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Cited by 104PARP inhibition in cancer therapyDNA Repair MechanismsUbiquitin and proteasome pathways

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The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors

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Peng, Yihan, Liao, Qingchao, Tan, Wei, Peng, Changmin, Hu, Zhaohua, Chen, Yali, Li, Zhuqing, Li, Jing, Zhen, Bei, Zhu, Wenge, Li, Xiangpan, Yao, Yi, Song, Qibin, Liu, Chengsheng, Qi, Xiangdong, He, Fuchu, Pei, Huadong·2019·Nature Communications
DOI 10.1038/s41467-019-09232-8PMC PMC6420636
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Abstract

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.

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Fig. 1 USP15 regulates homologous recombination (HR) and breast cancer response to poly-(ADP-ribose) polymerase (PARP) inhibitor. a USP15 depletion inhibits DNA damage repair. Representative γ-H2AX foci at indicated times after irradiation (2Gy) are presented (left panel). Quantification (right panel) are average of three independent experiments (100 cells each experiment), presented as mean±SD, two-tailed Student’s t test, *P < 0.05. Scale bar: 40 μm. b HR efficiency in USP15-depleted cells were determined using the direct repeat green fluorescent protein (DR-GFP) reporter assay, as described in the Methods section. c Non-homologous end-joining (NHEJ) efficiency in USP15-depleted cells were determined using the EJ5-GFP reporter assay, as described in the Methods section. CtIP or 53BP1 small interfering RNAs (siRNAs) were used as a positive control, respectively, in b, c. Data are shown as average of three independent experiments, presented as mean ± SD, two-tailed Student’s t test, *P < 0.01. d USP15 depletion sensitizes cancer cells to PARP inhibitor. The indicated MCF7 cells’ response to PARP inhibitor (AZD2281) was measured by MTS assay. Data are presented as mean± EM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. e–h Wild-type (Con) or USP15-knockout U2OS cells were subjected to micro-irradiation as described in the Method section; 3 h later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (e), BARD1 (f), RPA (g), and RAD51 (h) accumulation at sites of laser-induced DNA damage are shown. Scale bar: 10 μm. i Representative images of BARD1/BRCA1 foci at indicated times after irradiation (4Gy) are shown (left panel). Quantification (right panel) are average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean±SD, two-tailed Student's t test, *P < 0.01. Scale bar: 10 μm. Unprocessed scans of blots are provided in Supplementary Fig. 8
Fig. 1 USP15 regulates homologous recombination (HR) and breast cancer response to poly-(ADP-ribose) polymerase (PARP) inhibitor. a USP15 depletion inhibits DNA damage repair. Representative γ-H2AX foci at indicated times after irradiation (2Gy) are presented (left panel). Quantification (right panel) are average of three independent experiments (100 cells each experiment), presented as mean±SD, two-tailed Student’s t test, *P < 0.05. Scale bar: 40 μm. b HR efficiency in USP15-depleted cells were determined using the direct repeat green fluorescent protein (DR-GFP) reporter assay, as described in the Methods section. c Non-homologous end-joining (NHEJ) efficiency in USP15-depleted cells were determined using the EJ5-GFP reporter assay, as described in the Methods section. CtIP or 53BP1 small interfering RNAs (siRNAs) were used as a positive control, respectively, in b, c. Data are shown as average of three independent experiments, presented as mean ± SD, two-tailed Student’s t test, *P < 0.01. d USP15 depletion sensitizes cancer cells to PARP inhibitor. The indicated MCF7 cells’ response to PARP inhibitor (AZD2281) was measured by MTS assay. Data are presented as mean± EM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. e–h Wild-type (Con) or USP15-knockout U2OS cells were subjected to micro-irradiation as described in the Method section; 3 h later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (e), BARD1 (f), RPA (g), and RAD51 (h) accumulation at sites of laser-induced DNA damage are shown. Scale bar: 10 μm. i Representative images of BARD1/BRCA1 foci at indicated times after irradiation (4Gy) are shown (left panel). Quantification (right panel) are average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean±SD, two-tailed Student's t test, *P < 0.01. Scale bar: 10 μm. Unprocessed scans of blots are provided in Supplementary Fig. 8CC BY · PubMed Central
