Poly(ADP-ribosyl)ation of BRD7 by PARP1 confers resistance to DNA-damaging chemotherapeutic agents

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.

Keywords: BRD7; PARP1; PARylation; RNF146; ubiquitination.

Figure 1. Chemotherapeutic drugs and irradiation deplete BRD7

1. 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).

2. 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.

3. 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).

4. 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.

5. 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.

Figure 2. BRD7 interacts with PARP1

1. 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.

2. B

   Table summarizes proteins identified by mass spectrometry analysis.

3. 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).

4. 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).

5. 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).

6. 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.

Figure 3. BRD7 is ADP‐ribosylated by PARP1 in vitro and in vivo

1. 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).

2. 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).

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).

4. 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).

5. 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).

6. 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).

7. 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).

8. 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.

Figure EV1. The PARylation of BRD7 was analysed by mass spectrometry

1. 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.

2. BRD7 PARylation was analysed by mass spectrometry.

3. 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.

Figure 4. PARP1 regulates BRD7 protein stability through ubiquitination‐dependent pathway

1. A

   HeLa cells were transfected with Flag‐PARP1 and Myc‐BRD7 for 24 h, then treated with MG132 (10 μM) for additional 4 h, and proteins were detected by Western blot with indicated antibodies (n = 3).

2. B

   HeLa cells were transfected with either scrambled or PARP1 siRNAs for 48 h, and protein levels were detected by Western blot with the indicated antibodies (n = 3).

3. C

   Expression of BRD7, p21 and PARP1 measured in PARP1 wild‐type and knockout HeLa cells using PARP1 sgRNA by Western blot (n = 3).

4. D

   MDA‐MB‐231 cell was treated with different concentration of PARG inhibitor PDD0017273 for 48 h, and cell lysates were analysed by Western blot (n = 3).

5. E

   MDA‐MB‐231 cell was treated with 10 μM of PDD0017273 for indicated times, and cell lysates were analysed by Western blot (n = 3).

6. F, G

   Wild‐type and PARP1 knockout HeLa cells were incubated with 10 μg/ml cycloheximide (CHX) for the indicated times. Lysates were harvested and analysed by Western blot. Quantification of BRD7 protein is shown in (G) (n = 3), and results represent mean ± SEM. Relative amounts normalized to the BRD7 protein level at 0 h of Control or sgPARP1 cells, respectively.

7. H

   PARP1 wild‐type and knockout HeLa cells were transfected with HA‐ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, and cells were lysed with RIPA buffer, followed by anti‐BRD7 IP and analysed by Western blot with the indicated antibodies (n = 3).

8. I

   SFB‐BRD7 stably overexpressing HeLa cells transfected with scramble or PARP1 siRNA for 24 h were transfected with vector or HA‐ubiquitin (Lys48 or Lys63 only) for 24 h, and MG132 (10 μM) was added for an additional 4 h, lysed with RIPA, subjected to IP using S tag beads followed by Western blot (n = 3).

9. J

   HeLa cells were co‐transfected either wild‐type or BRD7 mutant with Flag‐PARP1 for 24 h and analysed by Western blot (n = 3).

10. K

    HeLa cells were co‐transfected with either wild‐type or BRD7 mutant plus HA‐ubiquitin for 24 h, and MG132 (10 μM) was added for an additional 4 h and lysed with RIPA buffer, followed by anti‐Myc agarose IP and analysed by Western blot with the indicated antibodies (n = 3).

Source data are available online for this figure.

Figure 5. E3 ligase RNF146 promotes BRD7 ubiquitination and degradation

1. HeLa and MDA‐MB‐231 cells were transfected with either scrambled or RNF146 siRNAs (48 h). Protein levels were detected by Western blot. PTEN, which is a known RNF146‐interacting protein, was included as a positive control (n = 3).

2. HeLa cells were transfected with either scrambled or RNF146 siRNAs for 48 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for the indicated periods of time. Lysates were harvested and analysed by Western blot (n = 3).

3. Quantification of BRD7 protein levels from (B), n = 3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.

4. Control and RNF146 stably overexpressing HeLa cells were transfected with Myc‐BRD7‐WT for 24 h, followed by incubation with 10 μg/ml cycloheximide (CHX) for indicated periods of time. Lysates were subjected to Western blot analysis (n = 3).

