAcontent similar to that of control cells. However, 6 days afterinfection, we observed a modest but consistent decrease in thepercentage of cells in the G1 phase and a corresponding in-crease in the fraction of cells with sub-G1 DNA content. WhenSnf5-depleted cells were cultured in low serum for 3 days, theapoptotic sub-G1 population increased significantly, reachingup to 30% of the total population. To examine whether Snf5-deficient cells proceed through DNA replication, we moni-tored BrdU incorporation. By 6 days postinfection, Snf5-nullcells exhibited a 50 to 70% decrease in the percentage of cellsreplicating their DNA (Fig. 2B). Since Snf5 depletion did notaffect the percentage of cells in S phase (Fig. 2A), this resultmay reflect a slower progression of the replication forks orunscheduled DNA replication with subsequent triggering of anS-phase checkpoint. Further, the fluorescence-activated cellsorter (FACS) profile of serum-starved, Snf5-depleted MEFsrevealed an increased subpopulation with S-phase DNA con-

FIG. 2. Snf5 depletion impairs cell cycle progression and increases the rate of apoptosis under growth-inhibiting conditions. (A) Cell cycleanalysis of mock- and AdCre-infected Snf5lox/� and Snf5lox/� MEFs, 6 days after infection. The cells were cultured in 10% serum-containingmedium or serum starved for 72 h before the analysis. DNA content was determined by propidium iodide staining and flow cytometric analysis.(B) BrdU incorporation assays of Snf5lox/�, Snf5lox/�, and Snf5lox/lox MEFs that were mock or AdCre infected. The cells were incubated with BrdUfor 30 min and processed for immunostaining. The graph is representative of three independent experiments performed on different preparationsof MEFs.



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tent yet no BrdU incorporation (Fig. 2A and data not shown),a profile consistent with a defective G1 arrest coupled withactivation of an S-phase checkpoint. The similar cell cycleprofiles and the unaltered BrdU incorporation of control- andAdCre-infected Snf5lox/� cells indicated that expression of Creitself had no significant effect on the ability of cells to prolif-erate or progress through the cell cycle. Therefore, the datapresented above indicate that loss of Snf5 in primary fibro-blasts impairs cell viability and cell cycle progression at multi-ple stages of the cell cycle.

Snf5 deletion does not result in increased expression of G1/SE2F target genes. Previous studies suggested that the SWI/SNFcomplex participates in the regulation of cell cycle progressionvia its role in pRb-mediated corepression of E2F1 target genes(39, 59). We reasoned that the phenotype observed mightresult from inappropriate activation of E2F1. We thereforeexamined the effect of Snf5 deletion on the expression of sev-eral E2F1-responsive genes. Interestingly, there were no de-tectable changes in the levels of cyclin E, E2F1 (Fig. 3A), andcyclin A mRNA (not shown). Furthermore, upon serum star-vation, cyclin E and cyclin A levels were reduced in bothSnf5-positive and -negative cells (data not shown), indicatingthat in primary fibroblasts, Snf5 is not required for Rb-medi-ated repression of G1/S-specific E2F target genes. These re-sults are consistent with a previous study showing that humanSNF5, unlike human BRG1, is not required for Rb-mediatedcell cycle arrest (6). DHFR and TK, both E2F1 target genesinvolved in the synthesis of deoxynucleotides essential forDNA synthesis, were, on the contrary, downregulated in Snf5-null cells (Fig. 3A). Real-time PCR confirmed that the tran-scriptional expression of both genes was decreased by 2-foldand 1.5-fold, respectively (data not shown). This finding isconsistent with Snf5-depleted cells being impaired in S-phaseprogression, as suggested by the decrease in BrdU incorpo-ration.

p53 accumulates in Snf5-depleted cells. We next investi-gated whether the increased apoptosis that we observed in theabsence of Snf5 could be related to an upregulation of pro-apoptotic genes. We therefore examined the expression of p53and several of its target genes in mock- and AdCre-infectedSnf5lox/�, Snf5lox/lox, and Snf5lox/� MEFs (Fig. 3B and C). By 4days after infection, Snf5 deletion led to an increase in p53protein levels compared to those of control cells. Serum with-drawal, between days 4 and 6, caused a further increase in thelevel of p53 protein in Snf5-depleted MEFs compared to thatof their wild-type or heterozygous counterparts (Fig. 3B). p53activation was accompanied by a two- to threefold transcrip-tional induction of the p53-responsive genes Mdm2 and Puma(which encodes a BH3-only proapoptotic Bcl-2 family mem-ber). In contrast, we did not observe effects of Snf5 depletionon the protein levels of p21 and Bax, two other p53 targetgenes that mediate the p53-induced G0/G1 arrest and apopto-sis, respectively (Fig. 3C). The absence of p21 induction mayexplain the reduction in the G1 population of Snf5-null cells, asdetected by FACS analysis. Additional p53-responsive genes(Gadd45, Noxa, Apaf1) involved in growth arrest or apoptosiswere unaffected by Snf5 loss despite the p53 accumulation(data not shown). We also verified the expression pattern ofthe p53 family member p73. The p73 gene contains two distinctpromoters, P1 and P2, that are used to generate transcripts

