p53 suppresses hyper-recombination by modulating BRCA1 function

Abstract

Both p53 and BRCA1 are tumor suppressors and are involved in a number of cellular processes including cell cycle arrest, apoptosis, transcriptional regulation, and DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair is still not clear. Therefore, we sought to determine whether there is a functional link between p53 and BRCA1 in DNA repair. Firstly, using a plasmid recombination substrate, pDR-GFP, integrated into the genome of breast cancer cell line MCF7, we have demonstrated that p53 suppressed Rad51-mediated hyper-recombinational repair by two independent cell models of HPV-E6 induced p53 inactivation and p53 knockdown assay. Our study further indicated that p53 mediated homologous recombination (HR) through inhibiting BRCA1 over-function via mechanism of transcription regulation in response to DNA repair. Since it was found p53 and BRCA1 existed in a protein complex, indicating both proteins may be associated at post-transcriptional level. Moreover, defective p53-induced hyper-recombination was associated with cell radioresistance and chromosomal stability, strongly supporting the involvement of p53 in the inhibition of hyper-recombination, which led to genetic stability and cellular function in response to DNA damage. In addition, it was found that p53 loss rescued BRCA1 deficiency via recovering HR and chromosomal stability, suggesting that p53 is also involved in the HR-inhibition independently of BRCA1. Thus, our data indicated that p53 was involved in inhibiting recombination by both BRCA1-dependent and -independent mechanisms, and there is a functional link between p53-suppression and BRCA1-promotion in regulation of HR activity at transcription level and possible post-transcription level.

Keywords: BRCA1; Chromosomal stability; Homologous recombination; Hyper-recombination; p53.

Fig. 1

p53 inhibits homologous recombination. (A) The establishment of MCF7 cells expressing the recombination substrate pDR-GFP. MCF7 cells were transfected with construct of pDR-GFP and further selected by puromycin to obtain a stable single clone. Flow cytometry was used for screening the positive clones expressing pDR-GFP. The pictures showed the positive cell clone expressing green fluorescent protein (GFP) induced by I-SceI falling above the diagonal. (B) The establishment of cell lines with or without HPV-E6 expression. MCF7/pDR-GFP cells were infected with the virus of E6 or LXSN to obtain stable clones expressing E6 (defective in p53) and LXSN (proficient in p53). At 8 h after exposure of the stable clones to 10 Gy of irradiation, p53 and p21 were detected by western blotting. β-actin was used as loading control. (C) At 24 h after exposure of MCF7/pDR-GFP cells expressing E6 or LXSN to 10 Gy of irradiation, the cells were detected to analyze cell cycle profile using flow cytometry. (D) MCF7/pDR-GFP cells expressing E6 or LXSN were used to analyze relative spontaneous recombination (SR) (left) and I-SceI-induced homologous recombination (HR) (right). (E) MCF7 cells were infected by p53 shRNA targeting 3′ untranslated regions of p53 (p53sh 3′UTR) or targeting coding region of p53 (p53sh CDS) to knockdown p53, at 48 h after infection relative mRNA of p53 expression was analyzed by RT-qPCR normalized against GAPDH (left). After restoring exogenous active p53 in the cells infected with p53sh 3′UTR, p53 and p21 protein expression in the cells were detected by western blotting and cell cycle profiling was analyzed by flow cytometry, β-actin was used as loading control (right). (F) MCF7/pDR-GFP cells were treated as in E left or E right, then the relative I-SceI-induced HR was analyzed by flow cytometry (left and right). Each data point in E and F was from three independent experiments (mean ± SD).P-values were calculated by Student's t-test (*P<0.01).

Fig. 2

p53 inhibits over-recruitment of Rad51 to DNA breaks. MCF7/E6 and MCF7/LXSN cells were fixed at 8 h after 10 Gy irradiation and stained with Rad51 antibody (A, B, C). DAPI was used for nuclear staining (A). (A) Representative example of a cell containing Rad51 foci formation. (B) The percentage of cells with Rad51 foci formation. (C) The percentage of cells with different patterns divided by the number of foci. (D) The dynamics of Rad51 foci formation in MCF7 cells with and without p53sh 3′UTR treatment over an 8 h period after 10 Gy irradiation. The percentage of the cells with more than 5 nuclear foci was calculated, at least 300 cells were counted per data point, each data point was from three independent experiments (mean ± SD). P-values were calculated by Student's t-test (**P< 0.05).

