A cooperative activation loop among SWI/SNF, γ-H2AX and H3 acetylation for DNA double-strand break repair
2010; Springer Nature; Volume: 29; Issue: 8 Linguagem: Inglês
10.1038/emboj.2010.27
ISSN1460-2075
AutoresHan-Sae Lee, Jihye Park, So Jung Kim, Su-Jung Kwon, Jongbum Kwon,
Tópico(s)DNA Repair Mechanisms
ResumoArticle11 March 2010free access A cooperative activation loop among SWI/SNF, γ-H2AX and H3 acetylation for DNA double-strand break repair Han-Sae Lee Han-Sae Lee Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Ji-Hye Park Ji-Hye Park Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author So-Jung Kim So-Jung Kim Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Su-Jung Kwon Su-Jung Kwon Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Jongbum Kwon Corresponding Author Jongbum Kwon Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Han-Sae Lee Han-Sae Lee Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Ji-Hye Park Ji-Hye Park Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author So-Jung Kim So-Jung Kim Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Su-Jung Kwon Su-Jung Kwon Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Jongbum Kwon Corresponding Author Jongbum Kwon Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea Search for more papers by this author Author Information Han-Sae Lee1,‡, Ji-Hye Park1,‡, So-Jung Kim1, Su-Jung Kwon1 and Jongbum Kwon 1 1Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea ‡These authors contributed equally to this work *Corresponding author. Division of Life and Pharmaceutical Sciences, Department of Life Science, Ewha Womans University, Science Building C-308, Seodaemun-gu, Daehyun-dong 11-1, Seoul 120-750, Republic of Korea. Tel.: +82 2 3277 4334; Fax: +82 2 3277 3760; E-mail: [email protected] The EMBO Journal (2010)29:1434-1445https://doi.org/10.1038/emboj.2010.27 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although recent studies highlight the importance of histone modifications and ATP-dependent chromatin remodelling in DNA double-strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser-139 (S139ph) after DNA damage, binds to γ-H2AX (the phosphorylated form of H2AX)-containing nucleosomes in S139ph-dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ-H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ-H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ-H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ-H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ-H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair. Introduction DNA double-strand breaks (DSBs) can be generated by environmental agents such as ionizing radiation (IR). Unless repaired accurately and efficiently, DSBs can lead to genome instability, cell death and cancer development. On DSB generation, a number of DSB response proteins are recruited to the site of a DSB, and stable accumulation of such proteins within the DNA lesion, manifested as microscopically visible nuclear foci, is important for efficient DSB repair and checkpoint activation (van Gent et al, 2001; Jackson, 2002; Harper and Elledge, 2007). The compaction of the eukaryotic genome into a highly ordered chromatin structure necessitates cellular mechanisms for allowing regulatory proteins to access their target DNA. Indeed, studies show that histone modifications and ATP-dependent chromatin remodelling are both important for DSB repair and damage response (Allard et al, 2004; Peterson and Cote, 2004; Bao and Shen, 2007; Downs et al, 2007; van Attikum and Gasser, 2009). Among the various histone modifications is S139ph, the most central for the cellular responses to DSBs. γ-H2AX, formed by ATM immediately after DNA damage, has an important function for the formation of stable repair foci in part by providing binding sites for DSB response proteins and has been shown to be important for efficient DSB repair and suppression of genome instability and cancer (Bonner et al, 2008; Kinner et al, 