ORIGINAL ARTICLE

The BRG1 ATPase of chromatin remodeling complexes is involved

in modulation of mesenchymal stem cell senescence through RB–P53

pathways

N Alessio1,2, T Squillaro1,3, M Cipollaro4, L Bagella1,2, A Giordano1,5,6 and U Galderisi1,4

1Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA,USA; 2Department of Biomedical Sciences, Division of Biochemistry and Biophysics, University of Sassari, Sassari, Italy; 3MedicalGenetics, University of Siena, Siena, Italy; 4Department of Experimental Medicine, Biotechnology and Molecular Biology Section,Second University of Naples, Naples, Italy; 5Human Pathology and Oncology Department, University of Siena, Siena, Italy and6Human Health Foundation, Spoleto, Italy

We focused our attention on brahma-related gene 1(BRG1), the ATPase subunit of the SWItch/SucroseNonFermentable (SWI/SNF) chromatin remodeling com-plex, and analyzed its role in mesenchymal stem cell(MSC) biology. We hypothesized that deviation from thecorrect concentration of these proteins, which act at thehighest level of gene regulation, may be deleterious forcells. We wanted to know what would happen if a cell hadto cope with altered regulation of gene expression, eitherby upregulation or downregulation of BRG1. We assumedthat cells would try to restore homeostasis or, alterna-tively, that the event could trigger senescence/apoptosisphenomena. To this end, in MSCs, we silencedBRG1gene. Knockdown of BRG1 expression induced asignificant increase in senescent cells and decrease inapoptotic cells. It is interesting that BRG1 downregula-tion also induced an increase in heterochromatin. At themolecular level, these phenomena were associated withactivation of retinoblastoma-like protein 2 (RB2)/P130-and P53-related pathways. Senescence was accompaniedby reduced expression of some stemness-related genes.This is consistent with our previous research, whichshowed that BRG1 upregulation by ectopic expressionalso induced senescence processes. Together, these datasuggest that BRG1 belongs to a class of genes whoseexpression is tightly regulated; hence, subtle alterations inBRG1 activity seem to negatively affect mechanismsregulating chromatin status and, in turn, impair cellularphysiology.Oncogene (2010) 29, 5452–5463; doi:10.1038/onc.2010.285;published online 9 August 2010

Keywords: stem cells; senescence; apoptosis; p53;retinoblastoma; chromatin

Introduction

Within normal tissues, stem cells are defined by commoncharacteristics: self-renewal to maintain the stem cellpool over time, regulation of stem cell number througha strict balance of proliferation, differentiation anddeath and the ability to give rise to a broad range ofdifferentiated cells (Morrison et al., 1997; Gage, 2000;Temple, 2001).

Alterations in stem cell function have been extensivelyreported in a variety of tissues and experimentalsystems. On one hand, impaired stem cell functionalitymay induce defective tissue regeneration and aging, andon the other, uncontrolled self-renewal and proliferationcan trigger tumorigenesis. The antiproliferative effect ofsenescence clearly indicates that this process is a tumorsuppressor mechanism. Early on, senescence was foundto be mediated by the two main tumor suppressorpathways of the cell: the ARF/p53 and the INK4a/RBpathways (Collado et al., 2007). For these reasons,studies on the mechanism of senescence in stem cells areof great interest to dissect the pathways that may controlaberrant cell proliferation and protect against thedevelopment of cancer.

A number of recent reports are beginning to define therole of chromatin organization in the regulation of stemcell biology, addressing the question of what gives stemcells these specific properties.

Modulations of chromatin structure that oftenaccompany regulation of transcription can be achievedby chromatin remodeling complexes. These complexescarry out enzymatic activities, changing chromatinstatus by altering DNA–histone contacts within anucleosome in an adenosine triphosphate (ATP)-depen-dent manner (Martens and Winston, 2003; Trotter andArcher, 2008). These ATP-dependent remodeling com-plexes are divided into at least five classes: SWI/SNF,ISWI, CHD(Mi-2), INO80 and SWR1. Each complex hasa catalytic ATPase subunit that is critical for allowing orimpairing access to nucleosomal DNA to promote orrepress gene transcription, respectively (Saha et al., 2006).

The mammalian SWItch/Sucrose NonFermentable(SWI/SNF) family includes several members that share

Received 3 December 2009; revised 27 May 2010; accepted 31 May 2010;published online 9 August 2010

Correspondence: Professor U Galderisi, Department of ExperimentalMedicine, Biotechnology and Molecular Biology Section, SecondUniversity of Naples, Via Costantinopoli 16, Napoli 80138, Italy.E-mail: umberto.galderisi@unina2.it

Oncogene (2010) 29, 5452–5463& 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10

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most of the same subunits, that is, the ATPase enzyme,either the brahma-related gene 1 (BRG1) or theBRM proteins and/or the presence of tissue-specificisoforms. Complexes containing BRG1 have beenshown to be required for cell cycle control, apoptosisand differentiation in several biological systems(Dunaief et al., 1994; Murphy et al., 1999; Bultmanet al., 2000; Reisman et al., 2002; Martens and Winston,2003; Hendricks et al., 2004; de la Serna et al., 2006).Homozygous knockout mice for the Brg1 gene dieduring the embryonic stage, whereas heterozygoticsurvivors are prone to tumors (Bultman et al.,2000; Saha et al., 2006; de la Serna et al., 2006).BRG1 protein can interact with different proteinsinvolved in regulation of transcription, such as factorsinvolved in myeloid, erythrocyte, lymphocyte,muscle, neural and adipocyte commitment and/ordifferentiation (Dunaief et al., 1994; Murphy et al.,1999; Bultman et al., 2000; Reisman et al., 2002;Martens and Winston, 2003; Hendricks et al., 2004;Saha et al., 2006; de la Serna et al., 2006). The BRG1chromatin remodeling protein can associate withnumerous chromatin-modifying complexes, includingtranscription co-activators and corepressors. Therefore,BRG1 contributes to distinct and even opposite func-tions in the regulation of cell biology, which depend onthe context (cell type, state of differentiation, timing,environmental cues, and so on).