Fig. 3

p53 inhibits BRCA1 over-function at protein and transcription level. (A) The knockdown of BRCA1: MCF7 cells were infected by BRCA1sh, and at 48 h after infection the cells were used for western blotting and cell cycle profiling analysis. β-actin was used as loading control. (B) The effect of p53 on BRCA1 protein levels. MCF7/E6 and MCF7/LXSN cell lines were infected with BRCA1sh or consh, at 48 h after infection, then BRCA1 and p53 protein expression in the cells were detected by western blotting. (C) H1299 cells were transfected with constructs of wtp53 or pcDNA3 as vector control, after 48 h-transfection, p53, p21 and BRCA1 were detected by western blotting. Under the same conditions, the cell cycle profiles were analyzed by flow cytometry. MCF7 cells were used as positive control for p53 expression. (D) MCF7 cells were treated as in Fig. 1E right, then BRCA1 and p53 protein expression in the cells were detected by western blotting. (E) p53 inhibits over-foci formation of BRCA1 in response to DNA damage. MCF7/E6 and MCF7/LXSN cells were treated as in Fig. 2 (A, B, C), and then cells were stained for BRCA1 (upper left and right, lower left). DAPI was used for nuclear staining (upper left). Upper left: the pictures displayed the cells containing BRCA1 foci formation. Upper right: the percentage of cells with BRCA1 foci formation. Lower left: the percentage of the cells with different patterns divided by foci number. Lower right: the dynamics of BRCA1 foci formation in MCF7 cells with and without p53sh 3′UTR treatment over an 8 h period after 10 Gy irradiation. The percentage of cells with more than 5 nuclear foci was calculated. In each experiment, at least 300 nuclei were counted per data point, each data point was from three independent experiments (mean±SD). P-values were calculated by Student's t-test (**P<0.05). (F) H1299 cells were transfected with the constructs of wtp53 or vector control, after 48 h-transfection, protein lysates were used for co-immunoprecipitation experiment. p53 antibody was employed to pull down the protein complex, then co-precipitated BRCA1 expression was detected in the complex. (G) MCF7 cells were treated as in Fig. 1E right. Relative mRNA expression of BRCA1 and p53 in MCF7 cells was analyzed by RT-qPCR normalized against GAPDH. Each data point was from three independent experiments (mean±SD). P-values were calculated by Student's t-test (*P<0.01).

Fig. 4

Disruption of p53 rescues HR in BRCA1-defective cells. MCF7/pDR-GFP cells expressing E6 or LXSN were infected with BRCA1sh, at 48 h after infection, the cells were analyzed for detecting relative recombination. (A) Relative SR in indicated cells. (B) Relative I-sceI-induced HR in the cells. Each data point was from three independent experiments (mean ± SD). P-values were calculated by Student's t-test (*P<0.01).

Fig. 5

Effect of p53 on IR sensitivity. Cytotoxicity was assayed following different doses of IR:0, 2, 4, 6 Gy. MCF7/E6 and MCF7/LXSN cells were treated with BRCA1sh as in Fig.4, then the cells were further irradiated by different doses for clonogenic assay. Each data point was from three independent experiments with standard deviations (mean ± SD). P-values were calculated by Student's t-test (*P< 0.01, ***P >0.05).

Fig. 6

The effect of p53 on chromosomal stability. MCF7/E6 and MCF7/LXSN cells were irradiated by 2 Gy, and the frequencies of spontaneous and IR-induced chromosomal aberrations were analyzed at 24 h after IR treatment. Fluorescence in situ hybridization using telomeric probe is indicated in red. Fifty metaphases for each sample were scored. Chromatid-type (A), chromosome (B) or radial structure (C) aberrations, respectively, were represented. FISH using telomeric probe reveals the pink color. Chromosomes were stained with DAPI (blue). Each data point was from three independent experiments (mean ± SD). P-values were calculated by Student's t-test (*P<0.01, ***P>0.05).

Fig. 7

Proposed model for the role of association between p53 and BRCA1 in the regulation of HR activity.