Fig. 2 USP15 interacts with BARD1 BRCT domain through its C-terminal region. a Reciprocal endogenous immunoprecipitation (IP) between USP15 and BARD1 were performed in HEK293T cells. (Left panel) IP with anti-BARD1 antibody and blot with anti-BARD1 or USP15 antibody, respectively. (Right panel) IP with anti-USP15 antibody and blot with anti-USP15 or BARD1 antibody, respectively. b Co-immunoprecipitation (co-IP) assays were performed to check the interaction between USP15 and BARD1 upon DNA damage. HEK293T cells treated as indicated were lysed and immunoprecipitated with anti-BARD1 antibody, and Western blot was performed with indicated antibodies. c Schematic representation of USP15-truncated mutants used in this study (upper panel). Plasmids encoding HA-tagged full-length or deletion mutants of USP15 were co-transfected with plasmids encoding FLAG-tagged full-length BARD1 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. d Schematic representation of BARD1-truncated mutants used in this study (upper panel). Plasmids encoding FLAG-tagged full-length or deletion mutants of BARD1 were co-transfected with plasmids encoding HA-tagged full-length USP15 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. e USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or D3 mutant and homologous recombination (HR) efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, *P < 0.05, **P < 0.01. f The indicated cells“ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were measured by colony formation assay. Data are presented as mean ± SEM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. Unprocessed scans of blots are provided in Supplementary Fig. 8
Fig. 2 USP15 interacts with BARD1 BRCT domain through its C-terminal region. a Reciprocal endogenous immunoprecipitation (IP) between USP15 and BARD1 were performed in HEK293T cells. (Left panel) IP with anti-BARD1 antibody and blot with anti-BARD1 or USP15 antibody, respectively. (Right panel) IP with anti-USP15 antibody and blot with anti-USP15 or BARD1 antibody, respectively. b Co-immunoprecipitation (co-IP) assays were performed to check the interaction between USP15 and BARD1 upon DNA damage. HEK293T cells treated as indicated were lysed and immunoprecipitated with anti-BARD1 antibody, and Western blot was performed with indicated antibodies. c Schematic representation of USP15-truncated mutants used in this study (upper panel). Plasmids encoding HA-tagged full-length or deletion mutants of USP15 were co-transfected with plasmids encoding FLAG-tagged full-length BARD1 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. d Schematic representation of BARD1-truncated mutants used in this study (upper panel). Plasmids encoding FLAG-tagged full-length or deletion mutants of BARD1 were co-transfected with plasmids encoding HA-tagged full-length USP15 into 293T cells. Immunoprecipitation and immunoblotting were performed 48 h post transfection as indicated. e USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or D3 mutant and homologous recombination (HR) efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, *P < 0.05, **P < 0.01. f The indicated cells“ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were measured by colony formation assay. Data are presented as mean ± SEM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. Unprocessed scans of blots are provided in Supplementary Fig. 8CC BY · PubMed Central
Fig. 3 USP15 deubiquitinates BARD1 and facilitates its interaction with HP1γ. a Wild-type (WT) or USP15-knockout 293T cells were transiently transfected with indicated plasmids and treated with or without mitomycin C (MMC) for 24 h. Cells were lysed, and ubiquitinated BARD1 proteins were pulled down and were detected by Western blot. b USP15 deubiquitinates BARD1 BRCT domain in vitro. BARD1-BRCT conjugated with indicated ubiquitin chains were purified from 293T cells and were used as substrates, glutathione S-transferase (GST)-USP15 WT or GST-USP15 C269A were purified from E. coli, and then the in vitro deubiquitination assay were performed, as described in the Methods section. Samples were separated by sodium dodecyl sulfate (SDS) gel and blotted with indicated antibodies. c USP15 deubiquitinates BARD1 BRCT domain upon DNA damage. WT or USP15-knockout 293T cells were transfected with the indicated plasmids and treated