Poly( ADP ‐ribosyl)ation of BRD 7 by PARP 1 confers resistance to DNA ‐damaging chemotherapeutic agents
2019; Springer Nature; Volume: 20; Issue: 5 Linguagem: Inglês
10.15252/embr.201846166
ISSN1469-3178
AutoresKaishun Hu, Wenjing Wu, Yu Li, Lehang Lin, Dong Chen, Haiyan Yan, Xing Xiao, Hengxing Chen, Zhen Chen, Yin Zhang, Shuangbing Xu, Yabin Guo, H. Phillip Koeffler, Erwei Song, Dong Yin,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle2 April 2019free access Source DataTransparent process Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA-damaging chemotherapeutic agents Kaishun Hu Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Wenjing Wu Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Breast Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yu Li Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Lehang Lin Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dong Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Interventional Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Haiyan Yan Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xing Xiao Department of Dermatology and Venerology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Search for more papers by this author Hengxing Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Zhen Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yin Zhang Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Shuangbing Xu Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yabin Guo Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author H Phillip Koeffler Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California Los Angeles School of Medicine, Los Angeles, CA, USA Search for more papers by this author Erwei Song Corresponding Author [email protected] orcid.org/0000-0002-5400-9049 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Breast Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dong Yin Corresponding Author [email protected] orcid.org/0000-0003-1494-7185 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Kaishun Hu Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Wenjing Wu Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Breast Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yu Li Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Lehang Lin Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dong Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Interventional Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Haiyan Yan Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Xing Xiao Department of Dermatology and Venerology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Search for more papers by this author Hengxing Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Zhen Chen Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Yin Zhang Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Shuangbing Xu Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yabin Guo Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author H Phillip Koeffler Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California Los Angeles School of Medicine, Los Angeles, CA, USA Search for more papers by this author Erwei Song Corresponding Author [email protected] orcid.org/0000-0002-5400-9049 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Department of Breast Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Dong Yin Corresponding Author [email protected] orcid.org/0000-0003-1494-7185 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Search for more papers by this author Author Information Kaishun Hu1,‡, Wenjing Wu1,2,‡, Yu Li1,‡, Lehang Lin1, Dong Chen1,3, Haiyan Yan1, Xing Xiao4, Hengxing Chen1, Zhen Chen1, Yin Zhang1, Shuangbing Xu5, Yabin Guo1, H Phillip Koeffler6,7, Erwei Song *,1,2 and Dong Yin *,1 1Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 2Department of Breast Oncology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 3Department of Interventional Radiology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China 4Department of Dermatology and Venerology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 5Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 6Cancer Science Institute of Singapore, National University of Singapore, Singapore City, Singapore 7Division of Hematology/Oncology, Cedars-Sinai Medical Center, University of California Los Angeles School of Medicine, Los Angeles, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +86 20 8133 2507; E-mail: [email protected] *Corresponding author. Tel: +86 20 8133 2405; E-mail: [email protected] EMBO Rep (2019)20:e46166https://doi.org/10.15252/embr.201846166 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The bromodomain-containing protein 7 (BRD7) is a tumour suppressor protein with critical roles in cell cycle transition and transcriptional regulation. Whether BRD7 is regulated by post-translational modifications remains poorly understood. Here, we