The BRG1 ATPase of chromatin remodeling complexes is ... - Nature

Oncogene (2010) 29, 5452–5463

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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 1

Sbarro 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; 3Medical Genetics, 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 and 6 Human Health Foundation, Spoleto, Italy

We focused our attention on brahma-related gene 1 (BRG1), the ATPase subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, and analyzed its role in mesenchymal stem cell (MSC) biology. We hypothesized that deviation from the correct concentration of these proteins, which act at the highest level of gene regulation, may be deleterious for cells. We wanted to know what would happen if a cell had to cope with altered regulation of gene expression, either by upregulation or downregulation of BRG1. We assumed that cells would try to restore homeostasis or, alternatively, that the event could trigger senescence/apoptosis phenomena. To this end, in MSCs, we silenced BRG1gene. Knockdown of BRG1 expression induced a significant increase in senescent cells and decrease in apoptotic cells. It is interesting that BRG1 downregulation also induced an increase in heterochromatin. At the molecular level, these phenomena were associated with activation of retinoblastoma-like protein 2 (RB2)/P130and P53-related pathways. Senescence was accompanied by reduced expression of some stemness-related genes. This is consistent with our previous research, which showed that BRG1 upregulation by ectopic expression also induced senescence processes. Together, these data suggest that BRG1 belongs to a class of genes whose expression is tightly regulated; hence, subtle alterations in BRG1 activity seem to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology. Oncogene (2010) 29, 5452–5463; doi:10.1038/onc.2010.285; published online 9 August 2010 Keywords: stem cells; senescence; retinoblastoma; chromatin

apoptosis;

p53;

Correspondence: Professor U Galderisi, Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Via Costantinopoli 16, Napoli 80138, Italy. E-mail: [email protected] Received 3 December 2009; revised 27 May 2010; accepted 31 May 2010; published online 9 August 2010

Introduction Within normal tissues, stem cells are defined by common characteristics: self-renewal to maintain the stem cell pool over time, regulation of stem cell number through a strict balance of proliferation, differentiation and death and the ability to give rise to a broad range of differentiated cells (Morrison et al., 1997; Gage, 2000; Temple, 2001). Alterations in stem cell function have been extensively reported in a variety of tissues and experimental systems. On one hand, impaired stem cell functionality may induce defective tissue regeneration and aging, and on the other, uncontrolled self-renewal and proliferation can trigger tumorigenesis. The antiproliferative effect of senescence clearly indicates that this process is a tumor suppressor mechanism. Early on, senescence was found to be mediated by the two main tumor suppressor pathways of the cell: the ARF/p53 and the INK4a/RB pathways (Collado et al., 2007). For these reasons, studies on the mechanism of senescence in stem cells are of great interest to dissect the pathways that may control aberrant cell proliferation and protect against the development of cancer. A number of recent reports are beginning to define the role of chromatin organization in the regulation of stem cell biology, addressing the question of what gives stem cells these specific properties. Modulations of chromatin structure that often accompany regulation of transcription can be achieved by chromatin remodeling complexes. These complexes carry out enzymatic activities, changing chromatin status by altering DNA–histone contacts within a nucleosome in an adenosine triphosphate (ATP)-dependent manner (Martens and Winston, 2003; Trotter and Archer, 2008). These ATP-dependent remodeling complexes are divided into at least five classes: SWI/SNF, ISWI, CHD(Mi-2), INO80 and SWR1. Each complex has a catalytic ATPase subunit that is critical for allowing or impairing access to nucleosomal DNA to promote or repress gene transcription, respectively (Saha et al., 2006). The mammalian SWItch/Sucrose NonFermentable (SWI/SNF) family includes several members that share