2008). Other types of histone modifications such as acetylation and methylation have also been shown to have important function in DSB response either individually or in conjunction with S139ph (Bird et al, 2002; Downs et al, 2004, 2007; Huyen et al, 2004; Sanders et al, 2004). In addition, a series of recent works have shown that S139ph facilitates the ubiquitination of histones H2A and H2B, which serves to recruit DSB response proteins to the sites of DSB, establishing crosstalk between histone modifications as an important mechanism for DSB response (Huen et al, 2007; Kolas et al, 2007; Mailand et al, 2007; Wang et al, 2007; Doil et al, 2009; Stewart et al, 2009). Several ATP-dependent chromatin remodelling complexes have been directly implicated in DSB response. In yeast, INO80, SWR1, SWI/SNF and RSC complexes have been shown to be recruited to a DSB and reconfigure the nucleosomes around the DSB in such a way to facilitate DNA repair and/or to modulate checkpoint activation (Downs et al, 2004; Morrison et al, 2004; van Attikum et al, 2004, 2007; Chai et al, 2005; Shim et al, 2005; Bao and Shen, 2007). Mammalian SWI/SNF complexes have also been shown to target the chromatin at DBS sites and facilitate DNA repair (Park et al, 2006). Although INO80 and Swr1 complexes have been suggested to be recruited to a DSB through interaction with γ-H2AX, the mechanisms for these interactions have remained unclear (Downs et al, 2004; Morrison et al, 2004; van Attikum et al, 2004). Similarly, BRG1 and hBrm, the mutually exclusive ATPase subunits of human SWI/SNF complexes, have been shown to interact with γ-H2AX nucleosomes; however, the nature of their interactions has remained elusive (Park et al, 2006). Interestingly, the yeast INO80 and mammalian SWI/SNF complexes interact with γ-H2AX and yet seem to stimulate γ-H2AX formation (Papamichos-Chronakis et al, 2006; Park et al, 2006), suggesting the existence of interdependence between chromatin remodelling and S139ph. However, how these distinct mechanisms of chromatin modifications work together during DBS repair has remained obscure. In this study, we investigated the specific interaction between SWI/SNF and γ-H2AX nucleosomes, and found an unexpected way of interaction between a chromatin remodeller and γ-H2AX nucleosomes; SWI/SNF binds to γ-H2AX nucleosomes by interacting with acetylated H3 rather than γ-H2AX itself. Through the course of further dissecting the responsible mechanisms for this interaction, we have revealed that SWI/SNF, γ-H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DBS repair. We propose a model for this novel mechanism and discuss its biological significance. Results SWI/SNF binds to γ-H2AX nucleosomes in S139ph-dependent manner We have earlier shown the interaction of SWI/SNF with γ-H2AX nucleosomes by immunoprecipitation (IP) using the cells that stably express flag-tagged H2AX (f-H2AX) (Park et al, 2006). Although this interaction was shown to be dependent on IR, whether it requires S139ph was not determined. We, therefore, generated the stable cells expressing non-phosphorylatable f-H2AX in which Ser-139 is changed to Ala (f-S139A) and performed the similar chromatin IP experiments. When flag-tagged nucleosomes were precipitated from the cells after irradiation, BRG1 and hBrm were co-precipitated with f-H2AX nucleosomes as shown earlier (Park et al, 2006), but barely detectable with f-S139A nucleosomes (Figure 1A). As control, Ku70, known to bind the ends of nucleosomal DNA (Park et al, 2003), was co-precipitated equally with f-H2AX and f-S139A nucleosomes (Figure 1A). Under our experimental conditions, immunoprecipitated nucleosomes are intact as they contain the four core histones plus f-H2AX (Figure 1B) and are typically dimers or trimers depending on the conditions for chromatin fragmentation (Figure 1C). These data formally show that SWI/SNF binding to γ-H2AX nucleosomes is dependent on S139ph. Figure 1.SWI/SNF binds to γ-H2AX nucleosomes in