encoding TAp73 and �Np73, respectively (77). TAp73 expres-sion is directly induced by E2F1 and contributes to E2F1-mediated apoptosis via activation of p53 target genes (19, 32,45, 79). The �Np73 promoter is activated by p53 and has been

FIG. 3. Expression analysis of E2F target genes and induction ofp53 in Snf5-deficient cells. Semiquantitative RT-PCR analysis of E2F-responsive genes (A) and p53 target genes (C). Total RNA was iso-lated from mock (�)- and AdCre (�)-infected Snf5lox/lox, Snf5lox/�,and Snf5lox/� MEFs, 6 days postinfection. PCR was carried out underlinear amplification conditions with 0.01 �l and 0.03 �l of the 25-�lcDNA sample. One representative experiment of four is shown. Sim-ilar results were obtained with AdCre-infected Snf5lox/lox and Snf5lox/�

MEFs (C). GAPDH expression is shown as a control. (B) Western blotanalysis of lysates prepared from mock- or AdCre-infected Snf5lox/�

and Snf5lox/� MEFs, 4 and 6 days after infection. The cells were grownin 10% serum containing medium or in 0.1% serum (low serum) for72 h as indicated.



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shown to act as a dominant inhibitor of p53 and TAp73 inseveral experimental systems (36, 70). �Np73 was strongly in-duced in SnfF5-null MEFs (Fig. 3C), whereas TAp73 tran-scripts were undetectable and were not affected by Snf5 abla-tion (not shown). We have confirmed by real-time PCR that�Np73 transcripts are undetectable in Snf5-positive MEFsand induced in Snf5-deficient MEFs (data not shown). Weconclude that the p53 protein that accumulates in Snf5-deficient MEFs selectively upregulates a specific subset ofgenes, among them Puma, which may contribute to apopto-sis, and �Np73, which could attenuate the p53-dependentresponse to Snf5 loss.

Mechanisms of p53 activation. Several types of cellularstress, including DNA damage, activate the p53 pathway andpromote cell cycle arrest or apoptosis (50, 56). In addition,

aberrant activation of oncogenes, such as Ras, E1A, or Myc,stimulates the transcription of the ARF tumor suppressor gene(5, 15, 52, 82). When induced, the p19ARF protein sequestersMdm2 to the nucleolus and blocks its shuttling to the cyto-plasm (66, 73). This prevents p53 degradation by Mdm2 andleads to increased p53 stability. We therefore examined a pos-sible effect of Snf5 loss on ARF gene expression at both theRNA and protein levels. When Snf5lox/lox and Snf5lox/� MEFswere infected with AdCre, we observed induction of ARF tran-scription by 4 days postinfection (Fig. 4A). This correlated withincreased accumulation of p19ARF protein at 6 dpi (Fig. 4B).ARF is encoded by the alternative reading frame of the INK4alocus, which also encodes the p16 cyclin-dependent kinase(Cdk4/Cdk6) inhibitor. In contrast to ARF, p16INK4a wasdownregulated in MEFs lacking Snf5, in agreement with pre-

FIG. 4. p19ARF induction and genotoxic stress in Snf5-depleted cells. (A, C) Semiquantitative RT-PCR analysis of Snf5, p19ARF (A), andp16-INK4A (C). Total RNA was isolated from mock (�)- and AdCre (�)-infected Snf5lox/lox and Snf5lox/� MEFs, 4 and 6 days after infection asindicated (A) and from Snf5lox/lox, Snf5lox/�, and Snf5lox/� MEFs 6 days after infection (C) and assayed as described in the legend to Fig. 3. GAPDHexpression is shown as a control (A). The GAPDH control for panel C is included in Fig. 3A. (B) Protein levels of p19ARF in mock- andAdCre-infected Snf5lox/� and Snf5lox/� MEFs, 4 and 6 days after infection, were determined by Western blot analysis. The cells were grown in 10%serum-containing medium or in 0.1% serum (low serum) for 72 h as indicated. (D) Phosphorylation of p53 at serine 18 is detected in Snf5-depletedMEFs. Lysates from mock- or AdCre-infected Snf5lox/lox and Snf5lox/� MEFs were analyzed by Western blot analysis. Extracts from cells harvested16 h after UV irradiation (10 J/m2) were included as a positive control.