BRG1 and senescence of mesenchymal stem cells N Alessio et al

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most of the same subunits, that is, the ATPase enzyme, either the brahma-related gene 1 (BRG1) or the BRM proteins and/or the presence of tissue-specific isoforms. Complexes containing BRG1 have been shown to be required for cell cycle control, apoptosis and differentiation in several biological systems (Dunaief et al., 1994; Murphy et al., 1999; Bultman et 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 die during the embryonic stage, whereas heterozygotic survivors are prone to tumors (Bultman et al., 2000; Saha et al., 2006; de la Serna et al., 2006). BRG1 protein can interact with different proteins involved in regulation of transcription, such as factors involved in myeloid, erythrocyte, lymphocyte, muscle, neural and adipocyte commitment and/or differentiation (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 BRG1 chromatin remodeling protein can associate with numerous chromatin-modifying complexes, including transcription co-activators and corepressors. Therefore, BRG1 contributes to distinct and even opposite functions in the regulation of cell biology, which depend on the context (cell type, state of differentiation, timing, environmental cues, and so on). A few reports have analyzed the role of SWI/SNF complexes in the biology of stem cells. For example, Matsumoto et al. (2006) showed that BRG1 is required for murine neural stem cell maintenance and gliogenesis. No data were found on the function of BRG1 in the biology of mesenchymal stem cells (MSCs), which have a key role in the body’s homeostasis. With the current and previous research, we have decided to address this issue (Napolitano et al., 2007). The bone marrow of mammals is composed of different elements that support hematopoiesis and bone homeostasis. Among these are MSCs, which are nonhematopoietic stem cells possessing multilineage potential (Muller-Sieburg and Deryugina, 1995; Zhang et al., 2003). MSCs are of interest because of the multiple roles they perform. Besides differentiation into mesenchymal tissues, MSCs support hematopoiesis and contribute to the homeostatic maintenance of many organs and tissues (Prockop, 1997; Beyer Nardi and da Silva Meirelles, 2006; Sethe et al., 2006). In our previous research, forced expression of BRG1 in MSCs triggered significant cell cycle arrest. This was associated with a large increase in apoptosis, along with senescence process. At the molecular level, these phenomena were associated with an activation of the retinoblastoma protein (RB)- and P53-related pathways (Napolitano et al., 2007). In this study, we decided to further analyze the role of BRG1 in MSCs, because studies on chromatin remodelers can be especially important for dissecting molecular pathways governing the biology of stem cells. Altogether, our studies suggest that BRG1 belongs to a class of genes having tightly regulated expression. For

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

Results Human MSCs were tested for BRG1 knockdown 48 h after adenovirus-delivered small interfering RNA (Ad-siRNA) transductions. We obtained a transduction rate of 80–90% with 50–100 multiplicities of infection of Ad-siRNAs (data not shown). We constructed several recombinant adenoviruses to silence BRG1. The Ad-siRNA named Ad-siRNA-2405 targeting nucleotides 2405–2423 of BRG1 mRNA (National Center for Biotechnology Information accession NM_003072.2) was effective in silencing and induced a 70% decrease of BRG1 mRNA, as detected by reverse transcriptase–PCR (Figure 1). Silencing was further verified by analyzing the protein levels of BRG1. We observed a 70% decrease in the target protein (Figure 1). BRG1 silencing was also obtained with another siRNA to rule out off-target effects (Supplementary File 5).

Figure 1 BRG1 silencing. (a) mRNA levels were normalized with respect to HPRT as internal control. The histogram shows the mean expression values (±s.d., n ¼ 3). The change in the mRNA levels of Ad-siRNA-BRG1-treated cells was compared with that of Ad-siRNA-CTRL-transduced cells, chosen as reference (**Po0.01). We used the comparative cycle threshold (Ct) method to quantify the expression levels. (b) Western blot analysis of MECP2 levels in MSCs treated with Ad-siRNA-BRG1 and Ad-siRNA-CTRL, respectively. The protein levels were normalized with respect to a-tubulin as the loading control. The table shows the mean expression values (±s.d., n ¼ 3) of protein densitometric analysis (*Po0.05). Oncogene


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BRG1 silencing reduced the percentage of S-phase cells and induced a decrease in apoptosis The downregulation of BRG1 did modify the cell cycle profile of MSCs in culture as determined by flow cytometry analysis. We observed a small increase of G1 in MSCs with silenced BRG1 compared with controls (67.61 versus 58.33%). It is noteworthy that MSCs with silenced BRG1 had a significant lower percentage of S-phase cells (Po0.05; 29.11 versus 40.23%) and an increase of G2/M cells (3.28 versus 1.43%; Figure 2a). Annexin assays evidenced a reduced percentage of apoptotic cells in cultures from BRG1-silenced samples when compared with the controls (Figure 2c). A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay also showed a decrease in the number of apoptotic cells in MSCs with silenced BRG1 (5.9±0.7% versus 2.7±0.6%). These results were confirmed in cells with BRG1 silenced by another siRNA to rule out off-target effects of the siRNA 2405 (Supplementary File 5). We did not detect a modification of caspase 9 activity in cells treated with Ad-siRNA targeting BRG1, suggesting that the decrease in apoptosis may occur independently of the cytochrome c/Apaf-1/caspase-9 apoptosome, as observed in other systems (Marsden et al., 2006; Figure 2b). It is interesting that in our previous research, we found that the upregulation of BRG1 expression in MSC cultures induced an increase in programmed cell death (Napolitano et al., 2007).