A few reports have analyzed the role of SWI/SNFcomplexes in the biology of stem cells. For example,Matsumoto et al. (2006) showed that BRG1 is requiredfor murine neural stem cell maintenance and gliogenesis.No data were found on the function of BRG1 in thebiology of mesenchymal stem cells (MSCs), which havea key role in the body’s homeostasis. With the currentand previous research, we have decided to address thisissue (Napolitano et al., 2007).

The bone marrow of mammals is composed ofdifferent elements that support hematopoiesis and bonehomeostasis. Among these are MSCs, which are non-hematopoietic stem cells possessing multilineage poten-tial (Muller-Sieburg and Deryugina, 1995; Zhang et al.,2003). MSCs are of interest because of the multiple rolesthey perform. Besides differentiation into mesenchymaltissues, MSCs support hematopoiesis and contribute tothe homeostatic maintenance of many organs andtissues (Prockop, 1997; Beyer Nardi and da SilvaMeirelles, 2006; Sethe et al., 2006).

In our previous research, forced expression of BRG1in MSCs triggered significant cell cycle arrest. This wasassociated with a large increase in apoptosis, along withsenescence process. At the molecular level, thesephenomena were associated with an activation of theretinoblastoma protein (RB)- and P53-related pathways(Napolitano et al., 2007). In this study, we decided tofurther analyze the role of BRG1 in MSCs, becausestudies on chromatin remodelers can be especiallyimportant for dissecting molecular pathways governingthe biology of stem cells.

Altogether, our studies suggest that BRG1 belongs toa class of genes having tightly regulated expression. For

these genes, even subtle alterations in their expressionmay disrupt the normal functioning of cells.

Results

Human MSCs were tested for BRG1 knockdown 48 hafter adenovirus-delivered small interfering RNA(Ad-siRNA) transductions. We obtained a transductionrate of 80–90% with 50–100 multiplicities of infection ofAd-siRNAs (data not shown).

We constructed several recombinant adenoviruses tosilence BRG1. The Ad-siRNA named Ad-siRNA-2405targeting nucleotides 2405–2423 of BRG1 mRNA(National Center for Biotechnology Information acces-sion NM_003072.2) was effective in silencing andinduced a 70% decrease of BRG1 mRNA, as detectedby reverse transcriptase–PCR (Figure 1). Silencing wasfurther verified by analyzing the protein levels of BRG1.We observed a 70% decrease in the target protein(Figure 1). BRG1 silencing was also obtained withanother siRNA to rule out off-target effects (Supple-mentary File 5).

Figure 1 BRG1 silencing. (a) mRNA levels were normalizedwith respect to HPRT as internal control. The histogram showsthe mean expression values (±s.d., n¼ 3). The change in themRNA levels of Ad-siRNA-BRG1-treated cells was compared withthat of Ad-siRNA-CTRL-transduced cells, chosen as reference(**Po0.01). We used the comparative cycle threshold (Ct) methodto quantify the expression levels. (b) Western blot analysis ofMECP2 levels in MSCs treated with Ad-siRNA-BRG1 andAd-siRNA-CTRL, respectively. The protein levels were normalizedwith respect to a-tubulin as the loading control. The table showsthe mean expression values (±s.d., n¼ 3) of protein densitometricanalysis (*Po0.05).

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BRG1 silencing reduced the percentage of S-phase cellsand induced a decrease in apoptosisThe downregulation of BRG1 did modify the cell cycleprofile of MSCs in culture as determined by flowcytometry analysis. We observed a small increase ofG1 in MSCs with silenced BRG1 compared withcontrols (67.61 versus 58.33%). It is noteworthy thatMSCs with silenced BRG1 had a significant lowerpercentage of S-phase cells (Po0.05; 29.11 versus40.23%) and an increase of G2/M cells (3.28 versus1.43%; Figure 2a).

Annexin assays evidenced a reduced percentage ofapoptotic cells in cultures from BRG1-silenced sampleswhen compared with the controls (Figure 2c). Aterminal deoxynucleotidyl transferase dUTP nick-endlabeling (TUNEL) assay also showed a decrease in thenumber of apoptotic cells in MSCs with silenced BRG1(5.9±0.7% versus 2.7±0.6%). These results wereconfirmed in cells with BRG1 silenced by anothersiRNA to rule out off-target effects of the siRNA 2405(Supplementary File 5).

We did not detect a modification of caspase 9 activityin cells treated with Ad-siRNA targeting BRG1,suggesting that the decrease in apoptosis may occurindependently of the cytochrome c/Apaf-1/caspase-9apoptosome, as observed in other systems (Marsdenet al., 2006; Figure 2b). It is interesting that in ourprevious research, we found that the upregulation ofBRG1 expression in MSC cultures induced an increasein programmed cell death (Napolitano et al., 2007).

