Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting γ-H2AX induction
2006; Springer Nature; Volume: 25; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7601291
ISSN1460-2075
AutoresJi‐Hye Park, Eun‐Jung Park, Han-Sae Lee, So Jung Kim, Shin-Kyoung Hur, Anthony N. Imbalzano, Jongbum Kwon,
Tópico(s)Cancer therapeutics and mechanisms
ResumoArticle24 August 2006free access Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting γ-H2AX induction Ji-Hye Park Ji-Hye Park Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Eun-Jung Park Eun-Jung Park Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Han-Sae Lee Han-Sae Lee Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author So Jung Kim So Jung Kim Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Shin-Kyoung Hur Shin-Kyoung Hur Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Anthony N Imbalzano Anthony N Imbalzano Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, Massachusetts, USA Search for more papers by this author Jongbum Kwon Corresponding Author Jongbum Kwon Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Ji-Hye Park Ji-Hye Park Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Eun-Jung Park Eun-Jung Park Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Han-Sae Lee Han-Sae Lee Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author So Jung Kim So Jung Kim Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Shin-Kyoung Hur Shin-Kyoung Hur Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Anthony N Imbalzano Anthony N Imbalzano Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, Massachusetts, USA Search for more papers by this author Jongbum Kwon Corresponding Author Jongbum Kwon Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea Search for more papers by this author Author Information Ji-Hye Park1, Eun-Jung Park1, Han-Sae Lee1, So Jung Kim1, Shin-Kyoung Hur1, Anthony N Imbalzano2 and Jongbum Kwon 1 1Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul, Korea 2Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, Massachusetts, USA *Corresponding author. Division of Molecular Life Sciences, Department of Life Science and Center for Cell Signaling Research, Ewha Woman's University, Seoul 120-750, Korea. Tel.: +82 2 3277 4334; Fax: +82 2 3277 3760; E-mail: [email protected] The EMBO Journal (2006)25:3986-3997https://doi.org/10.1038/sj.emboj.7601291 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double-strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (γ-H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA-PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB-surrounding chromatin via interaction with γ-H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for γ-H2AX in efficient DSB repair, these results suggest that the SWI/SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin. Introduction Eukaryotic DNA is organized into nucleosomes and higher-order structure that presents obstacles to the access of cellular proteins to their natural chromatin substrates. The process of modulating chromatin structure therefore must constitute a pivotal step in the regulation of the nuclear processes such as DNA repair (Kornberg and Lorch, 1999; Fyodorov and Kadonaga, 2001). One of two major mechanisms by which chromatin modulation is achieved involves reversible addition of an acetyl group to the lysine residues of the histones that comprise the nucleosomes. The other one is mediated by the family of ATP-dependent chromatin-remodeling complexes that utilize the energy of ATP hydrolysis to disrupt the histone–DNA interactions. While these two mechanisms have been well established to play crucial roles in the regulation of transcription (Narlikar et al, 2002), their role in chromosomal DNA repair is just beginning to be elucidated (Allard et al, 2004; Peterson and Cote, 2004; van Attikum and Gasser, 2005). Double-strand breaks (DSBs) of chromosomal DNA, the most destructive form of DNA damage, can be generated by exposure to ionizing radiation (IR), and also naturally occur during the nuclear processes such as DNA replication and V(D)J recombination. Unless accurately and efficiently repaired, DNA DSBs can result in chromosomal instability and cancer development (Hoeijmakers, 2001; Jackson, 2002). Upon generation of DSBs, cells activate the pathways leading to DNA repair as well as propagate the signals for checkpoint activation to arrest the cell cycle until the repair is completed. The three phosphatidylinositol-3 kinase-like kinases (PIKKs), ATM, ATR and DNA-PK, play a key role in initiating these