with or without MMC for 24 h. Cells were lysed and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. d USP15-knockout 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated as indicated for 24 h, cells were then lysed, and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. e USP15 facilitates BARD1’s interaction with HP1γ. The 293T cells were transfected with the indicated plasmids and treated with or without ionizing radiation (IR) (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with indicated antibodies. f USP15-depleted 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated with or without IR (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with the indicated antibodies. g USP15-mediated BARD1-BRCT deubiquitination promotes BARD1–HP1γ interaction in vitro. (Upper panel) Workflow of the in vitro binding assay. Briefly, ubiquitinated BARD1-BRCT were purified from 293T cells by tandem immunoprecipitation (IP) (first HA beads then FLAG beads) and were left on FLAG beads. Then one-half part was left untreated and one-half part was deubiquitinated by His-USP15 in vitro. Next, the immunoprecipitates were incubated with glutathione S-transferase (GST) or GST-HP1γ in vitro. (Lower panel) Samples were detected by Western blot with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Figs 8 and 9
Fig. 3 USP15 deubiquitinates BARD1 and facilitates its interaction with HP1γ. a Wild-type (WT) or USP15-knockout 293T cells were transiently transfected with indicated plasmids and treated with or without mitomycin C (MMC) for 24 h. Cells were lysed, and ubiquitinated BARD1 proteins were pulled down and were detected by Western blot. b USP15 deubiquitinates BARD1 BRCT domain in vitro. BARD1-BRCT conjugated with indicated ubiquitin chains were purified from 293T cells and were used as substrates, glutathione S-transferase (GST)-USP15 WT or GST-USP15 C269A were purified from E. coli, and then the in vitro deubiquitination assay were performed, as described in the Methods section. Samples were separated by sodium dodecyl sulfate (SDS) gel and blotted with indicated antibodies. c USP15 deubiquitinates BARD1 BRCT domain upon DNA damage. WT or USP15-knockout 293T cells were transfected with the indicated plasmids and treated with or without MMC for 24 h. Cells were lysed and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. d USP15-knockout 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated as indicated for 24 h, cells were then lysed, and ubiquitinated proteins were pull down by Ni-NTA. Samples were detected by Western blot with indicated antibodies. e USP15 facilitates BARD1’s interaction with HP1γ. The 293T cells were transfected with the indicated plasmids and treated with or without ionizing radiation (IR) (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with indicated antibodies. f USP15-depleted 293T cells reconstituted with HA-USP15 WT or C269A mutant were treated with or without IR (10Gy). Cell lysates were immunoprecipitated with FLAG M2 beads and subjected to immunoblot with the indicated antibodies. g USP15-mediated BARD1-BRCT deubiquitination promotes BARD1–HP1γ interaction in vitro. (Upper panel) Workflow of the in vitro binding assay. Briefly, ubiquitinated BARD1-BRCT were purified from 293T cells by tandem immunoprecipitation (IP) (first HA beads then FLAG beads) and were left on FLAG beads. Then one-half part was left untreated and one-half part was deubiquitinated by His-USP15 in vitro. Next, the immunoprecipitates were incubated with glutathione S-transferase (GST) or GST-HP1γ in vitro. (Lower panel) Samples were detected by Western blot with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Figs 8 and 9CC BY · PubMed Central
Fig. 4 USP15 is phosphorylated by ataxia telangiectasia-mutated (ATM) and recruited to DNA damage sites by MDC1. a The 293T cells were treated and immunoprecipitated as indicated, and then analyzed with anti-pSQ/TQ antibody. b Atm+/+ and Atm−/− mouse embryonic fibroblasts (MEFs) were treated with or without ionizing radiation (IR) (10 Gy). USP15 proteins were immunoprecipitated and blotted with anti-pSQ/TQ antibody. c The 293T cells transfected with the indicated plasmids were treated as indicated, and then immunoprecipitated and immunoblotted with indicated antibodies. d USP15 Ser678 site is conserved across species. e USP15-knockout U2OS cells were reconstituted with USP15 wild-type (WT) or S678A mutant. Then cells