Fig 1: BRD9 separately interacts with RAD51 and RAD54 through its C-terminal and bromodomain.a, b BRD9 interacts with RAD54 and RAD51. a 293T cells were transfected with HA-tagged BRD9 plasmid. Forty-eight hours after transfection, cells were exposed to 10-Gy IR and collected at the indicated time points. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. b OVCAR8 cells were exposed to 10-Gy IR. Lysates were collected after 8 h, and co-immunoprecipitation with anti-BRD9 antibody was performed. Blots were probed with the indicated antibodies. c–g BRD9 interacts with RAD51 through the C terminus, and its bromodomain mediates its interaction with RAD54. c Schematic diagram depicting a set of HA-tagged BRD9 expression constructs. d 293T cells were transfected with the indicated constructs of HA-BRD9 and exposed to 10-Gy IR. Lysates were collected after 8 h. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. e 293T cells were transfected with the indicated constructs. Forty-eight hours later, cells were treated with 10-Gy IR 8 h before immunoprecipitation with anti-HA beads. Blots were probed with indicated antibodies. f, g 293T cells were transfected with the indicated constructs. Twenty-four hours later, cells were exposed to BRD9i, 10-Gy IR, or both. Lysates were collected after 8 h. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. h, k Bromodomain of BRD9 is essential for its HR activity. h BRD9-knockdown 293T cells were infected with the indicated lentiviral plasmids. Twenty-four hours after transfection, cells were exposed to IR. Lysates were collected after 8 h, and immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. i BRD9-knockdown 293T cells expressing the DR-GFP reporter were infected with lentivirus expressing the indicated proteins. Twenty-four hours later, cells were subjected to flow cytometry. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. *p < 0.05, ***p < 0.001 by two-sided unpaired t test. j, k BRD9-knockdown OVCAR8 cells were infected with lentivirus expressing the indicated proteins, and subjected to colony-formation assay to assess the sensitivity to PARPi (j) and cisplatin (k). Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. *p < 0.05, **p < 0.01, ***p < 0.001 by two-sided unpaired t test.
Fig 2: RAD54 acetylation is important for BRD9 recognition and HR activity.a–c RAD54 is acetylated by GCN5/PCAF and deacetylated by HDAC 6/HDAC11 following induction of DNA damage. a 293T cells were transfected with control or RAD54-HA plasmid. Twenty-four hours after transfection, cells were exposed to the 10-Gy IR and harvested at the indicated time points. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. b 293T cells were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were exposed to 10-Gy IR, and lysates were collected after 8 h. Immunoprecipitation with anti-HA beads was performed. Blots were probed with the indicated antibodies. c 293T cells were transfected with the indicated plasmids, treated as indicated, and subjected to immunoprecipitation as outlined in b. Blots were probed with the indicated antibodies. d, e RAD54 K515 acetylation is important for RAD51–RAD54 complex formation. d 293T cells were transfected with the indicated plasmids, treated as indicated, and subjected to immunoprecipitation as outlined in b. Blots were probed with the indicated antibodies. e 293T cells were transfected with the indicated plasmids, exposed to either no IR or 10-Gy IR as indicated, and subjected to immunoprecipitation as outlined in b. Blots were probed with the indicated antibodies. f–h RAD54 K515 acetylation is essential for HR activity. f, g U2OS cells were transfected with the indicated plasmids and exposed to 2-Gy IR. Cells were fixed after 8 h and stained for the indicated proteins. Representative immunofluorescence images of RAD51 (green) and RAD54 (red) are shown in f. Quantification of the indicated foci is shown in g. Representative data (mean ± SEM) are shown from n = 50 cells examined over three independent experiments. **p < 0.01,***p < 0.001 by two-sided unpaired t test. NS not significant. Scale bar, 10 μm. h Survival curves of U2OS cells expressing the indicated constructs and exposed to the indicated doses of PARPi or cisplatin. Representative data (mean ± SEM) are shown from n = 3 biologically independent samples. *p < 0.05, ***p < 0.001 by two-sided unpaired t test.
Fig 3: BRD9 is required for interaction and co-localization of RAD51 and RAD54.a–d Representative immunofluorescence images of OVCAR8 cells demonstrating that BRD9 knockdown reduces RAD54 foci formation and RAD51/RAD54 co-localization. a–c OVCAR8 cells were infected with lentivirus-expressing control (Ctrl) or BRD9 shRNA and exposed to 2-Gy IR. Eight hours later, cells were stained with RAD51 (red) and RAD54 (green) antibodies. Representative immunofluorescence images are shown in a. RAD51 foci/cell (b), RAD54 foci/cell (c), and RAD51/RAD54 co-localized foci (d) after the indicated treatment were quantified. Representative data (mean ± SEM) are shown from three independent experiments. n = 50 cells examined over three independent experiments. ***p < 0.001 by two-sided unpaired t test. Scale bar, 10 μm. e, f DNA damage-induced RAD51–RAD54 interaction is dependent on BRD9. Endogenous BRD9 was knocked out in HEK293T cells using shRNA, and the indicated constructs were expressed. Cells were exposed to 10-Gy irradiation. Lysates were collected after 8 h, and immunoprecipitation with the indicated antibodies was performed. Blots were probed with the indicated antibodies. g RAD51 is necessary for BRD9 foci formation. OVCAR8 cells were infected with lentivirus-expressing RAD51 shRNA, exposed to 2-Gy IR, and fixed after 8 h. Cells were stained with BRD9 (green) and RAD51 (red) antibodies. Representative images of the indicated foci are shown. BRD9 foci/cell were quantified. Representative data (mean ± SEM) are shown from three independent experiments. n = 50 cells examined over three independent experiments. ***p < 0.001 by two-sided unpaired t test. Scale bar, 10 μm.
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