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vious studies that showed that SNF5 activates p16INK4a tran-scription (Fig. 4C) (6, 51). We have confirmed p16INK4a down-regulation (2.5-fold reduction) in Snf5-deficient MEFs by real-time PCR (data not shown). The fact that the delayed kineticsof p19ARF protein increase versus p53 accumulation in Snf5-depleted cells suggested that the initial activation of p53 mightinvolve an alternative pathway. Genotoxic stress induces p53via posttranslational modifications of the protein. A well-char-acterized modification triggered by DNA damage is the phos-phorylation of serine 15 (or the corresponding serine, serine18, in mice), which increases p53 protein stability and/or activ-ity (4, 64). To determine whether Snf5 loss-induced p53 acti-vation occurs through DNA damage signaling, we analyzed thephosphorylation status of Ser18 in Snf5-depleted MEFs. Asshown in Fig. 4D, phosphorylated p53 accumulated in AdCre-infected Snf5lox/lox MEFs but not in Cre-treated Snf5lox/� cells.p53 phosphorylation was induced by UV irradiation in bothnormal and Snf5-depleted cells. The level of phosphorylationcaused by Cre-mediated deletion of Snf5 roughly correspondedto that induced by 10 J/m2 of UV irradiation. Furthermore, thekinetics of phosphorylation coincided with p53 accumulation.Thus, our results suggest that the cellular response to DNAdamage followed by p19ARF upregulation activates p53 inSnf5-null MEFs.

Defective DNA repair in Snf5-null cells. The DNA damageresponse in Snf5-null MEFs could result from failure to repairspontaneous DNA damage caused by oxidative stress occur-ring under standard culture conditions (53), from defects inDNA replication, such as collapsed replication forks, or fromimpaired chromosome condensation or segregation.

Recent studies indicate that the yeast SWI/SNF complex canstimulate nucleotide excision repair (NER) of damaged chro-matin in vitro (20, 25, 26). This repair pathway is used for theremoval of UV-induced cyclobutane-pyrimidine dimers andpyrimidine (6–4) photoproducts. To test whether Snf5 wasindeed required for a proper cellular response to such dam-age, we examined the viability of AdCre-infected Snf5lox/lox,Snf5lox/�, and Snf5lox/� MEFs after irradiation with increasingdoses of UV irradiation. Snf5-null MEFs were 3.5- to 6-foldmore sensitive to UV radiation than Snf5-heterozygous MEFs(Fig. 5A). This finding indicates that Snf5 plays an essentialrole in the ability to survive UV-induced DNA damage, sug-gesting that it could function in DNA repair in vivo.

In normal MEFs, UV radiation triggers a p53-independentG1 checkpoint and a G2/M checkpoint activated by the ATR/Chk1 or p38 kinase pathway. Although our findings raise thepossibility that DNA excision pathways are impaired in theabsence of Snf5, they do not exclude a role for Snf5 in check-point control. Snf5-null cells also displayed increased sensitiv-ity, although less marked, to DNA double-strand breaks causedby the genotoxic agent doxorubicin (Fig. 5B). Thus, Snf5 deletioncauses increased sensitivity to distinct genotoxic agents, estab-lishing that the gene plays an essential role in the ability tosurvive DNA damage.

Aberrant mitotic features in Snf5-null cells. Snf5-null MEFsalso displayed aberrant nuclear morphology, which is charac-teristic of impaired mitosis and/or cytokinesis failure. We de-tected a two- to threefold increase in micronucleus formationin these cells in comparison with that of heterozygous MEFs(Fig. 5C). Micronuclei are indicative of lagging chromosomes

and can appear as a result of a failure to activate the G2/Mcheckpoint in the presence of incomplete replication or repairor from abnormal spindle organization. Furthermore, we coulddetect interphase nuclei with bridging chromatin, a featurethat results from unbalanced chromosomal segregation or de-fective cytokinesis. As centrosomes play a central role in spin-dle assembly, we determined the centrosome number in Snf5-depleted cells versus that of heterozygotes. The frequency ofSnf5-null cells containing more than two centrosomes wasmore than double that seen in heterozygous cells. Most Snf5-null cells with increased numbers of centrosomes had large,partially cleaved, bilobed nuclei or micronuclei, features highlysuggestive of aberrant cytokinesis (Fig. 5D). Consistently, arecent study reports that loss of SNF5 function promotespolyploidization and genomic instability in human cells (71).