find that chemotherapy-induced DNA damage leads to the rapid degradation of BRD7 in various cancer cell lines. PARP-1 binds and poly(ADP)ribosylates BRD7, which enhances its ubiquitination and degradation through the PAR-binding E3 ubiquitin ligase RNF146. Moreover, the PARP1 inhibitor Olaparib significantly enhances the sensitivity of BRD7-positive cancer cells to chemotherapeutic drugs, while it has little effect on cells with low BRD7 expression. Taken together, our findings show that PARP1 induces the degradation of BRD7 resulting in cancer cell resistance to DNA-damaging agents. BRD7 might thus serve as potential biomarker in clinical trial for the prediction of synergistic effects between chemotherapeutic drugs and PARP inhibitors. Synopsis PARP1 promotes the PARylation and degradation of the tumor suppressor BRD7 thereby enhancing survival of cancer cells in response to DNA-damaging agents. BRD7 interacts with PARP1 via three potential PAR-binding motifs. Inhibiting PARylation of PARP1 by Olaparib disrupted the interaction of BRD7 with PARP1. BRD7 protein is subjected to PARP1-mediated poly(ADP-ribosyl)ation and subsequent ubiquitylation by RNF146, leading to resistance to DNA-damaging agents of cancer cells. Introduction Protein post-translational modifications (PTMs) are known to be essential mechanisms exploited by eukaryotic cells to diversify their protein functions and modulate their cellular signalling networks. Emerging evidence suggests that PTMs play a key role in many cellular processes including protein–protein interactions, protein degradation, gene expression, signal transduction and cell differentiation 1, 2. These modifications including phosphorylation, acetylation, ubiquitination, methylation, glycosylation, oxidation and SUMOylation influence almost all aspects of normal cell biology and pathogenesis. Defect of PTMs leads to human disease including those associated with disorders of cell proliferation, highlighting the importance of PTMs in maintaining normal cellular state 3. Therefore, identifying and understanding PTMs are of biologically and clinically importance. Poly(ADP-ribosyl)ation, also termed PARylation, is a unique post-translational modification of proteins catalysed by ADP-ribosyltransferases and plays versatile roles in multiple biological processes including chromatin reorganization, DNA damage response, transcriptional regulation, apoptosis and mitosis 4-6. It causes the formation of poly-ADP-ribose (PAR) by transferring the ADP-ribose moiety from nicotinamide adenine dinucleotide (NAD+) to specific Lys/Glu/Asp/Arg/Cys amino acid residues on substrate proteins 4, 5, 7-12. Moreover, ADP-ribosylation is a reversible post-translational modification, which could be recognized and degraded by ADP-ribosylhydrolases including Poly(ADP-ribose) glycohydrolase (PARG), Terminal ADP-ribose protein glycohydrolase 1(TARG1), ADP-ribosylhydrolase 1 (ARH1), ARH3, Macro D1 and D2, Nudix-type-motif 9 and 16 (NUDT9 and NUDT16) 13-17. Poly(ADP-ribose) polymerases (PARPs) composed of 17 members play diverse roles in multiple cellular processes 5, 18. PARP1, the most characterized member of this family, regulates protein–protein interaction, protein stabilization, cellular localization, energy metabolism and cell fate determination through regulation of transcriptional and post-translational activity of its substrates 19-21. In response to DNA damage, PARP1 is activated and promotes the formation of poly(ADP-ribose) polymer (pADPr) on its substrates as well as itself, which provides a signal to recruit DNA double-strand break proteins to DNA-damaging site 4, 22. Through modulating histone modification and chromatin structure, PARP1 is also involved in the regulation of transcription process 23-27. Considering its diverse and important roles, PARP1 inhibitor Olaparib has been used in the treatment of ovarian and breast cancers 28-30. However, the mechanism that PARP1 inhibitor in combination with DNA-damaging reagents generates more inhibitory effects is still lacking. Bromodomain-containing protein 7 (BRD7) is a member of the family of bromodomain-containing proteins that contains a single bromodomain involved in multiple cellular processes including cell proliferation, apoptosis and epithelial–mesenchymal transition (EMT) 31-33. BRD7 has been reported to interact with p53 and is required for p53-dependent replicative senescence 34, 35. Moreover, as a component of chromatin remodelling of SWI/SNF complex, BRD7 has also been shown to function as either a transcriptional co-activator or co-repressor 34-37. For instance, BRD7 serves as a transcriptional co-activator through association with tumour suppressors BRCA1 and p53 to regulate downstream gene transcription 34, 35, 37. Conversely, BRD7 can interact with PRMT5 and PRC2 to be involved in transcriptional repression of their target genes 38. BRD7 also plays a