BRG1 silencing promotes senescence and affects stemnessof MSCsWe observed signs of senescence in cells treated withAd-siRNA-2405, as detected by acid-b-galactosidase,compared with cells transduced with control Ad-siRNA(Figure 3). This result was confirmed in cells with BRG1silenced by another siRNA (Supplementary File 5). Inagreement with these data, in cells with silenced BRG1,we detected a decrease in the level of telomerase reversetranscriptase mRNA (Figure 3).

In agreement with this result, colony-forming unitassays revealed a decrease in the clonogenic potential ofMSCs with silenced BRG1 when compared with thecontrol (Supplementary File 4).

To extend this finding, we analyzed the effect ofBRG1 downregulation on the expression of ‘stemness’genes. These are genes participating in the control ofstem cell properties such as self-renewal and retention ofan uncommitted state. Initially, these genes wereidentified in embryonic stem cells (Ramalho-Santoset al., 2002; Mikkers and Frisen, 2005; Takahashi andYamanaka, 2006). In adult stem cells, some ‘stemnessgenes’ are not expressed. We analyzed a panel ofembryonic stemness genes to evaluate which were activein MSCs and to observe the effects of BRG1 silencing ontheir expression. We did not detect any expression ofSOX2, GDF3 and ZFP42 in MSCs under the differentexperimental conditions used. For another group ofgenes (GDF3, UTF1, TCL1, DPPA2 and ERAS), weobserved negligible expression levels that did not allow

Figure 2 The effects of BRG1 silencing on cell cycle and apoptosis. (Top left) The histogram shows results (±s.d., n¼ 3, *Po0.05) offlow cytometry analysis of MSCs transduced with Ad-siRNAs. (Top right) Evaluation of caspase 9 activity by flow cytometry assay.The histogram shows the activity level in MSCs treated with Ad-siRNA-BRG1 and Ad-siRNA-CTRL, respectively. (Bottom)Detection of apoptotic cells by annexin assay. Fluorescence photomicrograph shows cells stained with annexin V (red), which binds tophosphatidylserine residues exposed on the outer layer of the cell membrane during the early stages of apoptosis. Nuclei werecounterstained with Hoechst 33342 (blue). The table shows the mean expression values (±s.d., n¼ 3). Culturing procedures can causeminimal cell membrane damage that produces background noise in annexin V-based assays. Based on our experience, we can say thatMSC cultures show background noise with annexin staining. This complication allows for the evaluation of the effects of pro-/anti-apoptotic agents but does not allow for the determination of the exact number of cells undergoing apoptosis. For this reason, we alsoevaluated apoptosis using the TUNEL assay (see text).

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reliable quantification. For a third group of genes, weobtained reproducible reverse transcriptase–PCR eva-luation (Figure 3). In particular, we detected theexpression of OCT3, NANOG and KLF4, which arepart of the core transcriptional circuitry for regulationof embryonic stem cell properties (Matoba et al., 2006;Masui et al., 2007). Of great interest is that BRG1downregulation completely abolished NANOG expres-sion (Figure 3).

Senescence is associated with changes in chromatin statusWe determined whether senescence induced by BRG1knockdown was associated with changes in chromatinorganization. It has been shown that distinct hetero-chromatin structures accumulate during senescence andcould represent a hallmark of this process. In particular,Narita et al. (2003) showed heterochromatin formationduring cellular senescence and showed that DNA fromsenescent cells was more resistant to limited micrococcal

nuclease digestion when compared with that fromnormal cells. In our experimental model, cells withsilenced BRG1 showed increased resistance to nucleasedigestion when compared with controls (Figure 4a).

The g-isoform of heterochromatic protein 1 (HP1-g)is a heterochromatic adaptor molecule involved inhigher-order chromatin structure (Maison and Almouzni,2004). It is widely used to identify heterochromatic fociin cell nuclei. In MSCs, after BRG1 downregulation, wedetected an increase in HP1-positive foci in the nuclei(Figure 4b).

Senescence is associated with damaged DNAThe imperfect maintenance of DNA represents a criticalcontributor to senescence (von Zglinicki et al., 2001;Lombard et al., 2005).

Indeed, after transduction with Ad-siRNA-2405, weobserved an increase in the number of cells labeled withanti phosphorylated-H2AX (g-isoform; Figure 5b). This

Figure 3 The effects of BRG1 silencing on cellular senescence. (Top) Senescence-associated b-galactosidase assay performed onMSCs. The histogram shows percentage of senescent cells (±s.d., n¼ 3; **Po0.01). Reverse transcriptase–PCR (RT–PCR) analysis ofstemness-related genes is shown in the table. The change in the mRNA of Ad-siRNA-BRG1-treated cells was compared with that ofAd-siRNA-CTRL-transduced cells, chosen as reference (**Po0.01). ND, not detected. The mRNA levels were normalized withrespect to HPRT and are expressed as ‘arbitrary units’. We used the comparative cycle threshold (Ct) method to quantify expressionlevels.

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histone is a key regulator of the cellular responses toDNA damage and is considered a hallmark of damagedDNA nuclear foci (Figure 5b).

Reactive oxygen species are among the most harmfulDNA-damaging agents. A major product of oxidativedamage to DNA is 8-oxo-20-deoxyguanosine (vonZglinicki et al., 2001; Lombard et al., 2005).