Growth properties of p53-null, Snf5-deficient MEFs. To ex-amine further the contribution of p53 induction to the growthand survival defects caused by Snf5 ablation, we preparedMEFs from p53�/�; Snf5lox/� mouse offspring and determinedthe growth rate of AdCre-infected p53�/�; Snf5lox/lox andp53�/�; Snf5lox/� cells over 10 days.

The absence of p53 did not alleviate the decreased prolifer-ation capacity of Snf5-depleted cells (Fig. 6A). By 12 dayspostinfection, AdCre-infected p53�/�; SNF5lox/lox cultures re-sumed a proliferation rate similar to that of Snf5lox/� cultures.However, Western blot analysis revealed that in the AdCre-infected p53�/�; Snf5lox/lox cells, the level of Snf5 increasedprogressively (Fig. 6B). Moreover, the cells remaining in cul-ture 15 days postinfection were heterozygous, harboring onefloxed and one deleted Snf5 allele (Fig. 6C). Thus, prolongedculture of AdCre-infected p53�/�; Snf5lox/lox MEFs led to theselective loss of Snf5-depleted cells and enrichment in thesmall cell population that had escaped biallelic deletion. How-ever, inactivation of p53 protected Snf5-deficient MEFs fromcell death. Cre-infected p53�/�; Snf5 lox/lox MEFs showed a2-fold decrease in the percentage of apoptotic cells (Fig.6D). This observation correlated with the absence of Pumainduction in these cells, in contrast with Cre-infected p53 wild-type; Snf5lox/lox MEFs (Fig. 6E). Further, the enhanced apop-tosis observed in serum-deprived Snf5�/� MEF cells was alsostrikingly reduced in p53-null, Snf5-depleted MEFs (Fig. 6D).From these experiments, we conclude that in this cellular con-text, Snf5 loss triggers a p53-dependent apoptotic response, inwhich Puma might be involved, and a p53-independent prolif-eration arrest.

p53 mutation accelerates rhabdoid tumor development inSnf5�/� mice. It was also interesting to monitor the conse-quence of the absence of p53 in our tumor-prone Snf5-het-erozygous mice. Would the absence of an apoptotic responseaccelerate tumor formation? A comparison of the tumor inci-dence of Snf5�/� mice with that of Snf5�/�; p53�/� com-pound-mutation mice revealed a strong genetic interactionbetween the inactivation of these two tumor suppressor genes(Fig. 7A). Whereas 28% of Snf5�/� mice developed tumorswith a mean latency of 42 weeks, 100% of the Snf5�/�; p53�/�

mice (n � 49) developed tumors within 19 weeks (2 monthsearlier than animals homozygous for the p53 mutation alone).Interestingly, while p53 deficiency accelerated tumorigenesis,the distribution of the tumors in the different anatomic loca-tions, as well as their type (sarcomas, some of them with



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rhabdoid features), was not altered. Fifty-five percent of thetumors developing in Snf5�/�; p53�/� mice arose in the ner-vous system (brain, cerebellum [shown in Fig. 7C], or spinalcord). Subcutaneous tumors developed in other sites, including

cheek, limb, eye, and axillary sites, as observed in Snf5-het-erozygous animals (Table 1). Twenty-five percent of the ani-mals developed two concurrent tumor foci affecting differenttissues, such as brain or cerebellum and cheek or limb; these

FIG. 5. Snf5-null cells display increased sensitivity to genotoxic agents, abnormal mitotic features, and amplification of centrosomes. Mock- andAdCre-infected MEFs with the indicated genotypes were plated in six-well culture dishes and exposed to increasing doses of UV light (A) ordoxorubicin (B) at day 3 after infection. The percentage of cell death in each well was assessed by trypan blue exclusion 72 h (A) or 24 h (B) aftertreatment. Each data point represents the mean of six observations. Similar results were obtained in three independent experiments. The differencein UV hypersensitivity between AdCre-infected Snf5lox/lox and Snf5lox/� MEFs is apparently due to experimental variability. All the Snf5-deficientclones (Cre-infected Snf5lox/lox or Snf5lox/�) tested were three- to sixfold more sensitive to UV radiation than their heterozygous counterparts,independently of whether the infected cells carried two floxed alleles or one floxed and one null allele. (C, D) Snf5lox/� and SNF5lox/� MEFs wereinfected with AdCre and immunostained 4 days after infection. (C) AdCre-infected Snf5lox/� MEFs were stained with anti-�-tubulin (green) andcounterstained with DAPI. White arrows indicate micronuclei, and yellow arrows point to interphase bilobed nuclei with bridging chromatin.(D) AdCre-infected Snf5lox/� MEFs were stained for centrosomes with anti-�-tubulin (green) and counterstained with DAPI. Micronuclei (C) andcentrosomes (D) of infected Snf5lox/� MEFs and Snf5lox/� MEFs were counted for 100 cells per sample in three independent experiments.