BRG1 silencing promotes senescence and affects stemness of MSCs We observed signs of senescence in cells treated with Ad-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 BRG1 silenced by another siRNA (Supplementary File 5). In agreement with these data, in cells with silenced BRG1, we detected a decrease in the level of telomerase reverse transcriptase mRNA (Figure 3). In agreement with this result, colony-forming unit assays revealed a decrease in the clonogenic potential of MSCs with silenced BRG1 when compared with the control (Supplementary File 4). To extend this finding, we analyzed the effect of BRG1 downregulation on the expression of ‘stemness’ genes. These are genes participating in the control of stem cell properties such as self-renewal and retention of an uncommitted state. Initially, these genes were identified in embryonic stem cells (Ramalho-Santos et al., 2002; Mikkers and Frisen, 2005; Takahashi and Yamanaka, 2006). In adult stem cells, some ‘stemness genes’ are not expressed. We analyzed a panel of embryonic stemness genes to evaluate which were active in MSCs and to observe the effects of BRG1 silencing on their expression. We did not detect any expression of SOX2, GDF3 and ZFP42 in MSCs under the different experimental conditions used. For another group of genes (GDF3, UTF1, TCL1, DPPA2 and ERAS), we observed 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., n ¼ 3, *Po0.05) of flow 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 to phosphatidylserine residues exposed on the outer layer of the cell membrane during the early stages of apoptosis. Nuclei were counterstained with Hoechst 33342 (blue). The table shows the mean expression values (±s.d., n ¼ 3). Culturing procedures can cause minimal cell membrane damage that produces background noise in annexin V-based assays. Based on our experience, we can say that MSC cultures show background noise with annexin staining. This complication allows for the evaluation of the effects of pro-/antiapoptotic agents but does not allow for the determination of the exact number of cells undergoing apoptosis. For this reason, we also evaluated apoptosis using the TUNEL assay (see text). Oncogene


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Figure 3 The effects of BRG1 silencing on cellular senescence. (Top) Senescence-associated b-galactosidase assay performed on MSCs. The histogram shows percentage of senescent cells (±s.d., n ¼ 3; **Po0.01). Reverse transcriptase–PCR (RT–PCR) analysis of stemness-related genes is shown in the table. The change in the mRNA of Ad-siRNA-BRG1-treated cells was compared with that of Ad-siRNA-CTRL-transduced cells, chosen as reference (**Po0.01). ND, not detected. The mRNA levels were normalized with respect to HPRT and are expressed as ‘arbitrary units’. We used the comparative cycle threshold (Ct) method to quantify expression levels.

reliable quantification. For a third group of genes, we obtained reproducible reverse transcriptase–PCR evaluation (Figure 3). In particular, we detected the expression of OCT3, NANOG and KLF4, which are part of the core transcriptional circuitry for regulation of embryonic stem cell properties (Matoba et al., 2006; Masui et al., 2007). Of great interest is that BRG1 downregulation completely abolished NANOG expression (Figure 3). Senescence is associated with changes in chromatin status We determined whether senescence induced by BRG1 knockdown was associated with changes in chromatin organization. It has been shown that distinct heterochromatin structures accumulate during senescence and could represent a hallmark of this process. In particular, Narita et al. (2003) showed heterochromatin formation during cellular senescence and showed that DNA from senescent cells was more resistant to limited micrococcal

nuclease digestion when compared with that from normal cells. In our experimental model, cells with silenced BRG1 showed increased resistance to nuclease digestion when compared with controls (Figure 4a). The g-isoform of heterochromatic protein 1 (HP1-g) is a heterochromatic adaptor molecule involved in higher-order chromatin structure (Maison and Almouzni, 2004). It is widely used to identify heterochromatic foci in cell nuclei. In MSCs, after BRG1 downregulation, we detected an increase in HP1-positive foci in the nuclei (Figure 4b). Senescence is associated with damaged DNA The imperfect maintenance of DNA represents a critical contributor to senescence (von Zglinicki et al., 2001; Lombard et al., 2005). Indeed, after transduction with Ad-siRNA-2405, we observed an increase in the number of cells labeled with anti phosphorylated-H2AX (g-isoform; Figure 5b). This Oncogene


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Figure 4 (a) Micrococcal nuclease assay. Agarose gel electrophoresis of genomic DNA from MSCs transduced with Ad-siRNACTRL and Ad-siRNA-BRG1. Same amounts of DNA were digested with micrococcal nuclease and electrophoresed. The image shows that nuclease digestion produced a laddering pattern. It is evident that DNA from sample with silenced BRG1 is more resistant to nuclease digestion. In fact, DNA bands from the control sample are dimmer than those present in the lane from Ad-siRNA-BRG1treated cells, because DNA underwent complete digestion that led to a progressive disappearance of DNA laddering. N.D. is control undigested 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 in HP1-positive cells (±s.d., n ¼ 3). In this assay we counted at least 500 HP1-positive cells for each treatment (cells infected with Ad-siRNA-BRG1 or Ad-siRNA-CTRL). The staining intensity for each positive cell was acquired with a CCD camera and analyzed with Quantity One 1-D analysis software (Bio-Rad Laboratories). We calculated the sum of the fluorescent pixel values of HP1-positive cells and then determined the average fluorescent pixel intensity, which was expressed in arbitrary units.