intracellular signaling pathways of DSB responses (Shiloh, 2003; Bakkenist and Kastan, 2004). Immediately after DSB generation, histone H2AX is phosphorylated at Ser-139 at the conserved SQ motif on the C-terminal tail, and the three PIKKs are all responsible for this phosphorylation. The phosphorylation of H2AX (termed γ-H2AX) occurs specifically at the DSB-surrounding chromatin encompassing hundreds of thousands of base pairs, resulting in organization into a poorly understood subnuclear chromosomal structure which can be visualized by immunostaining, termed as IR-induced nuclear foci (IRIF) or frequently referred to as repair foci. γ-H2AX is thought to serve as binding sites for repair and checkpoint proteins such as Nbs1 as well as to influence chromatin structure in such a way that DNA repair events are facilitated (Bassing and Alt, 2004; Fernandez-Capetillo et al, 2004). Studies using H2AX-deleted cells demonstrated that γ-H2AX is crucial for efficient repair of chromosomal DSBs and hence the maintenance of genome integrity (Downs et al, 2000; Bassing et al, 2002, 2003; Celeste et al, 2002, 2003a). Mammalian SWI/SNF complexes, belonging to the family of swi2/snf2-based ATP-dependent chromatin remodeling complexes, are capable of facilitating alterations of nucleosome structure to control the accessibility of chromatin substrates. The SWI/SNF complexes consist of at least eight subunits containing either BRG-1 or Brm as the catalytic core subunits with ATPase activity. It has been well established that the SWI/SNF complexes play an important role in transcription both in reconstituted in vitro system and within the cells (Narlikar et al, 2002). Studies also have implicated the SWI/SNF complexes in DNA processes other than transcription such as DNA replication (Flanagan and Peterson, 1999), V(D)J recombination (Kwon et al, 2000) and viral integration (Yung et al, 2001). In vitro studies have shown that yeast SWI/SNF complex can stimulate the nucleotide excision repair on reconstituted nucleosomal substrates (Hara and Sancar, 2002; Gaillard et al, 2003). However, the in vivo function of the SWI/SNF complexes in DNA repair has remained elusive. Here, we report a role for the mammalian SWI/SNF complexes in H2AX phosphorylation, DSB repair and cell survival after DNA damage. Results Inactivation of the SWI/SNF complexes results in increased DNA damage sensitivity and decreased DSB repair To investigate a potential role for the mammalian SWI/SNF complexes in DNA repair, we initially employed the NIH-3T3 cells, named B05-1, in which the SWI/SNF complexes can be conditionally inactivated by the expression of ATPase-defective dominant negative versions of BRG-1 under the control of tet-off system (de la Serna et al, 2000). Cells were cultured under the conditions with (as control) or without tetracycline (to induce the flag-tagged dominant negative BRG-1) for 4 days before being subjected to the experiments described below, and, for the sake of convenience, we often denote such cells as B05-1(+tet) and B05-1(−tet), respectively. In the first, we examined the effects of SWI/SNF inactivation on cell viability after DNA damage by colony formation assays. As expected, the expression of flag-tagged proteins was nicely induced by tetracycline depletion from B05-1 cells but not from tet-VP16 cells, the control NIH-3T3 cells expressing no flag-tagged proteins (de la Serna et al, 2000) (Figure 1A, top). When we exposed these cells to IR, we found that the viability of B05-1(−tet) cells was markedly decreased compared to B05-1(+tet) cells, whereas tet-VP16 cells showed the similar levels of viability regardless of tetracycline depletion (Figure 1A, bottom), indicating that the SWI/SNF complexes are important for cell survival after DNA damage. The cellular levels of flag-BRG-1 were not affected by irradiation (data not shown). It should be noted that SWI/SNF inactivation reduces clonogenic ability even in undamaged cells (data not shown), suggesting that the SWI/SNF complexes are important for maintaining cell health. Figure 1.Inactivation of the SWI/SNF complexes results in increased DNA damage sensitivity and decreased DSB repair. (A) (top) tet-VP16 and B05-1 cells were incubated with or without tetracycline for 4 days, and cell lysates were analyzed for the expression of flag-BRG-1 by immunoblottings. The expression of actin was also analyzed as internal control. (Bottom) Inactivation of the SWI/SNF complexes renders cells hypersensitive to IR. Cells grown as per in (A) were irradiated by various doses before the viability