FIG. 6. p53-null background does not rescue the slowed proliferation phenotype in Snf5-depleted cells but reduces apoptosis. (A) Growthcurves of mock- and AdCre-infected p53-null MEFs with the indicated genotype for the Snf5 locus. The experiment was performed as describedfor Fig. 1E, and the graph shows a representative of two independent experiments. Western blot (B) and Southern blot (C) analyses of DNA andlysates prepared from mock- and AdCre-infected p53-null MEFs with the indicated genotypes, 5, 12, and 15 days postinfection. (D) p53 wild-typeand p53-null MEFs with the indicated genotypes at the Snf5 locus were infected with AdCre and grown in 10% or 0.1% serum-containing mediumfor 72 h. The percentage of cell death was determined by trypan blue exclusion in three independent experiments. The percentages of cells witha sub-G1 content of DNA as determined by FACS analysis were similar. (E) Semiquantitative RT-PCR analysis of Puma. Total RNA was isolatedfrom mock (�)- and AdCre (�)-infected MEFs with the indicated genotype 6 days after infection and assayed as described in the legend to Fig.3. GAPDH expression is shown as a control.



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tumors displayed histological characteristics of Snf5-associatedlesions (undifferentiated sarcoma, some of them with rhabdoidfeatures). Histological examination of 22 tumors has been per-formed. Among these 22 tumor samples, 12 (60%) displayedrhabdoid features (presence of typical rhabdoid cells) (Fig.7D). The other lesions were defined as high-grade sarcomas(Table 1). Importantly, histological analysis revealed thatSnf5�/�; p53�/� mice did not develop thymic lymphoma, atumor type associated with mutation of p53. We believe thatthe early death of p53-null, Snf5-heterozygous mice due to thedevelopment of Snf5-associated tumors has precluded the fur-ther development of lymphomas. Southern blot analysis ofDNA from six tumors demonstrated that loss of the wild-typeSnf5 allele accompanied tumorigenesis in all tested tumors(Fig. 7E). Thus, loss of p53 accelerates Snf5-associated tumor-igenesis without altering the tumor spectrum in Snf5-hetero-

zygous mice, implying that inactivation of p53 and Snf5 coop-erate in tumorigenesis in specific tissues in mice.

DISCUSSION

The SNF5/INI1 protein was initially identified as a core subunitof the mammalian SWI/SNF complex (48, 55). Gene inactiva-tion in mice revealed that Snf5 and several other core subunitsof the SWI/SNF complex are essential for early mouse devel-opment (11, 22, 37, 38, 57). In culture, Snf5�/� cells from theinner cell mass and trophoblasts fail to proliferate and undergoapoptosis (38). Paradoxically, biallelic inactivation of the SNF5gene is associated with aggressive rhabdoid tumors in humansor mice (39, 59). These data suggest that Snf5 may be essentialin some cell types, whereas other cells become highly malig-nant in its absence. To unravel essential and growth-control-

FIG. 7. Snf5 and p53 deficiency cooperate in tumorigenesis in vivo. (A) Kaplan-Meier tumor-free survival curves of Snf5�/� (n � 32), p53�/�

(n � 25), and Snf5�/�; p53�/� (n � 49) mice (confidence interval [C.I.], 95%). The curves were compared pairwise by the log rank test (P � 0.0001).The mean age of survival was 12.5 weeks for Snf5�/�; p53�/� mice and 26 weeks for p53�/� mice. At 60 weeks, 72% of Snf5�/� mice were stillalive and tumor free. (B to D) Photomicrographs of hematoxylin- and eosin-stained slides from Snf5�/�; p53�/� mice (magnification, �400).Section of normal cerebellum (B) versus section showing infiltration of malignant cells into the molecular layer (C). Black and red arrows pointto mitotic and apoptotic figures, respectively. (D) Representative section of a brain rhabdoid tumor. The arrows point to rhabdoid cells with typicalprominent nucleoli and eosinophilic hyaline cytoplasmic inclusions. (E) LOH at the Snf5 locus is observed in tumors (T) from Snf5�/�; p53�/�

mice. Southern blot analysis of DNA extracted from tumors shows that the wild-type Snf5 allele is greatly reduced, while the ratio of mutant towild-type alleles is roughly 1 in adjacent normal tissues (N).