were treated with IR (4Gy), and 1 h later, stained with indicated antibodies. Scale bar: 5 μm. f Phosphorylated USP15 co-localizes with MDC1 upon DNA damage. Mdc1+/+ MEF cells were treated with IR (4Gy) and allowed to recover for 1 h. Then, cells were fixed and processed for immunostaining with the indicated antibodies. Scale bar: 5 μm. g MDC1 depletion abrogates USP15 foci formation upon DNA damage. Mdc1+/+ or Mdc1−/− MEF cells were treated with IR (4Gy) and allowed to recover for 1 h. Then, cells were fixed and processed for immunostaining with the indicated antibodies. Scale bar: 5 μm. h The 293T cells were transfected with indicated plasmids. The interaction between USP15 and MDC1 were investigated by immunoprecipitation and immunoblot with indicated antibodies. i Interactions between MDC1-FHA domain/MDC1-BRCT domain and USP15 were investigated by GST-pull down assay. j 293T cells were transfected with indicated plasmids and treated as indicated. The interaction between MDC1-FHA domain and USP15 was investigated by GST-pull down assay. k Mdc1−/− MEF cells transfected with indicated plasmids were irradiated, and 1 h later, stained with indicated antibodies. Scale bar: 5 μm. Unprocessed scans of blots are provided in Supplementary Fig. 9
Fig. 4 USP15 is phosphorylated by ataxia telangiectasia-mutated (ATM) and recruited to DNA damage sites by MDC1. a The 293T cells were treated and immunoprecipitated as indicated, and then analyzed with anti-pSQ/TQ antibody. b Atm+/+ and Atm−/− mouse embryonic fibroblasts (MEFs) were treated with or without ionizing radiation (IR) (10 Gy). USP15 proteins were immunoprecipitated and blotted with anti-pSQ/TQ antibody. c The 293T cells transfected with the indicated plasmids were treated as indicated, and then immunoprecipitated and immunoblotted with indicated antibodies. d USP15 Ser678 site is conserved across species. e USP15-knockout U2OS cells were reconstituted with USP15 wild-type (WT) or S678A mutant. Then cells were treated with IR (4Gy), and 1 h later, stained with indicated antibodies. Scale bar: 5 μm. f Phosphorylated USP15 co-localizes with MDC1 upon DNA damage. Mdc1+/+ MEF cells were treated with IR (4Gy) and allowed to recover for 1 h. Then, cells were fixed and processed for immunostaining with the indicated antibodies. Scale bar: 5 μm. g MDC1 depletion abrogates USP15 foci formation upon DNA damage. Mdc1+/+ or Mdc1−/− MEF cells were treated with IR (4Gy) and allowed to recover for 1 h. Then, cells were fixed and processed for immunostaining with the indicated antibodies. Scale bar: 5 μm. h The 293T cells were transfected with indicated plasmids. The interaction between USP15 and MDC1 were investigated by immunoprecipitation and immunoblot with indicated antibodies. i Interactions between MDC1-FHA domain/MDC1-BRCT domain and USP15 were investigated by GST-pull down assay. j 293T cells were transfected with indicated plasmids and treated as indicated. The interaction between MDC1-FHA domain and USP15 was investigated by GST-pull down assay. k Mdc1−/− MEF cells transfected with indicated plasmids were irradiated, and 1 h later, stained with indicated antibodies. Scale bar: 5 μm. Unprocessed scans of blots are provided in Supplementary Fig. 9CC BY · PubMed Central
Fig. 5 USP15 Ser678 phosphorylation is essential for its function in homologous recombination (HR). a USP15-depleted MCF7 cells were reconstituted with USP15 wild-type (WT) or S678A mutant. Cells’ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were analyzed by colony formation assay. Data are presented as mean±SEM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. b USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 WT or S678A mutant and HR efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, *P < 0.01. c–g USP15-knockout U2OS cells reconstituted with USP15 WT or S678A mutants were irradiated (4Gy), and 3 h later, BRCA1 foci (c), BARD1 foci (d), RPA foci (e), and RAD51 foci (f) are examined by indicated antibodies. Quantification (g) were average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01. Scale bar: 10 μm. Unprocessed scans of blots are provided in Supplementary Fig. 10