S139ph-dependent manner. (A) Flag-tagged nucleosomes were immunoprecipitated from the cells stably expressing either f-H2AX or f-S139A at 1 h after 10-Gy IR, and analysed for flag-tagged histones, γ-H2AX and associating proteins by immunoblot with specific antibodies. As control, the cells containing empty vector were subjected in parallel to the nucleosome immunoprecipitation. (B) Ponceau S stain of immunoblot shows that immunoprecipitated f-H2AX nucleosomes are intact. (C) DNA of fragmented chromatin was analysed along with a size marker (M) on agarose gel. Download figure Download PowerPoint S139ph stimulates H3 acetylation on γ-H2AX nucleosomes We wished to understand how SWI/SNF interacts with γ-H2AX nucleosomes in S139ph-dependent manner. Two protein domains, the fork-head associated (FHA) and BRCA1 C-terminal (BRCT), have been identified to specifically recognize the phosphorylated amino-acid residues that are frequently found in proteins involved in DNA damage response (Durocher et al, 1999; Manke et al, 2003; Yu et al, 2003). We studied the sequences of BRG1/hBrm and all the associating proteins of SWI/SNF, and found that none of these proteins has FHA, BRCT or any protein domains known to recognize phosphorylated amino-acid residues. Therefore, we reasoned that SWI/SNF binds to γ-H2AX nucleosomes indirectly through other protein(s), or alternatively, directly binds to γ-H2AX nucleosomes by interacting with other histones than γ-H2AX still in the S139ph-dependent manner. As BRG1 and hBrm both have a BRD known to recognize acetylated histones (Mujtaba et al, 2007), we hypothesized the latter possibility. As a step towards investigating our hypothesis, we asked whether S139ph influences acetylations of other histones on γ-H2AX nucleosomes. To answer this question, we examined the effects of S139A mutation on the acetylation of H3, the core histone most heavily subjected to post-translational modifications. When we analysed the flag-tagged nucleosomes from irradiated f-H2AX or f-S139A cells for the acetylation at the conserved Lys residues of the N-terminal tails of H3, we found that the acetylations at K9, K14, K18 and K23 was largely decreased by S139A mutation with that at K14 and K18 most severely affected (Figure 2A). Interestingly, the acetylation at K27 was not affected by S139A mutation, indicating that not all acetylations on H3 are influenced by S139ph. In contrast, none of the methylations at the Lys residues of H3 analysed, including K79 known to serve as the binding site of 53BP1 DNA damage checkpoint protein (Huyen et al, 2004), was affected by S139A mutation (Figure 2A). These data indicate that S139ph positively influences the acetylation of H3 on the same and/or neighbouring nucleosomes, suggesting for the first time the existence of a crosstalk between S139ph and histone acetylation. Figure 2.S139ph is required for the acetylation of H3 on γ-H2AX nucleosomes. (A) Flag-tagged nucleosomes were immunoprecipitated from the cells containing empty vector, or expressing either f-H2AX or f-S139A at 1 h after 10-Gy IR, and analysed for the acetylations and methylations of H3 by immunoblot with specific antibodies. The bands of acetyl-H3 on precipitated f-H2AX and f-S139ph nucleosomes were quantitated by densitometer, and after normalization to H3 bands, the fold reduction of H3 acetylation was calculated and shown at the right side of the corresponding gel. For the gel with star mark, the fold reduction was not calculable as the band intensity of f-S139A lane is lower than background. The nomenclature of histone modifications used in this paper was followed by Turner (2005); ac, acetylation; me, monomethylation; me2, dimethylation; ph, phosphorylation. (B) Vector and f-H2AX cells were transfected with either the siRNAs specific for BRG1 or hBrm (B/h), or non-specific control siRNA. Cells were collected at 1 h after 10-Gy IR for the analysis of BRG1 and hBrm knockdown by immunoblot with specific antibodies; α-tubulin