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ling functions of this gene, we studied the consequence ofSnf5/Ini1 deletion in primary mouse fibroblasts by using theCre/loxP system. The results presented here demonstrate thatSnf5 depletion affects several distinct pathways, including theDNA damage response pathway and cell proliferation.

Snf5 deletion causes an impaired response to genotoxicstress and growth arrest. By deleting Snf5 in primary MEFs,we have established that this gene is essential for cell growthand survival under normal conditions in culture. The growthdefect is attributable, at least in part, to an increase in theincidence of spontaneous apoptotic cell death, which was evenhigher under serum-starved conditions. This phenotype is con-sistent with the early embryonic death of Snf5-null mice andbone marrow failure in mice in which Snf5 was conditionallytargeted (58). However, increased cell death alone cannot ac-count for the proliferative defect of Snf5-deficient MEFs. Alarge number of cells arrested in the G1, S, and G2/M phasesstill remained viable 6 days after AdCre infection. Moreover,p53-null; Snf5-null cells did not exhibit significant apoptosisbut still failed to grow.

Growth inhibition in primary cells can be the result of de-fects in receiving growth- and survival-promoting signals or offailure to perform other essential functions of the cell. Thedata that we have obtained in this study suggest that Snf5-deficient cells accumulate DNA damage due to a defect intheir DNA repair process. Consistently, Snf5 loss was accom-panied by increased expression of phosphorylated p53, as dis-cussed below. This was corroborated by the higher sensitivityof Snf5-null MEFs to DNA-damaging UV irradiation. In-creased sensitivity to the double-strand-break-inducing agentdoxorubicin was also observed. These findings agree with pre-vious studies that showed that immobile nucleosomes inhibit invitro repair of UV-induced DNA lesions (24). Moreover, yeastSWI/SNF has been directly implicated in stimulating NER onnucleosomal substrates in vitro (20, 25, 26). It is plausible thatSWI/SNF enhances repair by facilitating access to UV-dam-

aged DNA sites. Loss of Snf5 might impair this function. Wecannot formally exclude the possibility that the increased UVradiation sensitivity of Snf5�/� cells reflects downregulation ofgenes involved in DNA repair. However, transcriptome anal-ysis following Snf5 deletion did not reveal any decrease in theexpression of known genes encoding NER components (datanot shown). Another link between the SWI/SNF complex andchromosome integrity was suggested by the isolation of a SWI/SNF-related complex containing the BRCA1 protein (9). Inthis context, it is important to recall recent studies that re-vealed an essential role for the INO80 chromatin-remodelingcomplex in double-strand break repair in yeast (18, 47, 67).However, unlike the INO80 complex, the SWI/SNF complexdoes not contain ATPases of the AAA� family, proteins re-lated to the bacterial RuvB protein, a double-hexameric DNAhelicase involved in Holliday junction resolution.

Increased sensitivity to genotoxic agents may be caused notonly by defects in DNA repair but also by disruption of cellcycle checkpoint responses to DNA damage. Pertinent exam-ples come from cells deficient in ATR or Chk1 genes, whichcode for DNA damage checkpoint proteins. These cells exhibitprogressive proliferative defects and are highly sensitive togenotoxic injury (10, 78). In addition, cell cycle arrest could betriggered by a p53-independent checkpoint response to spon-taneous DNA damage. Snf5-deficient cells showed signs ofsevere misregulation of mitosis, with an increase in micronu-cleus formation indicative of lagging chromosomes, and thepresence of lobulated nuclei connected by bridging chromatin,which reflects cytokinesis failure. These features are charac-teristic of DNA damage-induced mitotic catastrophe or couldreflect defects in the spindle attachment checkpoint and chro-mosome segregation (3, 12). Mitotic catastrophe can also betriggered by the DNA damage-induced, Chk1-dependentcheckpoint (30). Interestingly, the yeast SWI/SNF-related RSCcomplex has been implicated in chromatin structures at cen-tromeres and kinetochore function, and mutation of sfh1, anSnf5 homolog, causes chromosome missegregation (13, 28).Moreover, RSC has a role in cohesin loading onto chromo-some arms and establishment of arm cohesion (29). In humancells, the RSC-related SWI/SNF-B complex localizes at kinet-ochores and spindle poles in prometaphase cells, suggestingthat it could play a role there in mitosis (76). Finally, a studypublished recently shows that SNF5 mutation impairs chromo-some segregation in human cells and that loss of SNF5 resultsin elevated poly- and aneuploid cells (71). Thus, it is possiblethat SNF5 and the SWI/SNF complex play multiple roles incontrolling DNA damage and mitosis.