As such, we studied the effects of BRG1 silencing onthe level of damaged DNA by analyzing 8-oxo-20-deoxy-guanosine-positive cells. We did not detect significantchanges in the percentage of 8-oxo-20-deoxyguanosine-positive cells in MSC cultures transduced withAd-siRNA-2405 when compared with controls(Figure 5a). In addition, we found that BRG1 down-regulation did not affect the expression of manganese-dependent superoxide dismutase (SOD2), whereas it didincrease the protein level of catalase; both enzymes aretwo important reactive oxygen species-reducing agentsin cells (Figure 5c) (Giorgio et al., 2007). These datasuggest that H2AX activation was not associated withreactive oxygen species-induced DNA damage pathway.

Genes involved in DNA repairThe DNA repair system is one of the major mechanismsthat cells use to minimize DNA damage (Hoeijmakers,

2001; Khanna and Jackson, 2001; Ronen and Glickman,2001). We determined whether, after BRG1 silencing,the increase in MSCs with damaged DNA wasaccompanied by changes in the expression of genesinvolved in different types of DNA repair. We selected apanel of genes involved in the regulation of base andnucleotide excision repair (BER and NER, respectively),mismatch repair (MER) and double¼ strand breakrepair (DSBR) (Supplementary Table 1, panel A). It isinteresting that most of the analyzed genes showed asignificant upregulation (Po0.05) after the transductionof MSCs with Ad-siRNA-2405. In particular, weobserved a strong increase in POLD3 and MSH5,which belong to the MER pathway, RAD23A for theNER pathway and MPG for BER (SupplementaryTable 1, panel B).

We also analyzed the ability of MSCs to repairdouble-strand breaks, which represent the most danger-ous form of DNA damage. Plasmid-based assays forin vitro DNA repair activity showed strong repaircapacity in control cells (human embryonic kidney 293cells), whereas MSCs showed negligible activity that wasnot affected by BRG1 silencing (Figure 5). Altogether,our data suggest that BRG1 silencing altered thehomeostasis of DNA repair pathway. In fact, theH2AX activation and the upregulation in the expression

Figure 4 (a) Micrococcal nuclease assay. Agarose gel electrophoresis of genomic DNA from MSCs transduced with Ad-siRNA-CTRL and Ad-siRNA-BRG1. Same amounts of DNA were digested with micrococcal nuclease and electrophoresed. The image showsthat nuclease digestion produced a laddering pattern. It is evident that DNA from sample with silenced BRG1 is more resistant tonuclease digestion. In fact, DNA bands from the control sample are dimmer than those present in the lane from Ad-siRNA-BRG1-treated cells, because DNA underwent complete digestion that led to a progressive disappearance of DNA laddering. N.D. is controlundigested DNA. (b) Fluorescence photomicrographs show cells stained with anti-HP1-g (green) and with Hoechst 33342 (blue).A representative microscopic field for each treatment is shown. In the table is indicated the average fluorescence pixel intensity inHP1-positive cells (±s.d., n¼ 3). In this assay we counted at least 500 HP1-positive cells for each treatment (cells infected withAd-siRNA-BRG1 or Ad-siRNA-CTRL). The staining intensity for each positive cell was acquired with a CCD camera and analyzedwith Quantity One 1-D analysis software (Bio-Rad Laboratories). We calculated the sum of the fluorescent pixel values ofHP1-positive cells and then determined the average fluorescent pixel intensity, which was expressed in arbitrary units.

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of several genes involved in DNA repair did not improvethe ability of MSCs to repair DNA. This finding is inagreement with a recent report of Alves et al. (2009).They observed that loss of multipotency of humanMSCs was associated with both accumulation ofDNA damage and the activation of the DNA damagepathway.

BRG1, RB and P53 crosstalkSeveral reports suggested that BRG1 binds to andregulates the retinoblastoma protein and the tumorsuppressor protein p53 (Strobeck et al., 2000; Lee et al.,2002; Hendricks et al., 2004; Kang et al., 2004). Theseare master genes that control cell cycle arrest, differ-entiation, apoptosis and/or senescence (Felsani et al.,2006; Galderisi et al., 2006; Campisi and d’Adda diFagagna, 2007; Oberdoerffer and Sinclair, 2007). Wetried to evaluate whether the biological effects of BRG1silencing were related to RB- and P53-related pathways.

We observed no modification of RB mRNA level anda slight decrease in the corresponding protein. Incontrast, the expression of retinoblastoma-like protein2 (RB2)/P130 protein was strongly upregulated(Figures 7a and b). Upon in vitro BRG1 downregula-tion, we did not observe significant changes in P53mRNA, whereas only slight modification of proteinlevel occurred (Figures 7a and b).

In response to genotoxic stresses, P53 undergoes post-translational modifications that result in its activation.Phosphorylation of human P53 at serine 392 and/or

acetylation at lysine 382 occur after DNA damage (Haoet al., 1996; Lu et al., 1997; Sakaguchi et al., 1998).Silencing of BRG1 induced strong P53 activation, asobserved in its phosphorylated and acetylated forms(Figure 7b). It is noteworthy that the associationbetween senescence and acetylation of P53 at lysine382 has been suggested by several investigators (Bodeand Dong, 2004).

After P53 activation we did not observe a significantincrease in protein level. The activation of P53 affects itsconformation and capacity to bind to several proteins,resulting in its stabilization and enhanced DNA-bindingpotential (Wesierska-Gadek and Schmid, 2005; Lavinand Gueven, 2006; Rajagopalan et al., 2008). Anotherway to regulate the biological function of P53 involveschanges in its intracellular distribution. For this reason,in some experimental conditions, the activation of P53is associated with conformational changes and/orcellular distribution rather than on significant increasein protein level.

