NF-κB regulates DNA double-strand break repair in conjunction with BRCA1-CtIP complexes

Abstract

NF-κB is involved in immune responses, inflammation, oncogenesis, cell proliferation and apoptosis. Even though NF-κB can be activated by DNA damage via Ataxia telangiectasia-mutated (ATM) signalling, little was known about an involvement in DNA repair. In this work, we dissected distinct DNA double-strand break (DSB) repair mechanisms revealing a stimulatory role of NF-κB in homologous recombination (HR). This effect was independent of chromatin context, cell cycle distribution or cross-talk with p53. It was not mediated by the transcriptional NF-κB targets Bcl2, BAX or Ku70, known for their dual roles in apoptosis and DSB repair. A contribution by Bcl-xL was abrogated when caspases were inhibited. Notably, HR induction by NF-κB required the targets ATM and BRCA2. Additionally, we provide evidence that NF-κB interacts with CtIP-BRCA1 complexes and promotes BRCA1 stabilization, and thereby contributes to HR induction. Immunofluorescence analysis revealed accelerated formation of replication protein A (RPA) and Rad51 foci upon NF-κB activation indicating HR stimulation through DSB resection by the interacting CtIP-BRCA1 complex and Rad51 filament formation. Taken together, these results define multiple NF-κB-dependent mechanisms regulating HR induction, and thereby providing a novel intriguing explanation for both NF-κB-mediated resistance to chemo- and radiotherapies as well as for the sensitization by pharmaceutical intervention of NF-κB activation.

Figure 1.

Effect of TNFα on DSB repair. (A) Design of substrate Δ-EGFP/3′EGFP to monitor conservative and non-conservative homologous DSB repair. The fluorescence-based DSB repair assay monitors reconstitution of wild-type (wt) EGFP after I-SceI meganuclease-mediated cleavage of the mutated EGFP gene Δ-EGFP, which is under the control of a CMV promoter (black box and kinked arrow), via determination of the fraction of green fluorescent cells within the total population (9). Each measurement was accompanied by the analysis of split samples after transfection of the cells with the same DNA mixture as in DSB repair measurements, but replacing filler pBS plasmid by wtEGFP-expressing plasmid of the DSB repair substrate design. Fractions of green fluorescent cells obtained were thus normalized with the individual transfection efficiency for each sample to give DSB repair frequencies, thereby excluding differences in transfection, transcription, translation, proliferation and lethality. Substrate Δ-EGFP/3′EGFP enables the analysis of conservative HR plus non-conservative SSA repair processes. Mutated EGFP genes: grey boxes; spacer sequence (hygromycin resistance gene cassette): white box; I-SceI site: triangle; deleted EGFP sequence: cross. (B) Influence of TNFα on DSB repair was analysed in K562(Δ/3′) cells carrying stably integrated homologous DSB repair substrate Δ-EGFP/3′EGFP. Cells were electroporated with pCMV-I-SceI for targeted substrate cleavage together with expression plasmid for the NF-κB antagonist IκBα-SR (pcDNA3.0-IκBα-SR) or with pcDNA3.0 in controls. After 24 h cells were treated with 10 ng/ml TNFα and cultivated for another 24 h, when DSB repair frequencies were determined. DSB repair frequencies in H₂O-treated controls were defined as 100% each (absolute mean value 1 × 10⁻³). Mean values and SEMs from n = 9 (**P < 0.01). (C) In parallel, cells were treated with 25 μM caspase inhibitor z(VAD)-fmk or DMSO and results were evaluated as in (B). (D and E) Apoptosis (D) and cellular distribution (E) were determined by flow cytometry with propidium iodide-stained cells at the time point of repair measurements. Sub-G1 fractions were determined and viable cells were divided into G1, S and G2 cell cycle phases (presented in percentages). Mean values and SEMs from n = 6. (F) Expression levels of endogenous IκBα and exogenous IκBα-SR were examined by western blotting.

Figure 2.

DSB repair stimulation by chemotherapeutics as a function of NF-κB activity. (A) K562(Δ/3′) cells were co-transfected with pCMV-I-SceI and pcDNA3.0-IκBα-SR/pcDNA3.0 as indicated, cultivated for 24 h and then treated with 50 μM etoposide (Eto). Cells were further cultivated for 48 h, when HR assays were performed. DSB repair frequencies in DMSO-treated controls were defined as 100% (absolute mean value 2 × 10⁻³). Mean values and SEMs from n = 9 (*P < 0.05; **P < 0.01). (B and C) The percentages of cells with sub-G1 DNA content (B) in G1, S or G2 cell cycle phases (C) were determined with propidium iodide-stained cells at the time point of the repair measurements. Columns, mean values of n = 4; bars SEMs. (D) IκBα phosphorylation in cells exposed to 50 μM etoposide was analysed by western blotting (p- IκBα). (E–H) K562(Δ/3′) cells were co-transfected with pCMV-I-SceI and pcDNA3.0-IκBα-SR/pcDNA3.0, treated with 300 nM camptothecin (Cpt) and evaluated as in (A–D). Note that IκBα degradation interfered with p-IκBα detection 24 h after camptothecin addition in (H).

Figure 3.