Snf5 inactivation and the pRb-E2F pathway. Our under-standing of the molecular parameters involved in Brg1- orSnf5-mediated regulation of cellular proliferation has reliedmostly on overexpression experiments, namely, reintroducingthe missing gene into cell lines and analyzing the expression ofkey cell cycle regulators. These experiments led to the conclu-sion that both Brg1 and Snf5 induce G0/G1 arrest via down-regulation of a specific subset of E2F-regulated genes in col-laboration with pRb (39, 59). Additional studies showed thatSnf5 reexpression at physiological levels reinforces pRb-medi-ated transcriptional repression via induction of the cyclin-de-pendent kinase inhibitor p16INK4A (6, 51). Indeed, we foundthat Snf5 inactivation in MEFs caused p16INK4A downregula-

TABLE 1. Incidence and location of tumors in micewith mutations in Snf5 and p53

Tumor parameter

% found in mice of indicated genotype

Snf5�/�

(n � 37)Snf5�/�; p53�/�

(n � 22)p53�/�

(n � 15)

TypeRhabdoida 27 60 0Lymphoma 0 0 75Sarcoma 73 40 25

LocationNervous system 57 55 0Limb 5 8 0Cheek 8 10 0Thymus 0 0 68Other 30 27 32

a Rhabdoid tumors are diagnosed by the presence of typical rhabdoid cells inthe histological section as described in the legend to Fig. 7. The higher propor-tion of rhabdoid tumors in the Snf5�/�; p53�/� mice compared with that ofSnf5�/� mice might be biased as the histological data for the tumors developingin Snf5�/� mice have been taken from our previous study (38). It is worthmentioning that also in humans, the histological and immunohistochemical char-acteristics of rhabdoid tumors are imprecise and have led to diagnostic contro-versies.



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tion. However, the reduction in p16INK4A expression level wasnot associated with upregulation of E2F target genes and ac-celerated cell cycle progression. In contrast, inactivation ofSnf5 in primary MEFs impaired cell proliferation and survivalwhile the expression levels of the E2F-responsive genes, suchas cyclin A, cyclin E, cdc2, and E2F1, were not affected. More-over, we observed a decrease in the transcription of the TK andDHFR genes, which may contribute to the reduction in thefraction of surviving cells that replicate their DNA. It is pos-sible, however, that p16INK4A downregulation loosens the reg-ulation of cell cycle checkpoints through pathways that do notinvolve pRb-E2F. The discrepancy with previous studies couldalso result from cell-type-specific modes of action of Snf5.Alternatively, Snf5 could be required in our system, not onlyfor pRb-mediated repression but also for E2F-mediated tran-scriptional activation of these specific genes. The only E2Ftarget gene that we found induced by Snf5 depletion wasp19ARF. However, additional regulators of p19ARF have beenidentified, i.e., both activators (DMP-1, AP1) and transcrip-tional repressors (Bmi-1, Tbx2, Tbx3, and Twist) (2, 31, 34, 42,43). Thus, it remains unclear whether Snf5 disruption activatesp19ARF via E2F.

Snf5 and the p53 pathway. When ARF is induced, it activatesp53 indirectly, by binding to Mdm2 and inhibiting p53-directedE3 ubiquitin ligase activity and by sequestering Mdm2 into thenucleolus (73). Activated p53 induces transcription of genesinvolved in cell cycle arrest and apoptosis.

