J Biol Chem. 2017 May 19;292(20):8459-8471 

The chromatin-remodeling subunit Baf200 promotes homology-directed DNA repair and regulates distinct chromatin-remodeling complexes

Roberto J. Pezza


The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.To characterize the role of Baf200 in DNA repair, we analyzed the sensitivity of Baf200-depleted cells to the DNA-damaging agent etoposide (Fig. 1, A–C) and ionizing radiation (Fig. 1D) inducing DNA DSBs. U2OS cells treated with any of three different RNAi designed against Baf200 (Fig. 1, C and D) displayed increased sensitivity compared with control cells. This sensitivity is comparable with that observed upon depletion of Brg1 (Fig. 1A) and is apparently not caused by changes in topoisomerase II or Rad51 expression (Fig. 1B). Although this is consistent with a model in which PBAF complexes containing Brg1 play a central role in DNA repair, the mechanism of function of the PBAF-specific regulatory subunits such as Baf200 remains unclear.γH2AX accumulates rapidly after formation of a DSB, followed by reduction of the signal after DNA repair. To determine the effect of Baf200 and other PBAF components' depletion on DNA DSB repair, we tested whether RNAi-mediated depletion of Baf200, Baf180, and Brg1 in U2OS cells affected γH2AX foci number after DNA damage. The kinetics of γH2AX immunosignal was used to monitor foci formation and disappearance after inducing DNA damage using etoposide (Fig. 2, A and B) or ionizing radiation (Fig. 2C). A recent report has shown that siRNA depletion of Baf180 and Brg1 show higher γH2AX signals compared with control cells (21). Our experiments show that Baf200, Baf180, and Brg1 knockdown did not significantly induce γH2AX foci in nondamaged cells, as expected. Importantly, following etoposide or ionizing radiation exposure, cells transfected with siRNA control and cells depleted of PBAF components exhibited strong γH2AX, although Baf200-depleted cells (any of three different siRNAs targeting Baf200) showed reproducibly higher signals (Fig. 2, A–C). The number of γH2AX foci decreased in control cells at later time points (180 and 360 min), reflecting DNA repair. In contrast, there was a delay in the decrease of γH2AX foci number in cells depleted of Baf200, Baf180, and Brg1. Fig. 2, B and C, show the mean ± S.D. from three independent experiments initiated from a different set of cultured and treated cells. Statistical differences were examined using paired two-tailed Student's t test. For cells exposed to etoposide, comparison of control siRNA with all siRNA treatments for each time point, except siRNA Brg1 (360 min, p = 0.057) and siRNA Baf180 (10 min, p = 0.0002), resulted in p < 0.0001 (n = 150 cells; 95% confidence interval). For cells exposed to ionizing radiation, comparison of control siRNA with Baf200 siRNA treatments for each time point resulted in p < 0.0001 (n = 150 cells; 95% confidence interval). γH2AX kinetics analysis was performed with two additional siRNAs designed to target Baf200 (siRNA Baf200-2 and Baf200-3) (Fig. 2D). The results obtained were similar to those shown with siRNA Baf200-1 (Fig. 2B) in that siRNA Baf200 cell treatment results in increased γH2AX signal.We found that depletion of Baf200 or Brg1 did not alter the cell cycle distribution (Fig. 2E), indicating that these effects on DNA repair kinetics are not caused by changes in the cell cycle phase.In sum, we suggest a model in which Baf200 and Baf180 work together with Brg1 during the DNA damage response to stimulate DSB resolution.Previous work has shown that depletion of Baf200 results in reduction of Baf180 (26), a result that we observed as well (Fig. 3A), but had no effect on the levels of other subunits that are in common to both the PBAF and the BAF complexes (data not shown). It is possible that the DNA repair phenotype arising from Baf200 depletion is, at least in part, a consequence of reduced Baf180 levels. To investigate this possibility, we expressed Baf180-GFP (21) in Baf180- or Baf200-depleted U2OS cells (Fig. 3B). We found that expression of Baf180-GFP restored γH2AX foci numbers after etoposide treatment in Baf180-depleted cells, but not in Baf200-depleted cells. Thus, Baf200 is required to stimulate DNA repair (Fig. 3C).Although Baf200 is a recognized specific subunit of the PBAF chromatin-remodeling complex and has been implicated in gene regulation, its role in DNA repair is poorly understood. Our results suggest that Baf200 regulates the DNA damage response by promoting recombinational DNA repair dependent on Rad51 (Fig. 9).Antibodies against Baf200 were purchased from Abcam (ab510190), Bethyl (A302-230A), and Sigma (SAB2702507). We also generated polyclonal antibodies against human Baf200 using peptides directed against the N-terminal (RERRPSQPHTQSGGT) and C-terminal (PREEGKSKNNRPLRTSQC) sequences of the protein. The Baf180 (A301-591A) antibody was from Bethyl. Antibodies against α-tubulin (66031-1) and the Brg1 (21634-1) were from Proteintech Group. Baf155 (D7F8S), Baf170 (D8O9V), Baf250 (D2A8U), and Snf5 (D9C2) antibodies were from Cell Signaling. Anti-Rad51 (H-92) and anti-H2B (FL-126) were from Santa Cruz Biotechnology. Anti-RPA (Ab-3) was from Oncogene. The antibody against phospho-histone H2A.X (Ser-139) clone JBW301 was from Millipore (05-636) or from Cell Signaling (2577S). Lamin B1 antibody was from Abcam (ab16048). The HA antibody was from BioLegend (16B12). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories: HRP-conjugated goat anti-rabbit IgG Fc fragment specific (111-035-008), HRP-conjugated mouse anti-rabbit IgG light chain specific (211-032-171), HRP-conjugated goat anti-mouse IgG Fcγ fragment specific (115-035-071), ChromPure rabbit IgG whole molecule (011-000-003), rhodamine-conjugated goat anti-mouse IgG, F(ab′)2 (115-026-072), and FITC-conjugated goat anti-rabbit IgG, F(ab′)2 (115-096-047).We thank Dean Dawson and Linda Thompson for invaluable encouragement, advice, and discussion.
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