was also analysed for internal control. (C) Flag-tagged nucleosomes were immunoprecipitated from the aliquots of the cells prepared in (B) were analysed for the indicated modifications of H3 by immunoblot using specific antibodies. The fold reduction of H3 acetylation on precipitated f-H2AX nucleosomes by SWI/SNF knockdown was calculated as per in (A) and shown at the right side of the corresponding gel. (D) Cells were transfected with either control or BRG1/hBrm-specific siRNAs, and at 1 h after 10-Gy IR, whole cell lysates were prepared for the analysis of BRG1 and hBrm expression, and acid-extracted histones for the analysis of H3 acetylation as indicated. Download figure Download PowerPoint SWI/SNF stimulates H3 acetylation on γ-H2AX nucleosomes through S139ph The results in Figure 2A, together with our earlier finding that SWI/SNF facilitates γ-H2AX formation (Park et al, 2006), suggest that SWI/SNF stimulates H3 acetylation by facilitating S139ph. To determine whether this is the case, we knockdowned BRG1 and hBrm by specific siRNAs in f-H2AX cells (Figure 2B), and analysed the flag-tagged nucleosomes from these cells for H3 acetylation as described before. We found that the acetylations at K9, K14, K18 and K23, but not at K27, were largely decreased by SWI/SNF knockdown with K14 acetylation most dramatically affected (Figure 2C). Methylations at several lysine residues, including K79, were not affected by SWI/SNF knockdown (Figure 2C and data not shown). Importantly, the defect of H3 acetylation by SWI/SNF knockdown was not apparently detected when bulk chromatin was analysed, indicating that such effects occur preferentially on γ-H2AX nucleosomes (Figure 2D). These data, showing that SWI/SNF deficiency and S139A mutation result in the similar patterns of H3 acetylation defect, show that SWI/SNF stimulates H3 acetylation through S139ph. BRG1 binds to γ-H2AX nucleosomes by interacting with acetylated H3 through its bromodomain The results thus far raised the possibility that SWI/SNF may bind to γ-H2AX nucleosomes by interacting with acetylated H3 through the BRD of BRG1/hBrm. We investigated this possibility by focusing on BRG1. First, we examined whether the BRD is required for BRG1 binding to γ-H2AX nucleosomes. Flag-tagged nucleosomes were immunoprecipitated from the f-H2AX cells in which wild-type or BRD-deleted BRG1 (BRG1ΔBRD) was ectopically expressed and analysed for bound proteins. BRG1ΔBRD did not bind to γ-H2AX nucleosomes, whereas the wild-type BRG1 did as earlier seen (Figure 3A), indicating that BRD is required for BRG1 binding to γ-H2AX nucleosomes. Figure 3.BRG1 binds to γ-H2AX nucleosomes by interacting with acetylated H3 through its bromodomain. (A) f-H2AX cells were transfected with empty vector, or the expression vectors for the full-length BRG1 or the BRD-deleted BRG1 (BRG1ΔBRD). Flag-tagged nucleosomes were immunoprecipitated at 1 h after 10-Gy IR and analysed for associating proteins by immunoblot. (B) f-H2AX and f-S139A cells were transfected with empty vector (V) or the expression vector for myc-tagged BRG1 BRD (Myc-BRD, b). Flag-tagged nucleosomes were immunoprecipitated at 1 h after 10-Gy IR and analysed for associating proteins by immunoblot. Non-specific (NS) bands are also indicated. (C) f-H2AX (lane 1) and f-S139A (lane 2) cells were exposed to 10-Gy IR, and after 1 h, cells were collected and subjected to the affinity purification of flag-tagged nucleosomes. Coomassie stain gel of the purified nucleosomes is shown. (D) The GST proteins containing the 588–748 aa of BRG1 (lane 1) or the BRG1 BRD (lane 2) were expressed and purified from Escherichia coli, and analysed on an SDS gel with coomassie stain. (E) The purified flag-tagged nucleosomes shown in (C) were analysed for γ-H2AX and H3K14ac by immunoblot. (F) Affinity-purified f-S139A and f-H2AX nucleosomes were incubated with buffer only (lanes 1 and 4) or GST-BRD at increasing molar ratios of 1:2 (lanes 2 and 5) or 1:8 (lanes 3 and 