Volume 40, Issue 6, Pages 976-987 (December 2010) The ACF1 Complex Is Required for DNA Double-Strand Break Repair in Human Cells  Li Lan, Ayako Ui, Satoshi Nakajima, Kotomi Hatakeyama, Mikiko Hoshi, Reiko Watanabe, Susan M. Janicki, Hideaki Ogiwara, Takashi Kohno, Shin-ichiro Kanno, Akira Yasui  Molecular Cell  Volume 40, Issue 6, Pages 976-987 (December 2010) DOI: 10.1016/j.molcel.2010.12.003 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Accumulation of ACF1 and SNF2H at Laser-Irradiated Site in U2OS Cells (A) GFP-tagged ACF1 and SNF2H at laser-irradiated sites after irradiation. (B) Accumulation of endogenous ACF1 or SNF2H colocalizing with PCNA or γH2AX after laser microirradiation, respectively. (C) Cell cycle-independent accumulation of GFP-ACF1 and GFP-SNF2H at laser-irradiated site. (D and E) Schematic presentation of domains for ACF1 (D) and for SNF2H (E) and accumulation of GFP-tagged deletions (+, accumulation; and −, no accumulation) at laser-irradiated site. (F) Schematic presentation of the C-terminal domain of SNF2H and the response of GFP-tagged deletions to laser irradiation. Cellular localization of each portion tagged with GFP is indicated under Expression. Nuc and cyt represent nuclear and cytosol localization, respectively. For laser-irradiation experiments, five to ten cells were irradiated in the presence of BrdU in every experiment, and all the cells tested showed almost equivalent results. All the pictures were taken 5 min after irradiation. Representative responses of the cells are shown. See also Figure S1. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Establishment of U2OS Cell Line Harboring Multiple I-SceI Sites at a Genomic Locus and Visualization of Proteins Accumulated at the Sites after Expression of I-SceI (A) A 9.9 kb-long plasmid pTRE/I-SceI, harboring an I-SceI site in front of tetracycline-responsible elements (TRE), was constructed. (B) Establishment of human cell harboring TRE/I-SceI array; pTRE/I-SceI was introduced with pcDNA3.1(+)Zeo into U2OS cells and U2OS/TRE/I-SceI-19 cells harboring high copy number of the plasmid were isolated after selection for Zeocin resistance. (C) Southern blots of control cells with 200 copies of the probe site (Janicki et al., 2004) and two established cell lines, U2OS/TRE/I-SceI-11 and -19. (D) Experimental procedure; U2OS/TRE/I-SceI-19 cells were electroporated to introduce pCherry-tTA-ER, plasmid(s) for GFP-tagged protein(s) to be tested for its response to DSBs and the plasmid pCMV-NLS-I-SceI for expression of I-SceI. Focus was determined normally 6 hr after the electroporation. Tamoxifen was added immediately after electroporation. (E) Focus of γH2AX colocalized with that of Cherry-tTA-ER was found in cells electroporated with the plasmid pCMV-NLS-I-SceI (+I-SceI). (F and G) Focus of GFP-KU80 and GFP-KU70 (upper panels, F and G, respectively) colocalized with that of Cherry-tTA-ER, depends on electroporation with pCMV-NLS-I-SceI. (H and I) Focus of GFP-ACF1 and GFP-SNF2H (upper panels, H and I, respectively) colocalized with that of Cherry-tTA-ER. (J) Percentage of cells harboring GFP focus colocalized with Cherry-tTA-ER focus. Plasmids for GFP-KU70 and GFP-KU80, GFP-ACF1, or GFP-SNF2H were electroporated with pCMV-NLS-I-SceI. The rate of nonspecific accumulation of GFP proteins at Cherry-tTA-ER focus in the absence of I-SceI expression was subtracted. See also Figure S2. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 ACF1 and SNF2H Are Required for the Repair of DSBs (A) Effect of siACF1 and siSNF2H on the expression of ACF1 and SNF2H. There is no cross-action of gene suppression. (B) Effects of the siRNA on the colony-forming ability of U2OS cells treated or irradiated with methyl methane sulfonate (MMS), bleomycin, X-rays, camptothecin (CPT), or UVC. (C) Influence of siACF1 and siSNF2H on single-cell neutral gel electrophoresis (comet) assay of U2OS cells treated with bleomycin. Representative comet figures for without treatment, immediately (0 hr), and 4 hr after treatment are shown in the panels. The tail moments of 100 cells for 0, 2, and 4 hr after treatment or without treatment were measured, and mean values with standard deviations are shown in the graph below the panels. (D) Influence of siKU80, siSNF2H, or siACF1 on the retention of X-ray-induced γH2AX foci in U2OS cells 4 and 48 hr after X-ray irradiation. Smaller figures on the right show γH2AX foci in a single cell 48 hr after irradiation. Right graphs show percent of cells with foci (upper graph) in 100 cells and numbers of foci per cell in 50 cells (lower graph) before irradiation and 0.5, 4, 24, and 48 hr after irradiation. (E) Influence of suppression of ACF1 or/and KU80 on survival after bleomycin treatment or X-ray irradiation of U2OS cell. siACF1 or siKU80 did not suppress the expression of KU80 or ACF1, respectively. Data for survival curves (B and E), comet tail moments (C), and numbers of γH2AX foci (D) were obtained from three independent experiments, and standard deviations are shown in figures and graphs. See also Figure S3. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 NHEJ and HR Frequencies in Cells Depleted of ACF1 or SNF2H (A) Assay for NHEJ of chromosomal DSBs in H1299 cells (Ogiwara et al., in press). Two I-SceI sites in the reverse direction are shown with arrowheads. Transiently expressed I-SceI protein cleaves the I-SceI sites and produces DSBs with incompatible ends in the substrate. NHEJ of two broken DNA ends of chromosomal DNA results in deletion of the HSV-TK open reading frame and in EGFP expression. CMV, cytomegalovirus promoter/enhancer; TK, herpes simplex virus-thymidine kinase; pA, polyA signal. (B) GFP-positive cell fraction in cells depleted of ACF1 or SNF2H gene expression was determined and compared with those in cells treated with siCtrl or siKU80 for determination of NHEJ frequency. Results of western blot analysis after siRNA treatment are shown on the right. (C) Assay for HR frequency of chromosomal DNA containing a recombination substrate DR-GFP in HeLa cells (Pierce et al., 1999). Briefly, a gene conversion event within two differentially mutated GFP genes, a GFP gene harboring a single I-SceI site (arrowhead) with in-frame stop codons, SceGFP, and a downstream internal GFP fragment (iGFP) (shown on the top), results in the expression of intact GFP protein (shown on the bottom left). (D) GFP-positive cell fraction in cells depleted of ACF1 or SNF2H gene expression was determined. Results of western blot analysis after siRNA treatment are shown on the right. See also Figure S4. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Requirement of ACF1 and SNF2H for the Accumulation of KU at DSBs and DSB Repair (A) Accumulation of GFP-ACF1 or GFP-SNF2H at laser-irradiated (with BrdU) sites in wild-type and Ku80-defective CHO cells. (B) Influence of the suppression of ACF1 or SNF2H on the accumulation of coexpressed GFP-KU70 and GFP-KU80 at laser-irradiated (with 8-MOP) site in U2OS cells (representative data and mean intensity of coexpressed KU proteins in graph). (C) Suppression effect of siACF1 or siSNF2H on the accumulation of coexpressed GFP-KU70 and GFP-KU80 at multiple I-SceI sites in U2OS/TRE/I-SceI-19 cells. Twenty cells with Cherry-tTA-ER focus were examined in each experiment, and three independent electroporation experiments were done. (D) Kinetics of GFP-ACF1 (blue line) and GFP-SNF2H (black line) accumulated at laser-irradiated (with BrdU) sites compared with that of GFP-KU80 (red line) with error bars obtained from three independent experiments. (E) Effects of silencing with siUTR-SNF2H on the expression of SNF2H (western blots). (F) Influence of GFP-tagged wild-type or ATPase mutant SNF2H expression on single-cell neutral gel electrophoresis (comet) in cells depleted of endogenous SNF2H expression. Comets were determined 4 hr after treatment of U2OS cells with bleomycin. Controls are U2OS cells without siRNA treatment (WT) and cells suppressed with siUTR-SNF2H without additional SNF2H expression (−). Tail moments of 100 cells were measured and mean values of three experiments were given with standard deviations. Detailed comet data of one of the three experiments are presented in Figure S5C. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Interaction of KU Protein with Chromatin-Remodeling Factors of ISWI Family (A) Immunoprecipitation of FLAG-KU80 from cells treated with or without bleomycin. 293 cells stably expressing FLAG-KU80 were treated with or without bleomycin, and nuclear extracts were incubated with 20 μg/ml DNase I and 20 μg/ml RNase A 4 hr at 8°C before electrophoresis. Western blots of the precipitates obtained by anti-FLAG are shown. See also Figure S6. (B) Direct interaction of recombinant His-ACF1 with GST-KU70; His-ACF1 was identified by protein staining in the pull-down fraction of GST-KU70, but not in those of GST-tag alone or GST-KU80. Applied GST and fused proteins are shown on the bottom and His-ACF1 (1/20 of the amount used in the reaction) on the upper lane (left). (C) A model for interaction of KU complex with chromatin-remodeling factors in response to DSBs and three possible complexes accumulating at DSBs. (D) GFP-positive cell fractions in the cells depleted of CHRAC15 or CHRAC17 expression in the NHEJ assay shown in Figure 4A were significantly less than that in the cells treated with siCtrl. Western blots are shown on the right. (E) GFP-positive cell fraction in the cells depleted of CHRAC15 or CHRAC17 expression in the HR assay shown in Figure 4C. Western blots are shown on the right. Molecular Cell 2010 40, 976-987DOI: (10.1016/j.molcel.2010.12.003) Copyright © 2010 Elsevier Inc. Terms and Conditions