was determined by colony formation assays. Data are presented as mean±standard deviation (s.d.) from triplicates. (B) Neutral comet assays show that SWI/SNF inactivation leads to inefficient DSB repair. B05-1 (the first three graphs) and tet-VP16 cells (the last graph) grown as in (A) were irradiated by indicated doses, and the cells were collected immediately (0 h) or at the indicated time points after irradiation for comet assays. Each graph is depicted as mean±s.d. from three independent experiments. Immunoblot analysis for flag-BRG-1 expression in three independent experiments is shown next to the corresponding graphs. (C) The pictures show representative comet images of B05-1(+tet) and B05-1(−tet) cells untreated (0 Gy), or immediately (0 h) and 10 h after irradiation by 25 Gy. Download figure Download PowerPoint We then asked whether the SWI/SNF complexes are involved in DNA repair. We performed single-cell electrophoresis analysis under the neutral conditions (neutral comet assay) that specifically measures DNA DSBs. Immediately after irradiation (0 h) by 5, 10 and 25 Gy, approximately the same amounts of DNA fragments were generated from B05-1(+tet) and B05-1(−tet) cells at each dose (Figure 1B and C, and data not shown). When remaining unrepaired DNA fragments were monitored at various hours after irradiation, B05-1(−tet) cells exhibited significantly lower repair efficiency than B05-1(+tet) cells for all three doses tested, whereas tet-VP16 cells showed the same repair efficiency regardless of tetracycline depletion (Figure 1B and C). These results show that the SWI/SNF complexes are required for efficient DBS repair as well as cell survival after DNA damage. Analysis of the genes whose expression is affected by SWI/SNF inactivation To gain insight into whether the SWI/SNF complexes directly participate in DSB repair or contribute indirectly by transcription, we analyzed the genes that are differentially expressed between B05-1(+tet) and B05-1(−tet) cells using the mouse 11K microarray consisting of 11 376 genes that include 110 DNA repair genes. Out of the total 11 376 genes, 371 genes were induced or repressed at the significant levels (>1.8-fold) by SWI/SNF inactivation (Supplementary Table S1). Among them, only three genes were categorized into DNA repair, with two genes implicated in nucleotide excision repair and one in base excision repair (Supplementary Table S2). The human genome is estimated to contain about 150 genes encoding DNA repair enzymes and some proteins associated with cellular responses to DNA damage, among which 24 genes are directly implicated in DSB repair (Wood et al, 2005). The microarray data and RT–PCR experiments showed that none of the 24 DSB repair genes were significantly affected by SWI/SNF inactivation (Figure 2). These results raised a possibility that the SWI/SNF complexes may play a direct role in DSB repair. Figure 2.The expression of the known DSB repair genes is not significantly affected by SWI/SNF inactivation. (A) The fold changes of the expression of thus far known 24 DBS repair genes by SWI/SNF inactivation are summarized (these are all below the cutoff value). The data sources for the fold change of each gene are indicated in the last column. Note that the majority of the genes (17 genes) were changed by less than 1.2-fold by SWI/SNF inactivation, and that Rad51 and Rad51D were decreased by only 1.3-fold and the remaining five genes were rather increased by SWI/SNF inactivation. (B) (Top) The effects of SWI/SNF inactivation on the expression of the DSB repair genes that were not included in the mouse 11K gene chip. RT–PCR was performed using total RNA isolated from B05-1(+tet) and B05-1(−tet) cells. The mRNA expression of GAPDH was analyzed as internal control. The predicted sizes of the PCR products are as follow: RAD54B, 614 bp; EME1, 681 bp; LIG4, 641 bp; Artemis, 655 bp; GAPDH, 361 bp. The first lane (M) is 100-bp standard size marker (the most bottom band is 400 bp). (Bottom) The expression of flag-BRG-1 and α-tubulin (internal control) from the cells used in the top panel was analyzed by immunoblottings. Download figure Download PowerPoint Inactivation of the SWI/SNF complexes compromises the induction of γ-H2AX after DNA damage γ-H2AX is known to be essential for efficient repair of chromosomal DSBs (Bassing and Alt, 2004; Fernandez-Capetillo et al, 2004). The above results therefore prompted us to test whether the SWI/SNF complexes regulate γ-H2AX as one of the potential mechanisms to facilitate DBS repair. For this, we irradiated tet-VP16 and