critical role in regulation of endoplasmic reticulum (ER) stress and glucose metabolism via association with the p85 regulatory subunit of PI3K, resulting in suppression of PI3K signalling 39, 40. Inactivation or mutation of BRD7 increases sensitivity of tumour cell to interferon-γ, resulting in elevated sensitivity of cancer cells to PD-1 blockade, as well as, other forms of immunotherapy treatment 41. In this study, we identified BRD7 as a novel PARP1-binding protein and demonstrated that PARP1 directly ribosylated BRD7 and promoted BRD7 degradation, mediated by the E3 ubiquitin ligase RNF146, further enhancing survival of cancer cells. Furthermore, inhibition of PARP1 suppressed cell proliferation and promoted sensitivity of cancer cells to DNA damage chemotherapy through reduction of BRD7 PARylation. Therefore, our study uncovered a novel mechanism that PARP1 modulated resistance to DNA-damaging agents of cancer cells by promoting PARylation of BRD7, providing new insights into the molecular rationale for combination of chemotherapeutic drugs and PARP inhibitors in clinical treatment, and suggested that BRD7 may be served as a potential biomarker for predicting the synergistic effects of combining chemotherapeutic drugs and PARP inhibitors in clinical trial. Results Drug- or irradiation-induced DNA damage promoted BRD7 protein degradation To study the effect of BRD7 on the sensitivity of genotoxic drug in cancer cell, BRD7 protein levels were examined using cells resistant to either cisplatin (DDP), doxorubicin (ADR) or irradiation (IR). BRD7 protein levels significantly decreased in these cells (Fig 1A). Interestingly, quantitative real-time PCR data showed that mRNA level of BRD7 was not altered between wild-type and ADR or IR-resistant cells (Fig 1B). Notably, data from GEO database also revealed that mRNA level of BRD7 was slightly increased in MCF7/ADR-resistant cells compared with MCF7 normal cells (Appendix Fig S1). To confirm further these findings, cells were treated with either ADR or CPT for different time intervals. Both ADR and CPT treatments in a time-dependent manner caused a reduction of BRD7 at the protein levels in MDA-MB-468 and MDA-MB-231 cell lines (Fig 1C and D). To determine whether downregulation of endogenous BRD7 was not induced by cell death in response to DNA damage, we performed cell death detection assay measured by Fixable Viability Dye eFluor® 455UV reagent (eBioscience) according to manufacturer's instructions. As shown in Appendix Fig S2B–F, there was no dramatic increase in cell death within the treatment of indicated drugs and the ratio of dead cells was very low no more than 2% of total cells in both MDA-MB-231 and MDA-MB-468 cells. Consistent with the Western blot results, BRD7 fluorescence intensity was diminished dramatically in response to CPT treatment (Fig 1E and F, Appendix Fig S2A). Taken together, these findings suggested that BRD7 was involved in drug- and IR-induced DNA damage and was downregulated at the post-translational level rather than post-transcriptionally. Figure 1. Chemotherapeutic drugs and irradiation deplete BRD7 A. Western blot analysis of BRD7 and γH2AX protein levels in breast cancer cell lines: MCF7 parental or doxorubicin-resistant MCF7/ADR (ADR, 0.5 μg/ml) and irradiation-resistant MCF7/IR (IR, 10 Gy), MDA-MB-231 parental or doxorubicin-resistant MDA-MB-231/ADR (ADR, 0.5 μg/ml) or cisplatin-resistant MDA-MB-231/DDP (1 μg/ml), non-small lung cancer cell lines: A549 parental or cisplatin-resistant A549/DDP (1 μg/ml). B. MCF7, MCF7/ADR and MCF7/IR, MDA-MB-231 and MDA-MB-231/ADR cells were harvested and mRNA levels of BRD7 were determined by real-time PCR (n = 3). Values are mean ± SEM. C, D. Western blot analysis of BRD7 protein levels in MDA-MB-468 and MDA-MB-231 cell after treatment with ADR (5 μM) or camptothecin (CPT) (1 μM) for different intervals (n = 3). E. Representative images of endogenous BRD7 (green) and γH2AX foci (red) in paraformaldehyde-fixed MDA-MB-468 cells after treatment with CPT (1 μM) for different intervals. Visualized by immunofluorescence using anti-BRD7 and Alexa Fluor 555 anti-γH2AX antibodies. DNA staining with DAPI; Scale bars, 2 μm. F. Quantification of average fluorescence intensity of BRD7 of cells in (E). Error bars indicate SEM; n > 100. Source data are available online for this figure. Source Data for Figure 1 [embr201846166-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint BRD7 interacted with PARP1 To identify proteins that interact with BRD7 in response to DNA damage, proteomic analysis was performed. SFB-tagged (S-protein, Flag and streptavidin-binding peptide) BRD7 was stably expressed in HEK293T cell. After tandem affinity purification (TAP), proteins associated with BRD7 were subjected to silver staining and mass spectrometry (Fig 2A). In addition to known BRD7-binding proteins, such as TP53 34 and PIK3R 40, we identified