SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION SUPPLEMENTARY MATERIALS AND METHODS Antibodies and sirna The antibodies against BAF170, BAF155, BAF60a, BAF57 and BAF53 were purchased from Santa Cruz (TX), and the antibodies against p-sq/tq and GST from Cell Signaling (Danvers, MA). The sources of other antibodies, non-specific, BRG1- and Brm-specific sirnas have been described (1, 2). The sequences of the ATM-specific sirna are as follows: 5 - UCUGUACUUGAUAGACACU(dTdT), and 5 -AGUGUCUAUCAAGUACAGA(dTdT). Plasmid construction and site-directed mutagenesis The vector expressing GST-BRG1 ( ) (2) was subjected to site-directed mutagenesis according to the QuikChange mutagenesis protocol using Pfu turbo polymerase (Stratagene, CA) to generate vectors expressing S610A, S721A or S610/S721A mutants. The vector expressing Ha-BRG1 R (2) was subjected to site-directed mutagenesis to generate vectors expressing the mutant versions containing S721A or S721D. The vectors expressing f-brg1 R or f-brg1 R - S721A were generated by cloning the sequences PCR-amplified from the vectors expressing Ha- BRG1 R or Ha-BRG1 R -S721A into the EcoRV and XbaI sites of 3xFlag-CMV14 vector (Sigma). All the plasmid constructs generated in this work were verified by sequencing. The sequences of the oligonucleotides used in site-directed mutagenesis are as follows. GST-BRG1 ( ) S610A:

2 5'-GCCTCTGGACGAGACCGCTCAGATGAGCGACCTCCCGGTG-3' and 5'- CACCGGGAGGTCGCTCATCTGAGCGGTCTCGTCCAGAGGC-3' GST-BRG1 ( ) S721A and Ha-BRG1 R -S721A: 5'-ATGTCGATGATGAATATGGCGTGGCTCAGGCCCTTGCACGTGG-3' and 5'- CCACGTGCAAGGGCCTGAGCCACGCCATATTCATCATCGACAT-3'. Ha-BRG1 R -S721D: 5'-ATGTCGATGATGAATATGGCGTGGACCAGGCCCTTGCACGTGG-3' and 5'- CCACGTGCAAGGGCCTGGTCCACGCCATATTCATCATCGACAT-3' PCR primers for f-brg1 vectors: 5'-GCTCGATATCATGTCCACTCCAGACCCACCCCTGGGCGGAACTCCT-3' 5'-GCTCTCTAGAGTCTTCTTCGCTGCCACTTCCTGAGCGGTCCTCCTC-3' In vitro kinase assay In vitro kinase assay was modified from the procedures previously described (3). After wash with PBS, the cells were lysed in the lysis buffer containing 20 mm HEPES, 0.15 M NaCl, 1.5 mm MgCl 2, 1 mm EGTA ph 7.4, 1% triton X-100, 1 mm Na 3 VO 4, 1 mm NaF, 20 mm β- glycerophosphate and protease inhibitors (0.5 mm PMSF, 5 µg/ml pepstatin, 5 µg/ml leupeptin and 5 µg/ml aprotinin). After clarified by centrifugation, cell lysates were incubated with anti-p- ATM-Ser1981 antibody and protein A-sepharose beads for 4 h at 4 C. The beads were spun down by centrifugation, and resulting pellet (ATM immune complex) was washed twice with the lysis buffer, and wash once with high-salt buffer (0.1 M Tris-HCl, ph 7.4 and 0.6 M NaCl), and