B05-1 cells that had been cultured under the conditions with or without tetracycline to induce the expression of flag-BRG-1 (Figure 3A, top). To our surprise, immunoblot analysis using the specific antibodies showed that the phosphorylation of H2AX after irradiation (25 Gy) was severely compromised in B05-1(−tet) cells, whereas B05-1(+tet) cells exhibited a typical kinetics of γ-H2AX with its levels maximized at 30–60 min and declined afterwards, reflecting DSB generation and repair (Figure 3A, middle). tet-VP16 cells showed the similar kinetics as B05-1(+tet) cells regardless of tetracycline depletion (Figure 3A, bottom). The defect of H2AX phosphorylation in B05-1(−tet) cells was detected when the cells were irradiated by various doses from 0.5 to 100 Gy (Figure 3B and see below). The levels of H2AX proteins were not different between B05-1(+tet) and B05-1(−tet) cells (Figure 3B and also see below), indicating that the SWI/SNF complexes are critical for the efficient induction of H2AX phosphorylation after DNA damage. Figure 3.Cells inactivated for the SWI/SNF complexes are compromised in the induction of γ-H2AX after DNA damage. (A) (Top) Immunoblot analysis for the expression of flag-BRG-1 and actin (internal control) from the cells used below. (Middle and bottom) Indicated cells were untreated (0 Gy) or irradiated by 25 Gy, and histones were acid-extracted at various time points for analysis of the levels of γ-H2AX and H2A (loading control) by immunoblottings. (B) (Top) Immunoblot analysis for the expression of flag-BRG-1 and actin (internal control) from the cells used below. (Bottom) The effects of SWI/SNF inactivation on the induction of γ-H2AX after irradiation by various doses. The experiments were carried out as in (A) except that the expression of H2AX was analyzed as loading control. (C) (Top) Immunoblot analysis for the expression of flag-BRG-1 and actin (internal control) from the cells used below. (Bottom) The effects of SWI/SNF inactivation on the formation of γ-H2AX foci were analyzed by immunofluorescence microscopy. Indicated cells were untreated (0 Gy) or irradiated by 0.5, 1, 2 and 6 Gy, and the cells were fixed after 1 h for immunostaining with γ-H2AX antibodies. The graph shows the average number of γ-H2AX foci determined by counting about 50 nuclei per sample. The error bar indicates mean±s.d. from three independent experiments. (D) Representative confocal images of γ-H2AX foci taken 15, 30 and 60 min after irradiation by 2 Gy. The nuclei were visualized by DAPI staining. Download figure Download PowerPoint Next, we examined whether the SWI/SNF complexes also would affect the formation of γ-H2AX foci after DNA damage by immunofluorescence microscopy. For quantitative analysis of the foci, we irradiated cells by low-dose IR (0.5–6 Gy). When analyzed at 1 h after irradiation, the number of γ-H2AX foci of B05-1(−tet) cells was approximately 30% of that of the control cells (Figure 3C). The defect of γ-H2AX focus formation in B05-1(−tet) cells was manifested as early as 15 min postirradiation and detected throughout the tested time course up to 4 h (Figure 3D and data not shown). In addition, the majority of γ-H2AX foci detected from B05-1(−tet) cells appeared to be much smaller in size relative to those of control cells (Figure 3D). These results, together with the immunoblot data, suggest that the SWI/SNF complexes are critical for the formation of γ-H2AX foci as well as the phosphorylation of H2AX after DNA damage. Downregulation of BRG-1 and hBrm results in γ-H2AX defect, inefficient DSB repair and increased DNA damage sensitivity To further demonstrate specifically the role of the SWI/SNF complexes in γ-H2AX induction and DSB repair, we used small interference RNA (siRNA) approaches. When BRG-1 and human Brm (hBrm), the catalytic core subunits of the human SWI/SNF complexes, were downregulated by cotransfecting HeLa cells with the corresponding siRNAs, both H2AX phosphorylation (Figure 4A and 4B) and γ-H2AX focus formation (Figure 4C) following DNA damage were largely compromised. The cells downregulated for BRG-1 and hBrm also showed increased DNA damage sensitivity (Figure 4D) and decreased DSB repair (Figure 4E). These results, confirming the data from the dominant negative experiments, demonstrate that the SWI/SNF complexes are specifically responsible for the optimal induction of γ-H2AX, efficient DSB repair and cell survival after DNA damage. It is noted that, as individual knockdown of either BRG-1 or hBrm appears to influence each other in their cellular levels (HL and