The growth and survival defects that we observed in Snf5-depleted cells were indeed accompanied by p53 accumulation.The p53 protein that accumulates in Snf5-depleted MEFs isphosphorylated on serine 18. This modification plays an essen-tial role in both the stabilization and activation of the p53protein. Although p53 phosphorylation is typically induced bygenotoxic stress via ATM/ATR kinase activation, a recentstudy contradicts previous data and indicates that p19ARF canalso induce ATR- and Chk1-dependent phosphorylation ofp53 (60). However, since p53 phosphorylation and stabilizationpreceded the accumulation of p19ARF, we favor the first hypoth-esis. Interestingly, only a subset of p53-responsive genes, includ-ing Mdm2, Puma, and �N-p73, were activated following p53 in-duction in Snf5-null cells, whereas p21, Bax, and Gadd45atranscription levels remained unaltered. Chromatin structures atthe promoters of p53 target genes might dictate the differentialrequirement for Snf5. Promoters with an open configurationmight be activated by p53 in the absence of Snf5, whereas otherregulatory sequences might require chromatin-remodeling activ-ity to potentiate p53-mediated transcriptional activation. Consis-tently, research from several laboratories has implicated BRG1and SNF5 in the activation of p21 gene transcription through bothp53-dependent and -independent mechanisms (27, 35, 41). Theincreased expression of proapoptotic genes like Puma in the ab-sence of p21 activation, as well as the activation of p19ARF coupledwith the decrease in p16INK4A expression, could favor apoptosisover DNA repair and survival in Snf5-null cells.

In contrast with the enhanced apoptosis, the decreased pro-liferation of Snf5-null MEFs is p53 independent, as p53 inac-tivation did not abolish this phenotype. One could postulatethat the accumulation of spontaneous DNA damage and chro-mosome segregation defects are responsible for the prolifera-tion defect that is observed in the absence of p53. In vivo, p53

and Snf5 inactivation synergize to promote tumorigenesis suchthat all Snf5�/�; p53�/� mice succumb to Snf5-associated tu-mors before 4 months of age. The early development of thesetumors can account for the absence of p53-associated lesions inthe compound-mutation mice. It is worth noting that the tumorspectrum of our Snf5-heterozygous mutants (on both p53 wild-type and -null backgrounds) is different from that of the pre-viously published Snf5-conditional mice (i.e., mature T-celllymphomas) (58). Tumor development in our system dependson loss of heterozygosity at the Snf5 locus, whereas Snf5 inac-tivation in the conditional system is mediated by Cre recombi-nase, which has been shown to be highly expressed in thethymus and spleen. It is therefore plausible that the differentknockout systems generate distinct tumor spectrums. The ac-celeration of tumor onset by p53 deletion can be explained bydifferent mechanisms. First, p53 deficiency may increase theproportion of cells that lose the remaining wild-type Snf5 alleleor accelerate the timing of the loss of heterozygosity at the Snf5locus. Second, p53 inactivation could impair the differentiationof cells that become malignant upon Snf5 inactivation. Finally,p53 deficiency might enhance the survival of tumor cells afterthe LOH event at the Snf5 locus. Considering the fact thattumors in Snf5�/� mice arise primarily in the nervous system,it is interesting to note that p53 is involved in key survival/death checkpoints in both peripheral and central neurons (1,75). Moreover, p53 is involved in the differentiation of oligo-dendrocyte precursor cells, and overexpression of �N-p73, thedominant negative p53 family member that is strongly inducedupon Snf5 loss in MEFs, inhibits oligodendrocyte precursorcell differentiation (8). Thus, the cooperation between p53 andSnf5 inactivation in tumorigenesis may extend beyond thegenomic instability which is characteristic of p53-deficient cells.

In conclusion, our present study clearly demonstrates thatSnf5, a member of the core SWI/SNF chromatin remodelingcomplex, is essential for normal cell growth. Snf5 deletionresults in multiple dysfunctions, ranging from a failure to sur-vive DNA damage to an unbalanced transcription programdriven by activated p53. The failure to respond correctly toDNA damage and defects in chromosome segregation maygenerate additional mutations and explain why certain celltypes escape apoptosis and become malignant in the absenceof Snf5.

ACKNOWLEDGMENTS

We are grateful to Moshe Oren for helpful discussions and JonathanWeitzman for critical reading of the manuscript. We thank AharonRazin, Ruthy Shemer, and the friends from the Department of CellBiochemistry and Human Genetics (Hadassah Medical School, He-brew University) for their help and contribution to this work.

This work was supported by the Israel Cancer Research Fund (ICRFproject grant) and Association for International Cancer Researchgrants 00-221 and 03-109 (A.K.-Y.).

ADDENDUM IN PROOF

After submission of this paper, Isakoff et al. (Proc. Natl.Acad. Sci. USA 102:17745–17750, 2005) published a relatedstudy demonstrating apoptosis, polyploidy, and growth arrestin primary fibroblasts lacking Snf5/Ini1, as well as synergy withp53 inactivation in tumor formation.



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