histone is a key regulator of the cellular responses to DNA damage and is considered a hallmark of damaged DNA nuclear foci (Figure 5b). Reactive oxygen species are among the most harmful DNA-damaging agents. A major product of oxidative damage to DNA is 8-oxo-20 -deoxyguanosine (von Zglinicki et al., 2001; Lombard et al., 2005). As such, we studied the effects of BRG1 silencing on the level of damaged DNA by analyzing 8-oxo-20 -deoxyguanosine-positive cells. We did not detect significant changes in the percentage of 8-oxo-20 -deoxyguanosinepositive cells in MSC cultures transduced with Ad-siRNA-2405 when compared with controls (Figure 5a). In addition, we found that BRG1 downregulation did not affect the expression of manganesedependent superoxide dismutase (SOD2), whereas it did increase the protein level of catalase; both enzymes are two important reactive oxygen species-reducing agents in cells (Figure 5c) (Giorgio et al., 2007). These data suggest that H2AX activation was not associated with reactive oxygen species-induced DNA damage pathway. Genes involved in DNA repair The DNA repair system is one of the major mechanisms that cells use to minimize DNA damage (Hoeijmakers, Oncogene

2001; Khanna and Jackson, 2001; Ronen and Glickman, 2001). We determined whether, after BRG1 silencing, the increase in MSCs with damaged DNA was accompanied by changes in the expression of genes involved in different types of DNA repair. We selected a panel of genes involved in the regulation of base and nucleotide excision repair (BER and NER, respectively), mismatch repair (MER) and double ¼ strand break repair (DSBR) (Supplementary Table 1, panel A). It is interesting that most of the analyzed genes showed a significant upregulation (Po0.05) after the transduction of MSCs with Ad-siRNA-2405. In particular, we observed a strong increase in POLD3 and MSH5, which belong to the MER pathway, RAD23A for the NER pathway and MPG for BER (Supplementary Table 1, panel B). We also analyzed the ability of MSCs to repair double-strand breaks, which represent the most dangerous form of DNA damage. Plasmid-based assays for in vitro DNA repair activity showed strong repair capacity in control cells (human embryonic kidney 293 cells), whereas MSCs showed negligible activity that was not affected by BRG1 silencing (Figure 5). Altogether, our data suggest that BRG1 silencing altered the homeostasis of DNA repair pathway. In fact, the H2AX activation and the upregulation in the expression


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Figure 5 (a) Fluorescence photomicrographs show cells stained with anti-8-oxo-dG (green). A representative microscopic field for each treatment is shown. Mean expression values of 8-oxo-dG are indicated in the corresponding table (±s.d., n ¼ 3). (b) Fluorescence photomicrographs show merge of cells stained with anti-H2AX (green) and Hoechst 33342 (blue). A representative microscopic field for each treatment is shown. The degree of H2AX phosphorylation was evaluated by counting the number of H2AX foci/cell. We classified 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., n ¼ 3) (**Po0.01). Mild and strong activation of H2AX indicate cells with damaged DNA. (c) Western blot analysis of catalase and SOD2 levels in MSCs treated with Ad-siRNA-BRG1 and Ad-siRNA-CTRL, respectively. The protein levels were normalized with respect to a-tubulin as the loading control. The histogram shows protein mean expression values (±s.d., n ¼ 3).

of several genes involved in DNA repair did not improve the ability of MSCs to repair DNA. This finding is in agreement with a recent report of Alves et al. (2009). They observed that loss of multipotency of human MSCs was associated with both accumulation of DNA damage and the activation of the DNA damage pathway. BRG1, RB and P53 crosstalk Several reports suggested that BRG1 binds to and regulates the retinoblastoma protein and the tumor suppressor protein p53 (Strobeck et al., 2000; Lee et al., 2002; Hendricks et al., 2004; Kang et al., 2004). These are master genes that control cell cycle arrest, differentiation, apoptosis and/or senescence (Felsani et al., 2006; Galderisi et al., 2006; Campisi and d’Adda di Fagagna, 2007; Oberdoerffer and Sinclair, 2007). We tried to evaluate whether the biological effects of BRG1 silencing were related to RB- and P53-related pathways. We observed no modification of RB mRNA level and a slight decrease in the corresponding protein. In contrast, the expression of retinoblastoma-like protein 2 (RB2)/P130 protein was strongly upregulated (Figures 7a and b). Upon in vitro BRG1 downregulation, we did not observe significant changes in P53 mRNA, whereas only slight modification of protein level occurred (Figures 7a and b). In response to genotoxic stresses, P53 undergoes posttranslational modifications that result in its activation. Phosphorylation of human P53 at serine 392 and/or