JK, unpublished observations), we currently do not understand what relative extent to which each subunit would contribute to these functions. Figure 4.Downregulation of both BRG-1 and hBrm results in γ-H2AX defect, inefficient DSB repair and increased DNA damage sensitivity. (A) The effects of BRG-1/hBrm downregulation on the γ-H2AX induction after irradiation. HeLa cells were mock-transfected (−) or cotransfected with BRG-1 and hBrm siRNAs (+) for 48 h before irradiation by indicated doses. After 1 h, cells were collected and divided into two for preparation of whole-cell lysates and acid extracts of histones. The whole-cell lysates were analyzed for the expression of BRG-1, hBrm and actin (internal control) by immunoblottings. The acid extracts were divided into two for analyzing the levels of γ-H2AX and the expression of H2A and H2AX by immunoblottings in separate gels. The γ-H2AX blots show both short and long exposures for better comparison of γ-H2AX levels. A representative of five independent experiments is shown. (B) The effects of BRG-1/hBrm downregulation on the kinetics of γ-H2AX induction. HeLa cells transfected as in (A) were irradiated by 25 Gy and collected at various time points for analyzing the expression of indicated proteins. A representative of five independent experiments is shown. (C) The effects of BRG-1/hBrm downregulation on the formation of γ-H2AX foci. (Left) HeLa cells cotransfected with BRG-1 and hBrm siRNAs were irradiated by 2 Gy, and after 1 h cells were fixed and double stained with antibodies for BRG-1 and γ-H2AX. Confocal images were taken so as to capture both nontransfected and transfected cells in the same picture. The nuclei were visualized by DAPI staining. (Right) γ-H2AX foci were counted using the confocal image in the left panel and depicted as a graph. (D) The effects of BRG-1/hBrm downregulation on the viability after DNA damage. HeLa cells transfected as in (A) were irradiated by indicated doses before viability was evaluated by colony formation assays. Data are presented as mean±s.d. from triplicates. Immunoblot analysis of siRNA knockdown of BRG-1 is shown next to the graph. (E) The effects of BRG-1/hBrm downregulation on DSB repair. HeLa cells were transfected as in (A) and irradiated by 50 Gy and the cells were collected at the indicated time points before subjecting to neutral comet assays. Each graph is depicted as mean±s.d. from two independent experiments. Immunoblot analysis of siRNA knockdown of BRG-1 for the two independent experiments is shown next to the graph. Download figure Download PowerPoint The effects of the SWI/SNF complexes on γ-H2AX are independent of ATM, DNA-PK and ATR We then wished to understand how the SWI/SNF complexes regulate γ-H2AX. As a simple possibility, the SWI/SNF complexes could stimulate the phosphorylation of H2AX by regulating the expression of the kinase genes responsible for H2A phosphorylation. We found that this was not the case; the levels of ATM, DNA-PKcs and ATR proteins were not affected by inactivation of the SWI/SNF complexes (data not shown) or downregulation of BRG-1 and hBrm (Figure 5A). Next, we examined whether the SWI/SNF complexes are involved in the activation of ATM, the major player for the phosphorylation of H2AX after exposure to IR (Burma et al, 2001; Fernandez-Capetillo et al, 2002). ATM exists as an inactive dimer form before DNA damage, and, after DNA damage, ATM is autophosphorylated on Ser-1981 (p-ATM) and dissociated into active monomers (Bakkenist and Kastan, 2003). Using the specific antibodies recognizing this diagnostic autophosphorylation, we found that ATM was normally activated after irradiation in the SWI/SNF-inactivated cells (Figure 5B) and in the cells downregulated for BRG-1 and hBrm (Figure 5C). Further confirming the ATM activation, Nbs1, one of the major substrates of ATM, was phosphorylated in those cells after irradiation (Figure 5D and data not shown). These results suggest that the SWI/SNF complexes do not participate in the process of activating ATM following DNA damage. Figure 5.The effects of the SWI/SNF complexes on γ-H2AX are independent of ATM, DNA-PK and ATR. (A) siRNA knockdown of BRG-1 and hBrm has no effect on the expression of ATM, DNA-PKcs and ATR. HeLa cells mock transfected or cotransfected with BRG-1 and hBrm siRNA for 48 h were untreated (0 Gy) or irradiated by 10 Gy. After 1 h, cell lysates were analyzed for the expression of the indicated proteins by immunoblottings. (B) ATM activation after DNA damage normally occurs in SWI/SNF-inactivated cells. B05-1(+tet) and