6). Flag-tagged nucleosomes were immunoprecipitated and analysed for associating proteins by immunoblot. (G) Affinity-purified f-H2AX nucleosomes were incubated with buffer only (lanes 1 and 7) or indicated GST proteins at increasing molar ratios of 1:1 (lanes 2 and 8), 1:2 (lanes 3 and 9), 1:4 (lanes 4 and 10), 1:8 (lanes 5 and 11) or 1:16 (lanes 6 and 12). The nucleosomes were immunoprecipitated and analysed for associating proteins by immunoblot. (H) Verification of synthetic peptides by immunoblot analysis. Unmodified and K14-acetylated H3 peptides (left panel), and unmodified and S139-phosphorylated H2AX peptides (right panel) were run on 18% SDS gel and subjected to immunoblot with specific antibodies as indicated. (I) Indicated biotinylated peptides (5 μg/ml) were incubated with buffer only (lanes 1, 4, 7 and 10) or purified SWI/SNF complexes at the concentrations of 0.2 μg/ml (lanes 2, 5, 8 and 11) or 0.8 μg/ml (lanes 3, 6, 9 and 12). Peptide-protein complexes were pull downed by streptavidin-coated beads and the bead-bound proteins were analysed by immunoblot. Download figure Download PowerPoint To determine whether BRG1 BRD is alone able to bind γ-H2AX nucleosomes in S139ph-dependent manner, the flag-tagged nucleosomes from f-H2AX and f-S139A cells in which myc-tagged BRG1 BRD (Myc-BRD) was expressed were analysed for bound proteins. As shown in Figure 3B, Myc-BRD bound to f-H2AX, but not to f-S139A, nucleosomes, indicating that BRG1 BRD is sufficient to bind to γ-H2AX nucleosomes dependently on S139ph. Therefore, the BRD of BRG1 is necessary and sufficient for the S139ph-dependent binding of BRG1 to γ-H2AX nucleosomes. To directly show the S139ph-dependent interaction between BRG1 BRD and γ-H2AX nucleosomes, we performed in vitro pull-down experiments using affinity-purified f-H2AX and f-S139A nucleosomes (Figure 3C) and the GST proteins with BRG1 BRD (GST-BRD) purified from bacteria (Figure 3D). The purified flag-tagged nucleosomes contained the four core histones and the f-H2AX or f-S139A histones at stoichiometry. Immunoblot analysis verified that the levels of H3 acetylation were greatly reduced on f-S139A compared with f-H2AX nucleosomes as expected (Figure 3E). When incubated with purified flag-tagged nucleosomes, GST-BRD bound to f-H2AX much better than to f-S139A nucleosomes (Figure 3F). As a control, the GST proteins containing 588-748aa of BRG1 or GST alone did not bind to either nucleosomes (Figure 3G and data not shown), showing that BRG1 BRD specifically interacts with γ-H2AX nucleosomes. These data show that BRG1 BRD directly interacts with γ-H2AX nucleosomes in S139ph-dependent manner. The results described above strongly suggest that BRG1 binds to γ-H2AX nucleosomes by interacting with acetylated H3 instead of S139ph. To determine whether this is the case, we performed in vitro pull-down assays using purified human SWI/SNF complexes and the synthetic peptides containing the sequences corresponding to H3 in the form of either non-acetylated (H3) or acetylated at K14 (H3K14ac) (Figure 3H, left panel), or the sequences corresponding to H2AX in the form of either non-phosphorylated (H2AX) or phosphorylated at S139 (S139ph) (Figure 3H, right panel). As shown in Figure 3I, BRG1 in the form of SWI/SNF complex preferentially binds to H3K14ac over H3 peptides; however, it did not bind to H2AX or S139ph peptides. Taken all together, the results collectively show that SWI/SNF binds to γ-H2AX nucleosomes in S139ph-dependent manner by interacting with acetylated H3 through BRG1 BRD rather than by interacting with S139ph itself. BRG1 binding to γ-H2AX nucleosomes is important for S139ph and DSB repair Having found the mechanisms for the interaction between SWI/SNF and γ-H2AX nucleosomes, we wanted to investigate whether the interruption of this interaction would affect the earlier defined functions of SWI/SNF in γ-H2AX formation and DSB repair (Park et al, 2006). To this end, we generated the expression vector for