acetylation at lysine 382 occur after DNA damage (Hao et al., 1996; Lu et al., 1997; Sakaguchi et al., 1998). Silencing of BRG1 induced strong P53 activation, as observed in its phosphorylated and acetylated forms (Figure 7b). It is noteworthy that the association between senescence and acetylation of P53 at lysine 382 has been suggested by several investigators (Bode and Dong, 2004). After P53 activation we did not observe a significant increase in protein level. The activation of P53 affects its conformation and capacity to bind to several proteins, resulting in its stabilization and enhanced DNA-binding potential (Wesierska-Gadek and Schmid, 2005; Lavin and Gueven, 2006; Rajagopalan et al., 2008). Another way to regulate the biological function of P53 involves changes in its intracellular distribution. For this reason, in some experimental conditions, the activation of P53 is associated with conformational changes and/or cellular distribution rather than on significant increase in protein level. Some cyclin kinase inhibitors such as P21CIP1, P27KIP1 and P16INK4A have pathways that overlap with the RB family and P53. In particular, P21CIP1 and P16INK4A are often expressed in senescent cells (Campisi and d’Adda di Fagagna, 2007). After in vitro silencing of BRG1, we detected negligible increases in P21CIP1 and P27KIP1 expression, whereas we did not see evidence of modification of P16INK4A mRNA or protein (Figures 7a and b). These results suggest that the RB and P53 pathways do not rely upon these cyclin kinase inhibitors for their biological effects. Oncogene


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It is well known that some DNA-damaging agents, such as ultraviolet and ionizing radiations, can target P21CIP1 protein for degradation immediately after DNA damage (Fotedar et al., 2004; Hill et al., 2008). For this reason, in some experimental conditions, increased expression of P21CIP1 cannot be detected after P53 activation. BRG1 silencing was associated with an increase in damaged DNA and this, in turn, could affect the level of P21CIP1 protein. However, it is also possible that the duration of time that we selected to analyze the effects of BRG1 silencing was adequate to assess biological phenomena, but not to evaluate changes in the expression of cyclin kinase inhibitors. We decided to further assess the role of the RB family and P53 in the regulation of apoptosis and senescence after BRG1 knockdown. To this end, we used the E1A adenoviral protein that interacts with and inhibits both RB proteins and P53. We used adenovirus carriers expressing normal and mutated E1A proteins: AdCMV-E1A(YH47-928) and the Ad-CMV-E1A(RG2). The mutated E1A(YH47-928) encodes a protein that interacts with and inhibits P53 and not RB proteins, whereas E1A(RG2) has the opposite effect: it blocks RB activity and not that of P53 (Moran, 1993; Wang et al., 1993, 1995; Dornan et al., 2003). In MSCs transduced with Ad-siRNA2405 or with control virus, we inhibited P53 with Ad-CMVE1A(YH47-928) and RB family proteins with AdCMV-E1A(RG2). Both RB and P53 seemed to have a role in BRG1-mediated senescence. In situ acid-bgalactosidase staining showed that in cells with silenced BRG1 the inhibition of P53 and of RB reduced the percentage of senescent cells (Supplementary Table 2). In contrast, protection from apoptosis seemed to rely mainly upon the RB family, because inactivation of these proteins significantly increased the percentage of apoptotic cells, reaching a percentage higher than that observed when BRG1 was silenced (Supplementary Table 2).

Silencing of BRG1 induced the same effects of forced BRG1 expression, along with specific and opposite results In MSC cultures, BRG1 downregulation did affect cell cycle profile, induced a decrease in apoptosis and a significant augmentation of senescent cells (Figures 2 and 3). Notably, after BRG1 silencing we detected an increase in senescent cells as observed in MSCs overexpressing BRG1 (Figure 3; Napolitano et al., 2007). These data imply that each type of perturbation of mechanisms regulating chromatin status, as occurs either with up- or downregulation of BRG1 activity, may impair cellular physiology. Accordingly, our micrococcal nuclease digestion and HP1 distribution findings suggest that alteration of BRG1 levels induced heterochromatin formation (Figures 4a and b; Napolitano et al., 2007). This observation further confirms that nonphysiological modification of BRG1 expression triggers senescence. In fact, it has been shown that during senescence, distinct heterochromatin structures accumulate (Narita et al., 2003). Activation of H2AX may suggest that senescence induced by BRG1 silencing was associated with augmentation of damaged DNA, in spite of increased expression of several genes involved in DNA repair pathways. To reconcile these observations, it should be pointed out that increased expression of certain genes regulating DNA repair pathways does not produce an appreciable improvement in DNA repair capacity, at least for the removal of double-strand breaks (see Figure 6). Moreover, the upregulation of genes belonging to DNA repair programs may provoke an excess of damage signaling that, in turn, may perturb normal stem cell biology, driving stem cells to senescence. This hypothesis is in agreement with the observations of