3 once with kinase buffer (10 mm HEPES ph 7.5, 50 mm β-glycerophosphate, 50 mm NaCl, 10 mm MgCl 2, 10 mm MnCl 2, 1 mm DTT). ATM immune complex was resuspended in 30 µl of kinase buffer supplemented with 5 µm ATP, 10 µci - 32 P-ATP and 1 µg of substrate proteins. Kinase reaction was carried out for 30 min at 30 C and stopped by addition of SDS sample buffer. Where indicated, ATM immune complex was treated with 20 µm of wortmannin (Fluka Chemie AG, Switzerland) in the kinase buffer for 30 min at room temperature before being subjected to kinase assays. Protein samples of kinase reactions were separated by SDS-PAGE. Gels were either exposed to X-ray film followed by autoradiography or subjected to IB analysis. 2-D gel analysis The cells were suspended in the lysis buffer containing 8 M urea, 4% CHAPS, 2% 3-10 IPG (immobilized ph gradient) buffer, and 50 U/ l benzonase (Novagen, Denmark). Lysates were clarified by centrifugation at 12,000 g for 30 min, and the supernatants were subjected to 2-D gel analysis. Protein concentrations were determined using the 2-D Quant Kit (GE Healthcare) according to the manufacturer s protocol. Equivalent amounts of each protein sample were adjusted to a volume of 30 μl, mixed with 100 μl of rehydration buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 0.002% bromophenol blue, 20 mm dithiothreitol, and 0.8% (v/v) nonlinear IPG buffer (ph 3-10), and vortexed at room temperature for 30 min. The protein samples were subsequently subjected to isoelectric focusing using 7-cm Readystrip IPG strips (ph 3-10, non-linear; Bio-Rad) and IPGphor (GE Healthcare) under rehydration conditions for 10 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h and 5000 V for 7 h. The isoelectric-focused strips were equilibrated in buffer containing 50 mm Tris-HCl (ph 8.8), 6 M urea, 30% glycerol, 2% SDS,

4 1% DTT and a trace amount of bromophenol blue at room temperature for 15 min. The strips were loaded on a 1-mm-thick, 7% SDS gel and sealed using 1% low-melting agarose gel. ATPase assay A 20- l volume reaction containing the purified f-brg1, 12 mm HEPES ph 7.9, 60 mm KCl, 7 mm MgCl 2, 6% glycerol, 0.06 mg/ml BSA, 20 M ATP, 0.2 Ci -³²p-ATP and 20 nm plasmid DNA was incubated with at 30 C for 30 min. 2- l reaction was taken and mixed with 5- l stop solution (50 mm Tris-HCl ph 7.5, 100 mm EDTA, 3% SDS), aliquots of which were spotted on polyethyleneimine cellulose TLC plates (Sigma), which had been pre-run in distilled water and dried. Inorganic phosphate was separated from uncleaved ATP by running TLC plates in 1 M formic acid and 0.5 M LiCl. The ratio of inorganic phosphate to ATP was quantified using Bioimaging analyzer (Typhoon FLA 7000 IP, GE Healthcare Life Sciences). Biochemical fractionation Biochemical fractionation was performed as previously described with minor modifications (1). Briefly, approximately cells were suspended in 50 µl of fractionation buffer (50 mm HEPES ph 7.5, 150 mm NaCl, 1 mm EDTA, 0.2% NP-40, and protease inhibitors (5 µg/ml each of pepstatin, leupeptin and aprotinin), and 10 mm NaF)) for 5 min on ice and spun down by centrifugation at 16000g for 20 min to separate supernatant containing the cytoplasmic proteins from pellet containing the nuclear proteins. Luciferase reporter assay

5 SW13 cells were transfected with indicated vectors using PEI (polyethylenimine, Sigma). Cell lysates were prepared using standard method and analyzed for luciferase activity according to the manufacturer s protocol (Promega, WI). pgl4-hrluc plasmid was cotransfected and Renilla activity was measured to normalize the transfection efficiency.

6 SUPPLEMENTARY FIGURE LEGENDS Figure S1. (a) The extracts prepared from HeLa cells untreated or 1 h after 20 Gy were subjected to 2-D gel analysis as previously described (4). The separated proteins were analyzed by IB using anti-brg1 antibody. (b) The lysates prepared from HeLa cells untreated or 1 h after 20 Gy were subjected to immunoprecipitation using IgG or the anti-p-sq/tq antibody followed by immunoblot analysis for BRG1. Figure S2. (a) A coomassie stain gel of the purified GST-BRG1 fusion proteins. (b) The lysates were prepared from HeLa cells 1 h after 20 Gy and subjected to immunoprecipitation using antiphospho-atm-ser-1981 antibody. Aliquots were analyzed by immunoblot using anti-atm antibody. (c) (d) Dot blot analysis of the phospho-brg1 antibodies. Three-fold increasing amounts of the phospho- and non-phospho-peptides were spotted onto the nitrocellulose membranes, and probed with the antibodies raised against indicated phospho-peptides. (e) HeLa and SW13 cells were harvested before or 1 h after, and the cell lysates were prepared and subjected to IB. Figure S3. (a) HeLa cells were treated with vehicle (DMSO), 40 M of NU7026 (DNA-PKspecific inhibitor), 10 M of KU55933 (ATM-specific inhibitor), or 20 M of wortmannin ( nonselective PI-3 kinase inhibitor), and cells were then left untreated or irradiated. After 30 min, cells were harvested and subjected to IB analysis. (b) SW13 cells were fixed before or 30 min after for dual staining with the indicated antibodies. (c) HeLa cells prepared as per in Figure 2a were dually stained as indicated before confocal images were captured. While BRG1