PARP1 as a novel binding partner of BRD7 (Fig 2B, Dataset EV1). PARP1 is a key enzyme involved in DNA damage repair and cell survival 42. To confirm the interaction between BRD7 and PARP1, we performed transient transfection and co-IP experiments. As shown in Fig 2C and D, a complex containing BRD7 and PARP1 was clearly detected using either Flag agarose or Myc agarose in HEK293T cells expressing both Flag-tagged PARP1 and Myc-tagged BRD7. To examine further the interaction between endogenous BRD7 and PARP1, HeLa and MDA-MB-231 whole-cell extracts were prepared and subjected to immunoprecipitation assays in the presence of either control IgG, anti-BRD7 or anti-PARP1 antibody. Both BRD7 and PARP1 were clearly detected in the immunoprecipitated complex (Fig 2E and F). To further determine whether the interaction of BRD7 with PARP1 dependent on its poly-ADP-ribose (PAR) chains, we detected the association of BRD7 to PARP1 with or without PARP1 inhibitor Olaparib. As shown in Fig 2G, there was significant inhibition of the interaction of PARP1 with BRD7 upon Olaparib treatment, indicating that the PARylation of PARP1 is required to interact with BRD7. Most importantly, the interaction between BRD7 and PARP1 was greatly enhanced following exposure to CPT (Fig 2H and I). These data together suggest that BRD7 is a PAR-binding protein. Figure 2. BRD7 interacts with PARP1 A. Silver staining of the BRD7 complex separated by SDS–PAGE. HEK293T cells stably expressing SFB-tagged BRD7 were used for tandem affinity purification (TAP) of protein complexes. BRD7-interacting proteins, including PARP1 and PIK3R2, are indicated. B. Table summarizes proteins identified by mass spectrometry analysis. C, D. HEK293T cells transiently transfected with Flag-PARP1 and Myc-BRD7 for 24 h were lysed with RIPA buffer. Followed by immunoprecipitation (IP) using antibodies to either Myc (C) or Flag (D) conjugated to agarose followed by Western blot with the indicated antibodies (n = 3). E, F. HeLa and MDA-MB-231 cells were lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG, or BRD7 or PARP1 antibodies, and analysed by Western blot (n = 3). G. MDA-MB-231 cells were treated first with Olaparib (10 μM) for 6 h and lysed with RIPA buffer, and lysates were subjected to immunoprecipitation using either anti-IgG or PARP1 antibodies, and analysed by Western blot (n = 3). H, I. Association of endogenous BRD7 with PARP1 in HeLa cells was performed by co-immunoprecipitation using anti-BRD7 or anti-PARP1 antibody. HeLa cell was treated with CPT (1 μM, 1 h), followed by IP using indicated antibodies, and Western blot was performed. γH2AX was used as a marker of DNA damage induced by CPT (n = 3). Source data are available online for this figure. Source Data for Figure 2 [embr201846166-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Next, a serial of deletion mutants was generated to map the region of interaction of BRD7 and PARP1. PARP1 contains a DNA-binding domain, a BRCA1 C terminus, a tryptophan–glycine–arginine domain (WGR) and a catalytic domain (CA) (Appendix Fig S3A). As shown in Appendix Fig S3B, both the N-terminal DNA-binding/automodification domain and C-terminal catalytic domain of PARP1 were capable of binding BRD7. BRD7 has a conserved bromodomain (BD, 128–238 amino acids) which specifically binds to acetylated lysines on histones, and an uncharacterized conserved domain (361–651 amino acids) (Appendix Fig S3C). Deletion of individual regions of BRD7 demonstrated that the N-terminal bromodomain was not required for BRD7 binding to PARP1, but depletion of 361–651 domain significantly decreased the binding ability to PARP1 (Appendix Fig S3D). These results reinforce the hypothesis that BRD7 physically and specifically interacts with PARP1. BRD7 was ADP-ribosylated by PARP1 in vitro and in vivo Function of PARP1 is attaching the pADPr chain to specific glutamate, aspartate, arginine, lysine or cysteine residues of target proteins 4, 5, 7-12. We tested if BRD7 is ribosylated by PARP1 directly. First, we used anti-PAR antibody to immunoprecipitate the PARylated proteins in the absence and presence of CPT. As a positive control, we detected increased ribosylated PARP1 in response to CPT (Fig 3A). As expected, PARsylation of BRD7 was also greatly increased after 3 h CPT exposure (Fig 3A). Second, we used anti-BRD7 antibody to immunoprecipitate BRD7 followed by immunoblotting for PAR and further confirmed that BRD7 is ribosylated in vivo (Fig 3B). Moreover, to rule out the possibility of indirect binding of BRD7 to PARylated proteins, we performed a denaturing immunoprecipitation using either anti-BRD7 antibody or anti-PAR antibody. As shown in Appendix Fig S4A and B, a clear band of PARylated BRD7 was detected and suggested that BRD7 is covalently modified by poly-ADP-ribose (PAR) in vivo. To determine whether exogenous BRD7 could also be ribosylated, we used