Discussion It is becoming clear that several ATP-dependent chromatin remodeling factors have a major role in governing the biology of stem cells. The aim of our research was to investigate the role of these complexes in the biology of MSCs. To this end, in previous research, we triggered ectopic expression of the ATPase subunit of SWI/SNF (BRG1) in MSCs. Forced BRG1 expression induced significant cell cycle arrest in MSCs in culture. This was associated with a large increase in apoptosis accompanied by the senescence process. At the molecular level, these phenomena were related to activation of the RB- and P53-related pathways (Napolitano et al., 2007). To gain further insights in the role of BRG1, we adopted a complementary approach and used short hairpin RNA to silence its expression in MSC cultures. Oncogene

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


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Morales et al. (2005), who showed that overexpression of RAD50, a member of the MRE11 DNA repair complex, produced excess DNA damage signaling that compromised the functioning of hematopoietic stem cells. Forced BRG1 expression induced an increase in the number of apoptotic cells, whereas silencing caused a significant reduction in cells undergoing programmed cell death (Figure 2c; Napolitano et al., 2007). Many cell types acquire resistance to apoptosis when they become senescent (Campisi and d’Adda di Fagagna, 2007). As such, the reduction of apoptotic cells may be a consequence of senescence or, alternatively, a direct effect of BRG1 on genes involved in the regulation of programmed cell death. The observation that BRG1 overexpression induced apoptosis along with increased expression of pro-apoptotic genes supports the latter hypothesis (Napolitano et al., 2007). In agreement with the triggering of senescence, we observed modifications in the expression of some genes participating in the control of stem cell properties such as self-renewal ability and retention of an uncommitted state. It is noteworthy that BRG1 downregulation completely abolished expression of NANOG, which belongs to the OCT3/SOX2/NANOG/KLF4 stem cell

core circuitry (Figure 3). The suppression of NANOG expression may be related to activation of P53 because Lin et al. (2005) showed that P53 induces differentiation of embryonic stem cells by suppressing NANOG expression. Our data are consistent with the research of Kidder et al. (2009), who showed that RNA interferencemediated knockdown of BRG1 in embryonic stem cells resulted in a loss of pluripotency and self-renewal. RB and P53 pathways are involved in biological effects induced by BRG1 silencing RB and P53 pathways are part of the core circuitry that controls cell cycle arrest, differentiation, apoptosis and/or senescence (Galderisi et al., 2006; Campisi and d’Adda di Fagagna, 2007; Oberdoerffer and Sinclair, 2007). Several reports showed that BRG1 operates through regulation of these key cellular pathways (Strobeck et al., 2000; Lee et al., 2002; Hendricks et al., 2004; Kang et al., 2004). RB expression seemed to remain unaffected by BRG1 silencing. On the contrary, the expression of RB2/P130, another member of the RB gene 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 ‘arbitrary units’ (*Po0.05). We used the comparative cycle threshold (Ct) method as quantitative approach. (b) A representative western blot is shown. Protein levels were normalized with a-tubulin as the loading control. The table shows the protein mean expression values (±s.d., n ¼ 3) (*Po0.05; **Po0.01). ac-P53, anti-acetylated Lys 379-P53; p-P53, anti-phosphorylated Ser15-P53. Arrows indicate hyperphosphorylated form of RB2–P130. Oncogene