7 was stained evenly across the nucleus with the clear exclusion of nucleoli, the BRG1 staining pattern dramatically changed upon ATM depletion such as to exhibit irregularly shaped large granule-like structures at the nuclear periphery with the clear nucleolar boundary disappeared. This pattern of BRG1 staining in the ATM-deficient cells was same before and after irradiation. Although only speculative at the present time, these results suggest that ATM might control the nuclear distribution of BRG1 for efficient phosphorylation upon DNA damage. (d) Representative confocal images from the experiments in Figure 2f. (e) Representative confocal images from the experiments in Figure 2g. Figure S4. 293T cells were co-transfected as per in Figure 5a and collected before or 30 min after. The whole lysates and histone extracts were prepared for IB analysis. Figure S5. (a) 293T cells were co-transfected with BRG1-specific sirna and the indicated vectors. Ha-tagged proteins were immunoprecipitated and the various BRG1-associated proteins (BAFs) were analyzed by immunoblotting using specific antibodies. (b) 293T cells transfected as in (a) were subjected to detergent fractionation. The soluble cytoplasmic and insoluble nuclear fractions were subjected to IB. GAPDH and H2A were used as indicators of the cytoplasmic and nuclear fractions, respectively.

8 SUPPLEMENTARY REFERENCE 1. Park JH, Park EJ, Lee HS, Kim SJ, Hur SK, Imbalzano AN, et al. Mammalian SWI/SNF complexes facilitate DNA double-strand break repair by promoting gamma-h2ax induction. EMBO J Sep 6;25(17): Lee HS, Park JH, Kim SJ, Kwon SJ, Kwon J. A cooperative activation loop among SWI/SNF, gamma-h2ax and H3 acetylation for DNA double-strand break repair. EMBO J Apr 21;29(8): Park EJ, Chan DW, Park JH, Oettinger MA, Kwon J. DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. Nucleic Acids Res Dec 1;31(23): Lee SY, Park JH, Kim S, Park EJ, Yun Y, Kwon J. A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks. Biochem J May 15;388(Pt 1):7-15.

9 2 nd : SDS Input IgG p-sq/tq Supplementary Figure S1 a 1 st : IEF b IP - IR + IR IR BRG IB: BRG1

10 Marker WT S610A S721A Supplementary Figure S2 a GST-BRG1 ( ) b Input (5%) IP: p-atm Wortmannin + - IB: ATM (kda) GST-BRG1 ( ) c Antibody against p-brg1-s Peptides 1x 3x 9x 26 - Coomassie stain CLDETSQMSDL CLDETpSQMSDL d Peptides CEYGVSQALAR Antibody against p-brg1-s721 1x 3x 9x e HeLa SW13 IR IB: p-brg1-s610 IB: BRG1 CEYGVpSQALAR IB: a-tubulin

11 Supplementary Figure S3 d + IR Wortmannin - IR Wortmannin a p-brg1 g-h2ax Merge DAPI e 30 min - IR 1h 10 Gy + IR 2h KU Gy NU min Untreated 1 Gy KU min NU Gy Untreated 0 Gy p-brg1 BRG1 b SW13 p-brg1 g-h2ax Merge DAPI 4h c - IR p-brg1 si-control si-atm BRG1 + IR Merge DAPI p-brg1 BRG1 Merge DAPI p-brg1 g-h2ax Merge DAPI

12 Supplementary Figure S4 si-control si-brg Ha-BRG1 R Ha-BRG1 R -S721A IR p-brg1 Ha BRG1 WCL a-tubulin H2AX Histone extract

13 Vector WT S721A S721D Vector WT S721A S721D WT S721A S721D WT S721A S721D Supplementary Figure S5 a Input IP: Ha b Cytoplasm Nucleus Ha-BRG1 R (+si-brg1) Ha-BRG1 R (+si-brg1) Ha BAF170 BAF155 Ha GAPDH H2A BAF60a BAF57 BAF53