Some cyclin kinase inhibitors such as P21CIP1, P27KIP1

and P16INK4A have pathways that overlap with the RBfamily and P53. In particular, P21CIP1 and P16INK4A areoften expressed in senescent cells (Campisi and d’Addadi Fagagna, 2007). After in vitro silencing of BRG1, wedetected negligible increases in P21CIP1 and P27KIP1

expression, whereas we did not see evidence ofmodification of P16INK4A mRNA or protein (Figures 7aand b). These results suggest that the RB and P53pathways do not rely upon these cyclin kinase inhibitorsfor their biological effects.

Figure 5 (a) Fluorescence photomicrographs show cells stained with anti-8-oxo-dG (green). A representative microscopic field foreach treatment is shown. Mean expression values of 8-oxo-dG are indicated in the corresponding table (±s.d., n¼ 3). (b) Fluorescencephotomicrographs show merge of cells stained with anti-H2AX (green) and Hoechst 33342 (blue). A representative microscopic fieldfor each treatment is shown. The degree of H2AX phosphorylation was evaluated by counting the number of H2AX foci/cell. Weclassified cells in three groups: H2AX-negative cells (0 foci/cell); mild H2AX activation (1–10 foci/cell); and strong H2AX activation(410 foci/cell). Mean expression values of H2AX-positive cells are indicated in the corresponding table (±s.d., n¼ 3) (**Po0.01).Mild and strong activation of H2AX indicate cells with damaged DNA. (c) Western blot analysis of catalase and SOD2 levels in MSCstreated with Ad-siRNA-BRG1 and Ad-siRNA-CTRL, respectively. The protein levels were normalized with respect to a-tubulin as theloading control. The histogram shows protein mean expression values (±s.d., n¼ 3).

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It is well known that some DNA-damaging agents,such as ultraviolet and ionizing radiations, can targetP21CIP1 protein for degradation immediately after DNAdamage (Fotedar et al., 2004; Hill et al., 2008). For thisreason, in some experimental conditions, increasedexpression of P21CIP1 cannot be detected after P53activation. BRG1 silencing was associated with anincrease in damaged DNA and this, in turn, couldaffect the level of P21CIP1 protein.

However, it is also possible that the duration of timethat we selected to analyze the effects of BRG1 silencingwas adequate to assess biological phenomena, but not toevaluate changes in the expression of cyclin kinaseinhibitors.

We decided to further assess the role of the RB familyand P53 in the regulation of apoptosis and senescenceafter BRG1 knockdown. To this end, we used the E1Aadenoviral protein that interacts with and inhibits bothRB proteins and P53. We used adenovirus carriersexpressing normal and mutated E1A proteins: Ad-CMV-E1A(YH47-928) and the Ad-CMV-E1A(RG2).The mutated E1A(YH47-928) encodes a protein thatinteracts with and inhibits P53 and not RB proteins,whereas E1A(RG2) has the opposite effect: it blocks RBactivity and not that of P53 (Moran, 1993; Wang et al.,1993, 1995; Dornan et al., 2003).

In MSCs transduced with Ad-siRNA2405 or withcontrol virus, we inhibited P53 with Ad-CMV-E1A(YH47-928) and RB family proteins with Ad-CMV-E1A(RG2). Both RB and P53 seemed to have arole in BRG1-mediated senescence. In situ acid-b-galactosidase staining showed that in cells with silencedBRG1 the inhibition of P53 and of RB reduced thepercentage of senescent cells (Supplementary Table 2).In contrast, protection from apoptosis seemed to relymainly upon the RB family, because inactivation ofthese proteins significantly increased the percentage ofapoptotic cells, reaching a percentage higher than thatobserved when BRG1 was silenced (SupplementaryTable 2).

Discussion

It is becoming clear that several ATP-dependentchromatin remodeling factors have a major role ingoverning the biology of stem cells.

The aim of our research was to investigate the role ofthese complexes in the biology of MSCs. To this end, inprevious research, we triggered ectopic expression of theATPase subunit of SWI/SNF (BRG1) in MSCs.

Forced BRG1 expression induced significant cell cyclearrest in MSCs in culture. This was associated with alarge increase in apoptosis accompanied by the senes-cence process. At the molecular level, these phenomenawere related to activation of the RB- and P53-relatedpathways (Napolitano et al., 2007).

To gain further insights in the role of BRG1, weadopted a complementary approach and used shorthairpin RNA to silence its expression in MSC cultures.

Silencing of BRG1 induced the same effects of forcedBRG1 expression, along with specific and opposite resultsIn MSC cultures, BRG1 downregulation did affect cellcycle profile, induced a decrease in apoptosis and asignificant augmentation of senescent cells (Figures 2and 3). Notably, after BRG1 silencing we detected anincrease in senescent cells as observed in MSCs over-expressing BRG1 (Figure 3; Napolitano et al., 2007).These data imply that each type of perturbation ofmechanisms regulating chromatin status, as occurseither with up- or downregulation of BRG1 activity,may impair cellular physiology.

Accordingly, our micrococcal nuclease digestion andHP1 distribution findings suggest that alteration ofBRG1 levels induced heterochromatin formation(Figures 4a and b; Napolitano et al., 2007). Thisobservation further confirms that nonphysiologicalmodification of BRG1 expression triggers senescence.In fact, it has been shown that during senescence,distinct heterochromatin structures accumulate (Naritaet al., 2003).