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P53 protein, as observed upon examination of its phosphorylated and acetylated forms (Figures 7a and b). These results suggest that the biological effects of BRG1 knockdown may rely on RB and P53 pathways. To gain insight into the role of these proteins, we inhibited P53 or RB family proteins in MSC cultures with silenced BRG1. Both RB and P53 seemed to have a role in BRG1-mediated senescence. In situ acid-bgalactosidase staining showed that inhibition of P53 and of the RB family reduced the percentage of senescent cells in cultures with silenced BRG1 when compared with controls (Supplementary Table 2). This is in agreement with several reports showing that cellular senescence is controlled by the P53 and RB tumor suppressor proteins. Moreover, in agreement with our data, recent research showed that in several tumor cell lines, depletion of BRG1 induced the activation of endogenous wild-type P53 and cell senescence (Campisi and d’Adda di Fagagna, 2007; Naidu et al., 2009). The effects on apoptosis observed after BRG1 silencing seem to be related to RB pathways, because functional inactivation of RB family proteins eliminates the decrease in apoptotic cells observed after BRG1 depletion (Supplementary Table 2). On the other hand, it has become increasingly clear that RB has an antiapoptotic function, and that loss of RB function triggers the P53 apoptotic pathway (Harbour and Dean, 2000). Conclusion: BRG1 is a master gene in the regulation of MSC physiology Many studies have highlighted the key role of specific transcription factors to maintain stem cell characteristics. However, in recent years, it has become clear that stem cells have a specific chromatin organization that distinguishes them from more differentiated cells. This directed attention toward chromatin remodeling factors as 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 to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology. Several genes show tightly regulated expression, in which even subtle alterations may disrupt the normal functioning of cells (Kholodenko, 2000; Yu et al., 2008). One explanation for why certain genes require precise control is their potential to regulate, or be involved in balancing, disparate downstream pathways possessing mutually opposing activities (Yu et al., 2008). This may be the case with BRG1, which can modulate gene expression in either a positive or a negative manner (Trotter and Archer, 2008).

gradient (GE Healthcare, Milan, Italy), and the mononuclear cell fraction was collected and washed in phosphate-buffered saline. We seeded 1–2.5 105 cells/cm2 in a-minimum essential medium containing 10% fetal bovine serum and 2 ng/ml basic fibroblast growth factor. After 72 h, nonadherent cells were discarded, and adherent cells were further cultivated to carry out 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 were selected by the plastic-adherence procedure. More than 90% of the MSC population expressed the CD105, CD73 and CD90 antigens. In addition, we verified that the MSCs were able to differentiate 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) unless otherwise stated. Silencing siRNAs targeted to human BRG1 mRNA were designed following the procedure described by Reynolds et al. (2004). Selected siRNAs were inserted into an adenovirus vector (pSilencer-adeno, Ambion, Austin, TX, USA). We followed the manufacturer’s protocol to produce Ad-siRNAs. Once we obtained Ad-siRNAs targeting BRG1 and controls (Ad-siRNA-CTRL), MSC cell cultures were transduced at different multiplicities of infection to obtain a good silencing effect (a detailed protocol on the production, purification and titering 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 by Professor E Moran (University of Medicine, Newark, NJ, USA). Cell cycle analysis For each assay, 3 105 cells were collected and resuspended in a hypotonic buffer containing propidium iodide. Cells were incubated in the dark and then analyzed. Samples were acquired on a FACSCalibur flow cytometer using the Cell Quest software (BD, Franklin Lakes, NJ, USA) and analyzed with a standard procedure using Cell Quest and ModFitLT softwares (BD). Detection of apoptotic cells with Annexin V Apoptotic cells were detected using fluorescein-conjugated Annexin V (Roche, Monza (MI), Italy) following the manufacturer’s instructions. Apoptotic cells were observed through a fluorescence microscope (Leica Italia, Milan, Italy). In every experiment, at least 1000 cells were counted in different fields to calculate the percentage of dead cells in a culture.

Materials and methods

TUNEL assay The cells for TUNEL assays (Roche) were grown on glass coverslips. Cells were fixed for 15 min using 4% paraformaldehyde, and the TUNEL reaction was performed according to the manufacturer’s instructions. The apoptotic index was calculated by the number of positive TUNEL cells out of 1000 cells in five different microscope fields.

MSC cultures Bone marrow was obtained from healthy donors after informed consent. We separated cells on the Ficoll density

Evaluation of caspase 9 activity We evaluated caspase 9 activity following the manufacturer’s instructions (B-Bridge International, Mountain View, CA,

Oncogene


5461 USA). In brief, 2 105 cells were incubated with the caspase 9 substrate FAM-LEHD-FMK for 1 h, and were then were washed twice in phosphate-buffered saline and analyzed with fluorescence-activated cell sorting using a FACScalibur (BD) and Cell Quest Technology (BD). Senescence-associated b-galactosidase assay Cells were fixed using a solution of 2% formaldehyde and 0.2% glutaraldehyde. After this, cells were washed with phosphate-buffered saline and then incubated at 37 1C for at least 2 h with a staining solution (citric acid/phosphate buffer (pH 6), K4Fe(CN)6, K3Fe(CN)6, NaCl, MgCl2, X-Gal). The percentage of senescent cells was calculated by the number of blue, b-galactosidase-positive cells out of at least 500 cells in different microscope fields (Debacq-Chainiaux et al., 2009). 8-oxoguanine detection 8oxodG within DNA was detected by immunocytochemistry with the anti-8oxodG primary antibody (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Hoechst 33342 staining was performed, and cells were then observed through a fluorescence microscope (DC300F; Leica Italia). The percentage of 8oxodG-positive cells was calculated by counting at least 500 cells in different microscope fields.