B05-1(−tet) cells were untreated (0 Gy) or irradiated by 10 Gy, and the cell lysates were prepared at the indicated time points for analysis of the diagnostic autophosphorylation of ATM at Ser-1981 as well as the expression of ATM by immunoblottings. The expression of flag-tagged proteins was analyzed to ensure the induction of the dominant negative BRG-1, and the expression of α-tubulin was analyzed as internal control. (C) siRNA knockdown of BRG-1 and hBrm has no effect on the activation of ATM after DNA damage. HeLa cell lysates prepared as in (A) were analyzed for the expression of the indicated proteins. (D) Nbs1 is phosphorylated in SWI/SNF-inactivated cells after DNA damage. The expression of phospho-Nbs1(Ser-343) and Nbs1 was analyzed by immunoblottings at 1 h postirradiation (30 Gy). The expression of flag-BRG-1 and α-tubulin (internal control) was also analyzed. (E) ATM foci can be formed in SWI/SNF-inactivated cells after DNA damage. B05-1(+tet) and B05-1(−tet) cells were fixed at 1 h after irradiation by 2 Gy, and double stained by antibodies for γ-H2AX and p-ATM(Ser-1981) before confocal images were captured. The nuclei were visualized by DAPI staining. (F) siRNA knockdown of BRG-1/hBrm has no effect on the formation of ATM foci after DNA damage. HeLa cells cotransfected with BRG-1 and hBrm siRNAs for 48 h were untreated (0 Gy) or irradiated by 2 Gy. Irradiated cells were collected after 15, 30, 60 and 240 min for double staining with antibodies for BRG-1 and p-ATM(Ser-1981). Confocal images were taken so as to capture both nontransfected and transfected cells in the same picture for each sample. The nuclei were visualized by DAPI staining. (G, H) DNA-PKcs and ATR can form foci in SWI/SNF-defected cells after DNA damage. B05-1(+tet) and B05-1(−tet) cells were irradiated by 2 Gy (G) or 10 Gy (H) followed by incubation for 2 h. Cells were collected for double staining with antibodies for γ-H2AX and p-DNA-PKcs(Thr-2609) (G), or with antibodies for γ-H2AX and ATR (H). The nuclei were visualized by DAPI staining. Download figure Download PowerPoint A current model predicts that activated ATM is initially recruited to a DSB to phosphorylate nearby H2AX and that the phosphorylated H2AX recruit more ATM to the DSB site in a positive activation loop (Bakkenist and Kastan, 2004; Stavridi and Halazonetis, 2005). We therefore asked if the SWI/SNF complexes promote H2AX phosphorylation by regulating ATM recruitment. Immunofluorescence microscopy showed that p-ATM foci after irradiation were normally formed in B05-1(−tet) cells (Figure 5E and Supplementary Figure S1A) and in the cells downregulated for BRG-1 and hBrm (Figure 5F); their levels and time-course induction are not distinguishable from those of control cells, in keeping with the above results showing normal ATM activation in irradiated SWI/SNF-defective cells. We note that, as previously shown, γ-H2AX foci were barely detected from those SWI/SNF-defective cells (Figure 5E and Supplementary Figure S1A, and data not shown). These results were somewhat unexpected, however, given the number of studies suggesting the requirement of H2AX for ATM recruitment to DBS sites; cells lacking H2AX fail to form Nbs1 foci (Celeste et al, 2002, 2003b), and Nbs1 is required for ATM activation and recruitment to DSBs (Uziel et al, 2003; Kitagawa et al, 2004; Lee and Paull, 2005; Difilippantonio et al, 2005; Falck et al, 2005). We reasoned that the relative γ-H2AX levels (null versus downregulation) could make such differences. Indeed, after irradiation, cells lacking H2AX exhibited a pan-nuclear staining pattern of p-ATM in contrast to the control cells showing distinct p-ATM foci (Supplementary Figure S1B, and also shown by Stucki et al, 2005). The levels of γ-H2AX in SWI/SNF-defected cells, although largely decreased, might be still sufficient for supporting the accumulation of ATM into foci. Further, the recruitment of the phosphorylated DNA-PKcs on Thr-2609 (Chan et al, 2002) and ATR to DSBs was not affected by SWI/SNF inactivation (Figure 5G and H). These results suggest that the optimal phosphorylation of H2AX after DNA damage is not guaranteed just by ATM (and other PIKKs) activation and recruitment, but requires additional steps that likely involve the activity of the SWI/SNF complexes. The SWI/SNF complexes rapidly bind to DSB-surrounding chromatin via interaction with γ-H2AX The results thus far led us to hypothesize that the SWI/SNF complexes might promote H2AX phosphorylation by directly acting o
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