Activation of H2AX may suggest that senescenceinduced by BRG1 silencing was associated with aug-mentation of damaged DNA, in spite of increasedexpression of several genes involved in DNA repairpathways. To reconcile these observations, it should bepointed out that increased expression of certain genesregulating DNA repair pathways does not produce anappreciable improvement in DNA repair capacity, atleast for the removal of double-strand breaks (seeFigure 6). Moreover, the upregulation of genes belong-ing to DNA repair programs may provoke an excess ofdamage signaling that, in turn, may perturb normalstem cell biology, driving stem cells to senescence. Thishypothesis is in agreement with the observations of

Figure 6 In vitro DNA repair assay. The image shows agarose gelelectrophoresis of pcDNA3 plasmids that were digested withECoRI enzyme and then treated with protein lysates to allowin vitro DNA end-joining. Protein lysates were obtained fromMSCs treated with Ad-siRNA-BRG1 and ad-siRNA-CTRL,respectively. Human embryonic kidney 293 cells (HEK) werechosen as the positive control for the DNA-end joining activity.Repair of double-strand breaks produces circularized plasmids thatmigrate in agarose distinctly from linear DNAs of the same mass.Negative reaction controls indicate an EcoRI-digested plasmidtreated with denatured protein lysates. Linearized plasmid indicatesan EcoRI-digested plasmid.

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Morales et al. (2005), who showed that overexpressionof RAD50, a member of the MRE11 DNA repaircomplex, produced excess DNA damage signalingthat compromised the functioning of hematopoieticstem cells.

Forced BRG1 expression induced an increase in thenumber of apoptotic cells, whereas silencing caused asignificant reduction in cells undergoing programmedcell death (Figure 2c; Napolitano et al., 2007). Many celltypes acquire resistance to apoptosis when they becomesenescent (Campisi and d’Adda di Fagagna, 2007). Assuch, the reduction of apoptotic cells may be aconsequence of senescence or, alternatively, a directeffect of BRG1 on genes involved in the regulation ofprogrammed cell death. The observation that BRG1overexpression induced apoptosis along with increasedexpression of pro-apoptotic genes supports the latterhypothesis (Napolitano et al., 2007).

In agreement with the triggering of senescence, weobserved modifications in the expression of some genesparticipating in the control of stem cell properties suchas self-renewal ability and retention of an uncommittedstate. It is noteworthy that BRG1 downregulationcompletely abolished expression of NANOG, whichbelongs to the OCT3/SOX2/NANOG/KLF4 stem cell

core circuitry (Figure 3). The suppression of NANOGexpression may be related to activation of P53 becauseLin et al. (2005) showed that P53 induces differentiationof embryonic stem cells by suppressing NANOGexpression.

Our data are consistent with the research of Kidderet al. (2009), who showed that RNA interference-mediated knockdown of BRG1 in embryonic stem cellsresulted in a loss of pluripotency and self-renewal.

RB and P53 pathways are involved in biological effectsinduced by BRG1 silencingRB and P53 pathways are part of the core circuitrythat controls cell cycle arrest, differentiation, apoptosisand/or senescence (Galderisi et al., 2006; Campisi andd’Adda di Fagagna, 2007; Oberdoerffer and Sinclair,2007). Several reports showed that BRG1 operatesthrough regulation of these key cellular pathways(Strobeck et al., 2000; Lee et al., 2002; Hendrickset al., 2004; Kang et al., 2004). RB expression seemed toremain unaffected by BRG1 silencing. On the contrary,the expression of RB2/P130, another member of the RBgene family, was strongly upregulated. In addition,silencing of BRG1 seemed to induce an activation of the

Figure 7 Gene expression analysis of RB- and P53-related pathways in MSCs treated with Ad-siRNAs. (a) Reverse transcriptase–PCR (RT–PCR) of the indicated mRNAs. The mRNA levels were normalized with respect to HPRT and are expressed as ‘arbitraryunits’ (*Po0.05). We used the comparative cycle threshold (Ct) method as quantitative approach. (b) A representative western blot isshown. Protein levels were normalized with a-tubulin as the loading control. The table shows the protein mean expression values(±s.d., n¼ 3) (*Po0.05; **Po0.01). ac-P53, anti-acetylated Lys 379-P53; p-P53, anti-phosphorylated Ser15-P53. Arrows indicatehyperphosphorylated form of RB2–P130.

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P53 protein, as observed upon examination of itsphosphorylated and acetylated forms (Figures 7a and b).These results suggest that the biological effects of BRG1knockdown may rely on RB and P53 pathways.

To gain insight into the role of these proteins, weinhibited P53 or RB family proteins in MSC cultureswith silenced BRG1. Both RB and P53 seemed to have arole in BRG1-mediated senescence. In situ acid-b-galactosidase staining showed that inhibition of P53and of the RB family reduced the percentage ofsenescent cells in cultures with silenced BRG1 whencompared with controls (Supplementary Table 2). Thisis in agreement with several reports showing that cellularsenescence is controlled by the P53 and RB tumorsuppressor proteins. Moreover, in agreement with ourdata, recent research showed that in several tumor celllines, depletion of BRG1 induced the activation ofendogenous wild-type P53 and cell senescence (Campisiand d’Adda di Fagagna, 2007; Naidu et al., 2009).