Fig. 5 USP15 Ser678 phosphorylation is essential for its function in homologous recombination (HR). a USP15-depleted MCF7 cells were reconstituted with USP15 wild-type (WT) or S678A mutant. Cells’ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were analyzed by colony formation assay. Data are presented as mean±SEM of three independent experiments, two-way analysis of variance (ANOVA), *P < 0.05. b USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 WT or S678A mutant and HR efficiency were determined. Data are presented as mean±SD of three independent experiments. Two-tailed Student's t test, *P < 0.01. c–g USP15-knockout U2OS cells reconstituted with USP15 WT or S678A mutants were irradiated (4Gy), and 3 h later, BRCA1 foci (c), BARD1 foci (d), RPA foci (e), and RAD51 foci (f) are examined by indicated antibodies. Quantification (g) were average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01. Scale bar: 10 μm. Unprocessed scans of blots are provided in Supplementary Fig. 10CC BY · PubMed Central
Fig. 6 USP15 maintains genome stability in vivo. a Usp15 protein is absent in Usp15−/− mouse embryonic fibroblasts (MEFs). Cell lysates from Usp15+/+ and Usp15−/− MEFs were separated by sodium dodecyl sulfate (SDS) gel and blotted with indicated antibodies. b Representative γ-H2AX foci of untreated primary Usp15+/+ or Usp15−/− MEF cells (passage 2) are shown (left panel). Scale bar: 20 μm. Quantification are mean±SD of three independent experiments, and 100 cells per experiment were counted. Two-tailed Student's t test, *P < 0.05. c Five-week-old Usp15+/+ (n = 16), Usp15+/− (n = 19), and Usp15−/− (n = 25) littermates were subject to 8.5Gy X-ray radiation, mice were monitored daily, and survival was assessed by Kaplan–Meier curve and log-rank test. d Fourteen days after mice were treated as in c, they were dissected and tissues were subject to immunochemistry (IHC) with γ-H2AX antibody. Representative γ-H2AX IHC staining of indicated tissues are shown. e MTS assay of Usp15−/− MEFs compared with Usp15+/+ MEFs in response to increasing doses of camptothecin (CPT), mitomycin C (MMC), and poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were performed. Data are presented as mean ± SEM of three independent experiments. f Metaphase spreads to analyze chromosome aberrations in untreated primary Usp15+/+ or Usp15−/− MEFs (Passage 3) were performed (left panel). Quantification of indicated aberrations are mean of three independent experiments, and 100 metaphases per experiment were counted. Two-tailed Student's t test, *P < 0.05, **P < 0.01. Unprocessed scans of blots are provided in Supplementary Fig. 10
Fig. 6 USP15 maintains genome stability in vivo. a Usp15 protein is absent in Usp15−/− mouse embryonic fibroblasts (MEFs). Cell lysates from Usp15+/+ and Usp15−/− MEFs were separated by sodium dodecyl sulfate (SDS) gel and blotted with indicated antibodies. b Representative γ-H2AX foci of untreated primary Usp15+/+ or Usp15−/− MEF cells (passage 2) are shown (left panel). Scale bar: 20 μm. Quantification are mean±SD of three independent experiments, and 100 cells per experiment were counted. Two-tailed Student's t test, *P < 0.05. c Five-week-old Usp15+/+ (n = 16), Usp15+/− (n = 19), and Usp15−/− (n = 25) littermates were subject to 8.5Gy X-ray radiation, mice were monitored daily, and survival was assessed by Kaplan–Meier curve and log-rank test. d Fourteen days after mice were treated as in c, they were dissected and tissues were subject to immunochemistry (IHC) with γ-H2AX antibody. Representative γ-H2AX IHC staining of indicated tissues are shown. e MTS assay of Usp15−/− MEFs compared with Usp15+/+ MEFs in response to increasing doses of camptothecin (CPT), mitomycin C (MMC), and poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were performed. Data are presented as mean ± SEM of three independent experiments. f Metaphase spreads to analyze chromosome aberrations in untreated primary Usp15+/+ or Usp15−/− MEFs (Passage 3) were performed (left panel). Quantification of indicated aberrations are mean of three independent experiments, and 100 metaphases per experiment were counted. Two-tailed Student's t test, *P < 0.05, **P < 0.01. Unprocessed scans of blots are provided in Supplementary Fig. 10CC BY · PubMed Central