Immunocytochemistry for detection of H2AX and HP1 H2AX and HP1 were detected according to the manufacturer’s protocol. In brief, cells were grown on coverslips, fixed with 4% formaldehyde and permeabilized with methanol. Blocking was carried out with 5% serum for 60 min at room temperature. Slides were incubated overnight with antiH2AX primary antibody (1:50) or anti-HP1-a (1:400) (Millipore Italia, Milan, Italy). Afterwards, the slides were incubated with goat anti-rabbit secondary antibodies conjugated to FITCT (Jackson Immunoresearch, Milan, Itlay) for 45 min at room temperature. Hoechst 33342 staining was performed, and cells were then observed through a fluorescence microscope (Leica Italia). The percentage of H2AX- or HP1-positive cells was calculated by counting at least 500 cells in different microscope fields.

Plasmid-based assay for in vitro DNA repair activity The 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 ethanol precipitation. MSCs transduced either with Ad-siMECP2 or Ad-siCTRL were lysed in 50 mM Tris–HCl pH 7.5, 1 M KCl, 2 mM EDTA and 1 mM DTT. Protein lysates were added to digested and purified pcDNA3 in a reaction buffer that allowed in vitro DNA end-joining of the digested plasmid, carried out for 2 h at 37 1C. End-joined plasmids were treated with proteinase K and sodium dodecyl sulfate. The reactions were phenol-purified and then electrophoresed on agarose gels. RNA extraction, reverse transcriptase–PCR and real-time PCR Total RNA was extracted from cell cultures using OMNIZOL (Euroclone, Milan, Italy) according to the manufacturer’s protocol. mRNA levels were measured by reverse transcriptase–PCR amplification, as previously reported (Galderisi et al., 1999). Real-time PCR assays were run on an Opticon4 machine (Bio-Rad, Hercules, CA, USA). The reactions were performed according to the manufacturer’s instructions using SYBRGreen PCR Master mix. The mRNA levels were normalized with respect to HPRT, chosen as an internal control. Each experiment was repeated at least three times. The variations in gene expression are given as arbitrary units. Western blotting Cells were lysed in a buffer containing 0.1% Triton for 30 min at 4 1C. The lysates were then centrifuged for 10 min at 10 000 g at 4 1C. After centrifugation, 10–40 mg of each sample was loaded, electrophoresed in a polyacrylamide gel and electroblotted onto a nitrocellulose membrane. All the primary antibodies were used according to the manufacturer’s instructions. Immunoreactive signals were detected with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and reacted with ECL plus reagent (GE Healthcare). Statistical analysis Statistical significance was evaluated using analysis of variance analysis, followed by Student’s t-test and Bonferroni’s tests.

Conflict of interest Micrococcal nuclease assay Cells were permeabilized with 0.01% L-a-lysophosphatidylcholine (Sigma-Aldrich, Milan, Italy) in 150 mM sucrose, 80 mM KCl, 35 mM HEPES pH 7.4, 5 mM K2HPO4, 5 mM MgCl2 and 0.5 mM CaCl2 for 90 s, followed by digestion for 60 s with 2 U/ml micrococcal nuclease (Sigma-Aldrich) in 20 mM sucrose, 50 mM Tris–HCl pH 7.5, 50 mM NaCl and 2 mM CaCl2 at room temperature for various durations. Digestion of the DNA was arrested by adding 50 mM EDTA. DNA was then purified by Tris-buffered phenol/chloroform/isoamyl alcohol extraction. DNA was precipitated using 0.3 M NaOAc (pH 6.5) and two volumes of ethanol on dry ice for 30 min, and then resuspended in Tris-EDTA (pH 8.0). DNA concentration was evaluated with a spectrophotometer. DNA separation was performed by agarose (1%) gel electrophoresis with SYBRGreen I staining (Stratagene, Milan, Italy). The data were collected using a Chemidoc XRF (Bio-Rad Italia, Milan, Italy) and Quantity One version 4.6.3 software (Bio-Rad Italia).

The authors declare no conflict of interest.

Acknowledgements This work was partially supported by SHRO funds to UG and AG. We thank Maria Rosaria Cipollaro for technical assistance. We thank Dr Francesca Pentimalli for helpful discussion. Author contributions: Tiziana Squillaro: conception and design, performed experiments; Nicola Alessio: conception and design, performed experiments; Marilena Cipollaro: assembly of data, data analysis and interpretation; Luigi Bagella: provision of study material, data analysis and interpretation and financial support; Antonio Giordano: conception and design, data analysis and interpretation and financial support; Umberto Galderisi: conception and design, data analysis and interpretation, paper writing and financial support. Oncogene


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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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