The effects on apoptosis observed after BRG1silencing seem to be related to RB pathways, becausefunctional inactivation of RB family proteins eliminatesthe decrease in apoptotic cells observed after BRG1depletion (Supplementary Table 2). On the other hand,it has become increasingly clear that RB has anantiapoptotic function, and that loss of RB functiontriggers the P53 apoptotic pathway (Harbour and Dean,2000).

Conclusion: BRG1 is a master gene in the regulationof MSC physiologyMany studies have highlighted the key role of specifictranscription factors to maintain stem cell characteri-stics. However, in recent years, it has become clear thatstem cells have a specific chromatin organization thatdistinguishes them from more differentiated cells. Thisdirected attention toward chromatin remodeling factorsas key players in the regulation of stem cell identity.

Our data are in good agreement with these hypotheses.BRG1 seemed to be actively involved in these processes.In fact, each type of alteration of BRG1 activity seems tonegatively affect mechanisms regulating chromatin statusand, in turn, impair cellular physiology.

Several genes show tightly regulated expression, inwhich even subtle alterations may disrupt the normalfunctioning of cells (Kholodenko, 2000; Yu et al., 2008).One explanation for why certain genes require precisecontrol is their potential to regulate, or be involved inbalancing, disparate downstream pathways possessingmutually opposing activities (Yu et al., 2008). This maybe the case with BRG1, which can modulate geneexpression in either a positive or a negative manner(Trotter and Archer, 2008).

Materials and methods

MSC culturesBone marrow was obtained from healthy donors afterinformed consent. We separated cells on the Ficoll density

gradient (GE Healthcare, Milan, Italy), and the mononuclearcell fraction was collected and washed in phosphate-bufferedsaline. We seeded 1–2.5� 105 cells/cm2 in a-minimum essentialmedium containing 10% fetal bovine serum and 2 ng/ml basicfibroblast growth factor. After 72 h, nonadherent cells werediscarded, and adherent cells were further cultivated to carryout experiments.We verified that under our experimental conditions, MSC

cultures fulfilled the three proposed criteria to define MSCs: (1)adherence to plastic, (2) specific surface antigen expression and(3) multipotent differentiation potential. First, MSCs wereselected by the plastic-adherence procedure. More than 90% ofthe MSC population expressed the CD105, CD73 and CD90antigens. In addition, we verified that the MSCs were able todifferentiate into osteoblasts, adipocytes and chondroblasts(Dominici et al., 2006).All cell culture reagents were obtained from Euroclone

Life Sciences (Milan, Italy) and Hyclone (Milan, Italy) unlessotherwise stated.

SilencingsiRNAs targeted to human BRG1 mRNA were designedfollowing the procedure described by Reynolds et al. (2004).Selected siRNAs were inserted into an adenovirus vector(pSilencer-adeno, Ambion, Austin, TX, USA). We followedthe manufacturer’s protocol to produce Ad-siRNAs. Oncewe obtained Ad-siRNAs targeting BRG1 and controls(Ad-siRNA-CTRL), MSC cell cultures were transduced atdifferent multiplicities of infection to obtain a good silencingeffect (a detailed protocol on the production, purification andtitering of adenoviruses is found in Supplementary File 3).The activity of P53 and of RB family proteins was inhibited

with Ad-CMV-E1A(YH47-928) and the Ad-CMV-E1A(RG2),respectively. These adenoviruses were kindly provided byProfessor E Moran (University of Medicine, Newark, NJ,USA).

Cell cycle analysisFor each assay, 3� 105 cells were collected and resuspended ina hypotonic buffer containing propidium iodide. Cells wereincubated in the dark and then analyzed. Samples wereacquired on a FACSCalibur flow cytometer using the CellQuest software (BD, Franklin Lakes, NJ, USA) and analyzedwith a standard procedure using Cell Quest and ModFitLTsoftwares (BD).

Detection of apoptotic cells with Annexin VApoptotic cells were detected using fluorescein-conjugatedAnnexin V (Roche, Monza (MI), Italy) following the manu-facturer’s instructions. Apoptotic cells were observed througha fluorescence microscope (Leica Italia, Milan, Italy). In everyexperiment, at least 1000 cells were counted in different fieldsto calculate the percentage of dead cells in a culture.

TUNEL assayThe cells for TUNEL assays (Roche) were grown on glasscoverslips. Cells were fixed for 15min using 4% paraform-aldehyde, and the TUNEL reaction was performed accordingto the manufacturer’s instructions. The apoptotic index wascalculated by the number of positive TUNEL cells out of 1000cells in five different microscope fields.

Evaluation of caspase 9 activityWe evaluated caspase 9 activity following the manufacturer’sinstructions (B-Bridge International, Mountain View, CA,

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USA). In brief, 2� 105 cells were incubated with the caspase 9substrate FAM-LEHD-FMK for 1 h, and were then werewashed twice in phosphate-buffered saline and analyzed withfluorescence-activated cell sorting using a FACScalibur (BD)and Cell Quest Technology (BD).

Senescence-associated b-galactosidase assayCells were fixed using a solution of 2% formaldehyde and0.2% glutaraldehyde. After this, cells were washed withphosphate-buffered saline and then incubated at 37 1C forat least 2 h with a staining solution (citric acid/phosphatebuffer (pH 6), K4Fe(CN)6, K3Fe(CN)6, NaCl, MgCl2, X-Gal).The percentage of senescent cells was calculated by thenumber of blue, b-galactosidase-positive cells out of at least500 cells in different microscope fields (Debacq-Chainiauxet al., 2009).