the silent mutant form of BRG1 whose expression is resistant to the BRG1-specific siRNA (BRG1R), as well as the expression vector for BRG1R lacking BRD (BRG1RΔBRD). First, we verified that BRG1R could be expressed in the cells in which BRG1 was knockdowned by siRNA and that the expression levels of BRG1R were similar to those of the endogenous BRG1 (Figure 4A). We then examined the effects of BRD deletion on the ability of BRG1 to stimulate S139ph after DNA damage by immunoblot. As shown in Figure 4B, BRG1RΔBRD failed to rescue the defect of S139ph induced by BRG1 knockdown, whereas BRG1R completely rescued this defect. Virtually, the same results were obtained when the effects of BRD deletion on the formation of γ-H2AX foci were examined using immunofluorescence microscopy (Figure 4C and D). Similar rescue experiments showed that BRG1R completely rescued the defect of cell survival after DNA damage in BRG1-knockdowned cells, whereas BRG1RΔBRD did not (Figure 4E). These data show that the BRD is critical for the BRG1's functions for γ-H2AX formation and DSB repair. Figure 4.BRG1 binding to γ-H2AX nucleosomes is important for S139ph and DSB repair. (A) Cells were cotransfected with non-specific (lane 1) or BRG1-specific siRNAs (lanes 2 and 3), plus either empty vectors (lanes 1 and 2) or the expression vectors for HA-tagged siRNA-resistant BRG1 (BRG1R) (lane 3). Whole cell lysates were analysed for the expression of BRG1 and BRG1R by immunoblot. (B) Cells were cotransfected with non-specific (lanes 1 and 2) or BRG1-specific siRNAs (lanes 3–5), plus either empty vectors (lanes 1–3) or the expression vectors for BRG1R (lane 4) or BRD-deleted BRG1R (BRG1RΔBRG1, lane 5). At 1 h after untreated (lane 1) or 10-Gy IR (lanes 2–5), cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-BRG1 or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. (C) Cells were transfected as described in lanes 2–5 of (B) and exposed to 2-Gy IR. After 1 h, cells were fixed and dually stained with anti-BRG1 or anti-γ-H2AX antibodies before confocal images were captured. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 cells. The error bar indicates mean±s.d. of three independent experiments. (D) Representative confocal images from the experiments in (C) are shown. (E) Cells transfected as per in (D) were untreated (0 Gy) or exposed to 1–5 Gy IR before the viability was determined by colony formation assays with triplicates per sample. The graph shows average number of colonies with mean±s.d. of four independent experiments. (F) Cells were transfected with empty or Myc-BRD expression vectors. At 1 h after 10-Gy IR, cells were collected to prepare whole cell lysates and acid-extracted histones for immunoblots with anti-Myc or anti-γ-H2AX antibodies, respectively; α-tubulin and H2A were also analysed for loading control. (G) Cells transfected with empty, Myc-BRD or Myc-BRG1(588–748) vectors were irradiated by 2 Gy, and after 1 h, cells were fixed for dual staining with the antibodies against Myc or γ-H2AX. The Myc-BRG1(588–748) vector expresses the sequences of 588–748 amino acids of BRG1, outside the BRD, and was used as a control. Average number of γ-H2AX foci per cell was depicted as graph by counting at least 50 each of untransfected and transfected cells. The error bar indicates mean±s.d. of three independent experiments. (H) Representative confocal images from the experiments in (G) are shown. (I) Cells were transfected with indicated vectors, and after irradiation, cells were subjected to colony formation assays as described in (E). The graph shows average number of colonies with mean±s.d. of three independent experiments. Download figure Download PowerPoint Finally, we determined whether the BRD of BRG1, when overexpressed in the cells, could interfere with the BRG1's functions for γ-H2AX formation and DSB repair. We found that overexpression of Myc-BRD in the cells compromises S139ph (Figure 4F) and γ-H2AX focus formation (Figure 4G and