anti-myc agarose to immunoprecipitate myc-tagged BRD7. Consistently, exogenous BRD7 was also ribosylated, and the ribosylation could be enhanced in the cells treated with either CPT or ADR (Fig 3C and D). Next, we examined if BRD7 could be ribosylated by PARP1 in vivo. As shown in Fig 3E, depletion of PARP1 profoundly decreased BRD7 ribosylation levels in vivo. Moreover, we identified nine ADP-ribosylation sites between aspartic acid and glutamic acid resides on BRD7 by mass spectrometric analysis (Fig EV1 and Dataset EV2). Hence, a novel post-translational modification of BRD7 has been discovered. Figure 3. BRD7 is ADP-ribosylated by PARP1 in vitro and in vivo HeLa cells were untreated or treated with CPT (1 μM) for 1 h followed by lysing with RIPA buffer, and lysates were then immunoprecipitated using anti-IgG or anti-PAR antibodies and immunoblotted with the indicated antibodies (n = 3). HeLa cells were untreated or treated with CPT (1 μM) for 1 h, and cellular lysates were immunoprecipitated using anti-IgG or anti-BRD7 antibodies and immunoblotted using the indicated antibodies (n = 3). HeLa and 293T cells transfected with Myc-BRD7 plasmid for 24 h were lysed with RIPA buffer. Lysates were then immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies. Ribosylation levels of exogenous BRD7 were detected using anti-PAR antibody (n = 3). HeLa cells transfected with Myc-BRD7 plasmid. After 24 h, cells were treated with either CPT (1 μM) or ADR (5 μM) combined with MG132 (10 μM) for indicated times. Cellular lysates were immunoprecipitated using anti-Myc agarose and immunoblotted using the indicated antibodies (n = 3). HeLa PARP1 wild-type and PARP1 knockout cells were transfected with Myc-BRD7 for 24 h, and lysates were subjected to immunoprecipitation using anti-Myc agarose and analysed by Western blot (n = 3). HeLa was transfected with BRD7 wild-type and various BRD7-mutant plasmids for 24 h, lysed with RIPA, followed by anti-Myc IP and Western blot with indicated antibody (n = 3). Ribosylation of BRD7 by PARP1 in vitro. Recombinant BRD7 was subjected to in vitro ribosylation either in absence or presence of biotin-labelled NAD+. Recombinant proteins were detected by indicated antibodies, and ribosylated proteins were determined with anti-biotin antibody (n = 3). PAR-binding motif of BRD7 is required for its ribosylation by PARP1. Recombinant Myc-BRD7-WT and Myc-BRD7-mutant were subjected to in vitro ribosylation assay and analysed by Western blot as indicated (n = 3). Source data are available online for this figure. Source Data for Figure 3 [embr201846166-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The PARylation of BRD7 was analysed by mass spectrometry HeLa cells stably expressing SFB-BRD7 were treated with 10 μM of PARG inhibitor PDD0017273 for 6 h, and SFB-BRD7 was pulled down by Flag beads from cell lysates and subjected to mass spectrometry analysis. BRD7 PARylation was analysed by mass spectrometry. Distribution of ADP-ribosylation sites of BRD7 between aspartic acid (Asp) and glutamic acid (Glu) resides. Asp- and Glu-ADP-ribosylation is susceptible to NH2OH attack, generating a hydroxamic acid (H) derivative, allowing easy localization of the ADP-ribosylation sites. The site of modification is indicated by an H.m. Download figure Download PowerPoint The above data demonstrated in vivo PAR-binding activity of BRD7 prompted us to search for potential PAR-binding motif in BRD7 (Fig 2G). PAR-binding proteins commonly contain a conserved PAR-binding motif, consisting of eight amino acids [HKR]-X-X-[AIQVY]-[KR]-[KR]-[AILV]-[FILPV] 43. Through sequence alignment, we identified three highly conserved residues 222Lys/223Lys, 545Arg/546Lys and 613Arg/614Lys in the BRD7 protein as potential PAR-binding motifs (Appendix Fig S4C and D). To investigate whether the interaction of BRD7 against PARP1 and subsequent ribosylation of BRD7 through its potential PAR-binding motif, co-IP was performed. Unlike wild-type BRD7, each mutant of these three candidate sites decreased the binding affinity for PARP1 and subsequent suppression of its ribosylation, and 613Arg/614Lys may be the major PAR-binding motif responsible for association of BRD7 with PARP1 (Fig 3F). To verify further that whether BRD7 could be ribosylated by PARP1 in vitro, an in vitro ribosylation assay was performed using recombinant BRD7 and biotin-labelled NAD+. As shown in Fig 3G, PARP1 ribosylated not only itself but also BRD7. Indeed, we generated 6A point mutations (222/223/545/546/613/614A) on the PAR-binding motif of BRD7 (BRD7-6A) and the ribosylation on BRD7-6A by PARP1 was significantly inhibited both in vivo and in vitro (Fig 3H and Appendix Fig S4C), caused by dramatic reduction of binding affinity for BRD7 against PARP1. Taken together, BRD7 binds PARylated PARP1 non-covalently via its PAR-binding motifs an
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