Expression of the NF-κB subunit p65 enhances DSB repair. (A and E) Homologous DSB repair was analysed in K562(Δ/3′) cells 48 h (A) and 72 h (E) after cells had been electroporated with pCMV-I-SceI, pcDNA3.0-p65/pcDNA3.0 and pcDNA3.0-IκBα-SR/pcDNA3.0 as indicated. Mean values from controls were defined as 100% (absolute mean values: 0.4 × 10⁻³ after 48 h, 0.9 × 10⁻³ after 72 h); Mean values and SEMs from n = 6; *P < 0.05. (B and F) In parallel experiments cells were treated with 25 μM z(VAD)-fmk. (C, D, G and H) Apoptosis (C and G) and cell cycle distribution (D and H) were analysed under the same conditions as in (A) and (E). Mean values and SEMs from n = 4. (I) Western blot analysis verifying p65 expression.

Figure 4.

NF-κB-dependent DSB repair stimulation affects mostly HR. (A) Substrate design for the assessment of different DSB repair pathways. In plasmid substrate EJ-EGFP, the I-SceI site (triangle) is flanked by 5 bp microhomologies for detection of microhomology-mediated NHEJ. Substrate HR-EGFP/5′EGFP enables the analysis of conservative HR (mostly gene conversion) and 5′EGFP/HR-EGFP assessment of non-conservative homologous repair (mostly SSA after I-SceI-mediated cleavage). Mutated EGFP genes: grey boxes; spacer sequence: white box; deleted EGFP sequence: cross; (B–D) K562 cells were co-transfected with pCMV-I-SceI plus pcDNA3.0-p65/pcDNA3.0 and pcDNA3.0-IκBα-SR/pcDNA3.0 plus repair substrates to assess NHEJ (B), SSA (C) or HR (D). DSB repair was measured 48 h after transfection. Mean values and SEMs from n = 6 (*P < 0.05), controls were defined as 100% each (absolute mean value for NHEJ: 2.7 × 10^-3, SSA: 2.7 × 10⁻², HR: 0.4 × 10⁻³).

Figure 5.

Role of Bcl2-family proteins in NF-κB-mediated DSB repair regulation. (A, D and G) K562(Δ/3′) cells were co-transfected with pCMV-I-SceI, pcDNA3.0-p65 or empty vector plus a mixture of two different shRNA expression plasmids directed against Bcl2, Bcl-xL or BAX or empty vector. DSB repair frequencies were measured 72 h post-transfection; mean values of controls were taken as 100% each (absolute mean value 1 × 10⁻³). Columns, mean values of n = 6–12; bars, SEM; *P < 0.05; ***P < 0.001. (B, E and H) Cells were treated with 25 μM z(VAD)-fmk and homologous DSB repair was assayed as in (A, D and G). (C, F and I) Immunoblot analysis showing Bcl2, Bcl-xL and BAX knockdown.

Figure 6.

p65-dependent DSB repair involves the classical homologous DSB repair proteins ATM, BRCA2, BRCA1 and CtIP. (A, C, E and G) HeLa control and ATM, BRCA2, BRCA1 or Mre11 HeLa knockdown cell lines (ATM kd, BRCA2 kd, BRCA1 kd, Mre11 kd) were transfected with pCMV-I-SceI plus pcDNA3.0-p65/pcDNA3.0 and repair substrate to assess HR. After cultivation for 48 h, cells were FACS analysed for green fluorescence. Mean DSB repair frequencies with controls were set to 100% each (absolute mean value 0.5 × 10⁻³). Columns, mean values of n = 6; bars, SEM; *P < 0.05; **P < 0.01. (B, D, F and H) Western blots confirming ATM, BRCA2, BRCA1 or Mre11 knockdown and p65 expression. (I, K and M) Homologous DSB repair in K562(Δ/3′) cells was analysed after p65 expression with or without transient BRCA1, CtIP, or Ku70 knockdown by use of shRNA plasmids. Columns, mean values of n = 9–12; bars, SEM; *P < 0.05; **P < 0.01; ***P < 0.001. (J, L and N) Western blots demonstrating BRCA1, CtIP or Ku70 knockdown and p65 expression. (O) K562(Δ/3′) cells were transfected with pCMV-I-SceI and 24 h later treated with 10 ng/ml of TNFα for 4 h, when immunoprecipitation was performed with antibodies directed against BRCA1 or p65 followed by immunoblotting with antibodies to detect BRCA1, CtIP and p65. (P) K562(Δ/3′) cells were transfected with pCMV-I-SceI, cultivated for 24 h, then, treated with 7 μM cycloheximide for 45 min and after that exposed to TNFα for 24 h (10 ng/ml). Cells were further cultivated for 24 h followed by protein extraction and immunoblotting analysis with antibodies against BRCA1, CtIP, Ku70 and tubulin.

Figure 7.

Influence of TNFα on 53BP1, RPA and Rad51 foci assembly. SaOS cells were exposed to bleomycin (7.5–100 mU) alone or in combination with TNFα 10 ng/ml (A–L). In Figure 7M–O cells were transfected with pcDNA3.0 or pcDNA3.0-p65, cultivated for another 24 h and then treated with bleomycin. (A–C) At the indicated incubation times after treatment with 15 mU bleomycin, cells were processed for immunolabeling of 53BP1 (A), RPA (B), or Rad51 (C) to visualize foci assembly. (D–O) Immunolabeled foci from two independent experiments were scored by automated quantification in 50 nuclei from two separate slides for each time point. Bleomycin concentrations: 7.5 mU (D, G and J), 15 mU (E, H and K), 30–100 mU (F, I, L and M–O). Maximum scores were set to 100%. (P) Model of NF-κB-dependent DSB repair regulation.