H) as well as the cells’ ability to survive DNA damage (Figure 4I), indicating that BRG1 BRD can function as dominant-negative inhibitors of BRG1 in γ-H2AX formation and DSB repair. Therefore, blocking the interaction between BRG1 and γ-H2AX either by deletion or overexpression of the BRG1 BRD leads to loss of the BRG1's ability to facilitate S139ph and DSB repair, showing the importance of SWI/SNF binding to γ-H2AX nucleosomes for these processes. H3 acetylation is required for BRG1 binding to γ-H2AX nucleosomes As our results showed that BRG1 binds to γ-H2AX nucleosomes through acetylated H3, we wanted to ask whether H3 acetylation is indeed required for this binding. For this, we generated a pair of plasmid constructs expressing flag-tagged H3 (f-H3) either in wild-type or the mutant forms in which the four N-terminal Lys residues (K9, K14, K18 and K23) were all changed to Gln, and also generated a construct expressing myc-tagged H2AX (myc-H2AX). We transfected 293T cells with myc-H2AX together with wild-type or mutant f-H3 vectors, and 1 h after irradiation, we sequentially immunoprecipitated the nucleosomes containing both f-H3 and myc-H2AX from these cells and analysed them for bound BRG1 (Figure 5A). Strikingly, the BRG1 binding to these nucleosomes was largely diminished by the mutation of the four acetylation sites of H3 (Figure 5B). These data, therefore, show that H3 acetylation is crucial for the BRG1 binding to γ-H2AX nucleosomes. Figure 5.H3 acetylation is required for the binding of BRG1 to γ-H2AX nucleosomes. (A) The experimental procedure used in (B) is represented as a schematic flow chart. See Materials and methods for details. (B) 293T cells were transfected with myc-H2AX plus either f-H3-wt (lanes 1 and 3) or f-H3-K9/14/18/23Q vectors (lanes 2 and 4), and irradiated by 10 Gy 1 h before harvest. f-H2AX nucleosomes were immunoprecipitated using anti-Flag M2 beads and eluted with Flag peptides, and the Flag-eluted nucleosomes were then subjected to the second immunoprecipitation by Myc antibody followed by immunoblot analysis as indicated. The K to Q mutations for the four acetylation sites were verified by DNA sequencing (Materials and methods) as well as by immunoblot analysis with specific antibodies (here and data not shown). H4 was also analysed to monitor the integrity of the precipitated nucleosomes. Download figure Download PowerPoint Gcn5 is responsible for the H3 acetylation on γ-H2AX nucleosomes To understand how γ-H2AX stimulates H3 acetylation, we sought for the histone acetyltransferases (HATs) that are responsible for this mechanism. We reasoned that such HATs are likely to interact with γ-H2AX nucleosomes in S139ph-dependent manner, and examined several HATs with respect to this characteristic. We initially considered the HATs that have activity towards H3 and also has been shown to be implicated in DNA repair; those are Gcn5/KAT2A, p300/KAT3B and their closely related HATs, PCAF/KAT2B and CBP/KAT3A, respectively (Tamburini and Tyler, 2005; Allis et al, 2007; Das et al, 2009). We immunoprecipitated flag-tagged nucleosomes from irradiated f-H2AX and f-S139A cells, and analysed these nucleosomes for bound HATs by immunoblot using specific antibodies. As shown in Figure 6A and B, among the four HATs tested, only Gcn5 binds to γ-H2AX nucleosomes in S139ph-dependent manner, making this protein a strong candidate for the right HAT. Figure 6.Gcn5 is responsible for the γ-H2AX-mediated H3 acetylation. (A, B) To search for the HATs that bind to γ-H2AX nucleosomes in the S139ph-specific manner, all the HATs earlier shown to be implicated in DNA repair were examined by the similar experiments as described in Figure 1. Several independent sets of experiments were performed to test some subgroups of HATs in each of which the specific interaction between BRG1 and γ-H2AX nucleosomes was always monitored to control the chromatin IP experiments. The results
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