5. Quantification of BRD7 protein levels from (D), n = 3. Error bars indicate SEM. Relative amounts normalized to the BRD7 protein level at 0 h.

6. HeLa cells transfected with either RNF146 or control siRNA for 24 h followed by transfection of HA‐ubiquitin for another 24 h; MG132 (10 μM) was added for 4 h and lysed with RIPA, followed by anti‐BRD7 IP and analysed by Western blot with the indicated antibodies (n = 3).

7. HeLa cells stably expressing RNF146 were transfected with HA‐ubiquitin for 24 h; MG132 (10 μM) was added for an additional 4 h, followed by anti‐BRD7 IP and Western blot (n = 3).

Source data are available online for this figure.

Figure 6. E3 ligase RNF146 interacted with BRD7 in a PARP1‐dependent manner

1. A, B

   HeLa cells transfected with the indicated plasmids; after 24 h, cells were lysed with RIPA buffer followed by immunoprecipitation (IP) using either anti‐Myc or anti‐Flag agarose and Western blot with indicated antibody (n = 3).

2. C

   HeLa and MDA‐MB‐231 cells were lysed with RIPA buffer, and lysates were subjected to IP using either anti‐IgG, anti‐BRD7 or anti‐RNF146 antibodies followed by analysis with Western blot (n = 3).

3. D

   Association of endogenous BRD7 with RNF146 in HeLa cells was performed by co‐IP using anti‐RNF146 antibody. Cell lysates were incubated with protein G agarose beads conjugated with indicated antibodies, and Western blot was performed (n = 3).

4. E

   HeLa cells with wild type or PARP1 knockout were transfected with Myc‐BRD7‐WT for 24 h, and their lysates were subjected to IP using anti‐Myc agarose and analysed by Western blot with indicated antibodies (n = 3).

5. F

   HeLa cells stably expressing Flag‐RNF146 were transfected with either Myc‐BRD7‐WT or Myc‐BRD7‐mutant for 24 h, and lysates were subjected to IP using anti‐Flag agarose and analysed by Western blot with indicated antibodies (n = 3).

Source data are available online for this figure.

Figure 7. Inhibiting PARylation of BRD7 enhanced sensitivity of cancer cells to DNA‐damaging agents

1. MDA‐MB‐231 cells were transfected twice with indicated siRNAs, and the absorbance values of indicated cell lines were measured at different time points by CCK8 assay (n = 3).

2. Cell lysates treated in (A) were analysed by Western blot with indicated antibodies (n = 3).

3. MDA‐MB‐231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM), CPT (0.5 μM) or VP16 (10 μM) for 72 h, and CCK8 activity of the cells was detected (n = 3).

4. MDA‐MB‐231 cells were treated with Olaparib (10 μM) combined with ADR (0.5 μM) for 72 h, and their lysates were subjected to IP using anti‐BRD7 antibodies and analysed by Western blot with indicated antibodies (n = 3).

5. MDA‐MB‐231 cells were transfected twice with BRD7 siRNAs, and these cells were subjected to CCK8 assay after treatment with Olaparib (10 μM) combined with either ADR (0.5 μM) or VP16 (10 μM) for 72 h (n = 3).

6. Resistance of BRD7‐depleted cells to Olaparib was reversed by the expression of wild‐type BRD7 but not mutant BRD7. shRNA‐resistant BRD7 wild‐type and BRD7‐mutant plasmids were transduced into BRD7‐depleted MDA‐MB‐231 cells, and cell viability was determined as indicated above (n = 3).

Data information: In (A, C, E, F), results represent mean ± SEM of three experiments. N.S., not significant; **P < 0.01, Student's t‐test.Source data are available online for this figure.

Figure 8. Schematic model of the PARP1‐RNF146‐BRD7 pathway in response to DNA damage

BRD7 was ribosylated by PARP1 after DNA damage, promoting recognization by E3 ligase RNF146 which targeted BRD7 for ubiquitination–proteasome‐dependent degradation enhancing cell survival.