Fig. 7 The cancer-associated USP15 mutation resulted in homologous recombination (HR) defect. a A schematic representation of breast cancer-associated USP15 mutations. b USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or deletion mutant (DM) and HR efficiency were determined. Data are presented as mean±SD of three independent experiments. Student's t test, *P < 0.05, **P < 0.01. c–e USP15-knockout U2OS cells stably reconstituted with USP15 WT or DM mutants were subjected to IR (4Gy). Representative immunostaining of BARD1 (c), RPA (d), and RAD51 (e) are shown. Scale bar: 10 μm. f USP15-depleted MCF7 cells were reconstituted with USP15 WT or DM mutant. Cells’ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were performed by colony formation assay. Data are presented as mean±SEM of three independent experiments. Two-way analysis of variance (ANOVA), **p < 0.01. g The 293T cells transfected with FLAG-USP15 WT or indicated mutants were lysed and immunoprecipitated by FLAG M2 beads. Samples were analyzed by Western blot with indicated antibodies. h USP15-knockout 293T cells were reconstituted with HA-USP15 WT or DM, and treated as indicated for 24 h. Cells were lysed and ubiquitinated BARD1 were pulled down by Ni-NTA under denatured condition and blotted with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Fig. 10. NS not significant
Fig. 7 The cancer-associated USP15 mutation resulted in homologous recombination (HR) defect. a A schematic representation of breast cancer-associated USP15 mutations. b USP15-knockout U2OS direct repeat green fluorescent protein (DR-GFP) cells were reconstituted with HA-USP15 wild-type (WT) or deletion mutant (DM) and HR efficiency were determined. Data are presented as mean±SD of three independent experiments. Student's t test, *P < 0.05, **P < 0.01. c–e USP15-knockout U2OS cells stably reconstituted with USP15 WT or DM mutants were subjected to IR (4Gy). Representative immunostaining of BARD1 (c), RPA (d), and RAD51 (e) are shown. Scale bar: 10 μm. f USP15-depleted MCF7 cells were reconstituted with USP15 WT or DM mutant. Cells’ response to poly-(ADP-ribose) polymerase (PARP) inhibitor (AZD2281) were performed by colony formation assay. Data are presented as mean±SEM of three independent experiments. Two-way analysis of variance (ANOVA), **p < 0.01. g The 293T cells transfected with FLAG-USP15 WT or indicated mutants were lysed and immunoprecipitated by FLAG M2 beads. Samples were analyzed by Western blot with indicated antibodies. h USP15-knockout 293T cells were reconstituted with HA-USP15 WT or DM, and treated as indicated for 24 h. Cells were lysed and ubiquitinated BARD1 were pulled down by Ni-NTA under denatured condition and blotted with indicated antibodies. Unprocessed scans of blots are provided in Supplementary Fig. 10. NS not significantCC BY · PubMed Central

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Authors
Peng, Y., Liao, Q., Tan, W., Peng, C., Hu, Z., Chen, Y., Li, Z., Li, J., Zhen, B., Zhu, W., Li, X., Yao, Y., Song, Q., Liu, C., Qi, X., He, F., & Pei, H.
Affiliations
  • George Washington University (US)
  • Tianjin University of Science and Technology (CN)
  • Beijing Proteome Research Center (CN)
  • Renmin Hospital of Wuhan University (CN)
Corresponding author
  • Qi, Xiangdong
  • He, Fuchu
  • Pei, Huadong
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-03-15
Volume
10
Issue
1
Pages
1224-1224
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-09232-8
PMC
PMC6420636
OpenAlex
W2920931417
Cited by
104
References
69
Citations by year
2026: 11 · 2025: 21 · 2024: 17 · 2023: 15 · 2022: 13 · 2021: 10 · 2020: 11 · 2019: 6
FWCI
4.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (31571463, 2015CB910601, 31371433, 81572740)
  • National Key Research and Development Program of China (2015CB910601, 2018YFC0910303)
  • National Cancer Institute (R01CA184717, R01CA177898)
Topics
  • Health Sciences › Medicine › Oncology › PARP inhibition in cancer therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
Keywords
USP15, deubiquitinase, homologous recombination repair, PARP inhibitors, BARD1, BRCA1, MDC1, DNA double-strand breaks, deubiquitination, genomic instability, breast cancer, ovarian cancer, biomarkers
MeSH terms
Poly(ADP-ribose) Polymerase Inhibitors, Animals, Humans, Mice, Inbred C57BL, Mutation, Neoplasms, Neoplasms, Experimental, Nuclear Proteins, Whole-Body Irradiation, Trans-Activators, Treatment Outcome, Mice, Knockout, Cell Cycle Proteins, Drug Resistance, Neoplasm, Tumor Suppressor Proteins, RNA, Small Interfering, Ubiquitin-Protein Ligases, Intracellular Signaling Peptides and Proteins, Adaptor Proteins, Signal Transducing, Mice, Kaplan-Meier Estimate, DNA Breaks, Double-Stranded, HEK293 Cells, Recombinational DNA Repair, MCF-7 Cells, Ubiquitin-Specific Proteases

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