8-oxoguanine detection8oxodG within DNA was detected by immunocytochemistrywith the anti-8oxodG primary antibody (Trevigen, Gaithers-burg, MD, USA) according to the manufacturer’s protocol.Hoechst 33342 staining was performed, and cells werethen observed through a fluorescence microscope (DC300F;Leica Italia). The percentage of 8oxodG-positive cells wascalculated by counting at least 500 cells in different microscopefields.

Immunocytochemistry for detection of H2AX and HP1H2AX and HP1 were detected according to the manufacturer’sprotocol. In brief, cells were grown on coverslips, fixed with4% formaldehyde and permeabilized with methanol. Blockingwas carried out with 5% serum for 60min at roomtemperature. Slides were incubated overnight with anti-H2AX primary antibody (1:50) or anti-HP1-a (1:400) (Milli-pore Italia, Milan, Italy). Afterwards, the slides were incubatedwith goat anti-rabbit secondary antibodies conjugated toFITCT (Jackson Immunoresearch, Milan, Itlay) for 45minat room temperature.Hoechst 33342 staining was performed, and cells were

then observed through a fluorescence microscope (LeicaItalia). The percentage of H2AX- or HP1-positive cells wascalculated by counting at least 500 cells in different microscopefields.

Micrococcal nuclease assayCells were permeabilized with 0.01% L-a-lysophosphatidyl-choline (Sigma-Aldrich, Milan, Italy) in 150mM sucrose, 80mM

KCl, 35mM HEPES pH 7.4, 5mM K2HPO4, 5mM MgCl2and 0.5mM CaCl2 for 90 s, followed by digestion for 60 s with2U/ml micrococcal nuclease (Sigma-Aldrich) in 20mM

sucrose, 50mM Tris–HCl pH 7.5, 50mM NaCl and 2mM

CaCl2 at room temperature for various durations. Digestion ofthe DNA was arrested by adding 50mM EDTA. DNA wasthen purified by Tris-buffered phenol/chloroform/isoamylalcohol extraction. DNA was precipitated using 0.3M NaOAc(pH 6.5) and two volumes of ethanol on dry ice for 30min, andthen resuspended in Tris-EDTA (pH 8.0). DNA concentrationwas evaluated with a spectrophotometer. DNA separationwas performed by agarose (1%) gel electrophoresis withSYBRGreen I staining (Stratagene, Milan, Italy). The datawere collected using a Chemidoc XRF (Bio-Rad Italia, Milan,Italy) and Quantity One version 4.6.3 software (Bio-RadItalia).

Plasmid-based assay for in vitro DNA repair activityThe assay was carried out according to Diggle et al. (2003)with modifications. In brief, the pcDNA3 plasmid (Promega,Milan, Italy) was digested with ECoRI enzyme (Invitrogen,Milan, Italy), and then purified using phenol and ethanolprecipitation. MSCs transduced either with Ad-siMECP2 orAd-siCTRL were lysed in 50mM Tris–HCl pH 7.5, 1M KCl,2mM EDTA and 1 mM DTT. Protein lysates were added todigested and purified pcDNA3 in a reaction buffer thatallowed in vitro DNA end-joining of the digested plasmid,carried out for 2 h at 37 1C. End-joined plasmids were treatedwith proteinase K and sodium dodecyl sulfate. The reactionswere phenol-purified and then electrophoresed on agarose gels.

RNA extraction, reverse transcriptase–PCR and real-time PCRTotal RNA was extracted from cell cultures using OMNIZOL(Euroclone, Milan, Italy) according to the manufacturer’sprotocol. mRNA levels were measured by reverse transcrip-tase–PCR amplification, as previously reported (Galderisiet al., 1999). Real-time PCR assays were run on an Opticon-4 machine (Bio-Rad, Hercules, CA, USA). The reactions wereperformed according to the manufacturer’s instructions usingSYBRGreen PCR Master mix. The mRNA levels werenormalized with respect to HPRT, chosen as an internalcontrol. Each experiment was repeated at least three times. Thevariations in gene expression are given as arbitrary units.

Western blottingCells were lysed in a buffer containing 0.1% Triton for 30minat 4 1C. The lysates were then centrifuged for 10min at 10 000 gat 4 1C. After centrifugation, 10–40mg of each sample wasloaded, electrophoresed in a polyacrylamide gel and electro-blotted onto a nitrocellulose membrane. All the primaryantibodies were used according to the manufacturer’s instruc-tions. Immunoreactive signals were detected with a horse-radish peroxidase-conjugated secondary antibody (Santa CruzBiotechnology, Santa Cruz, CA, USA) and reacted with ECLplus reagent (GE Healthcare).

Statistical analysisStatistical significance was evaluated using analysis of varianceanalysis, followed by Student’s t-test and Bonferroni’s tests.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This work was partially supported by SHRO funds to UG andAG. We thank Maria Rosaria Cipollaro for technicalassistance. We thank Dr Francesca Pentimalli for helpfuldiscussion.

Author contributions: Tiziana Squillaro: conception and de-sign, performed experiments; Nicola Alessio: conception anddesign, performed experiments; Marilena Cipollaro: assembly ofdata, data analysis and interpretation; Luigi Bagella: provision ofstudy material, data analysis and interpretation and financialsupport; Antonio Giordano: conception and design, dataanalysis and interpretation and financial support; UmbertoGalderisi: conception and design, data analysis and interpreta-tion, paper writing and financial support.

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