Essential roles of Jab1 in cell survival, spontaneous DNA damage and DNA repair

Abstract

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 overexpression has been implicated in the pathogenesis of human cancer. JAB1 regulates several key proteins and thereby produces varied effects on cell cycle progression, genome stability and cell survival. However, the biological significance of JAB1 activity in these cellular signaling pathways is unclear. Therefore, we developed mice that were deficient in Jab1 and analyzed the null embryos and heterozygous cells. This disruption of Jab1 in mice resulted in early embryonic lethality due to accelerated apoptosis. Loss of Jab1 expression sensitized both mouse primary embryonic fibroblasts and osteosarcoma cells to γ-radiation-induced apoptosis, with an increase in spontaneous DNA damage and homologous recombination (HR) defects, both of which correlated with reduced levels of the DNA repair protein Rad51 and elevated levels of p53. Furthermore, the accumulated p53 directly binds to Rad51 promoter, inhibits its activity and represents a major mechanism underlying the HR repair defect in Jab1-deficient cells. These results indicate that Jab1 is essential for efficient DNA repair and mechanistically link Jab1 to the maintenance of genome integrity and to cell survival.

Figure 1

Jab1^+/+ and Jab1^−/− embryos at E6.5. (a) Expression of JAB1 and other related targets were analyzed by immunohistochemical staining with antibodies to JAB1 (a and b), p27 (c and d), c-Jun (e and f), p53 (g and h), and c-Myc (i and j) at E6.5. Black and blue arrows point to positive staining of the embryonic cells and the surrounding cells from the uterus, respectively. (b) Analysis of apoptosis by TUNEL assay in E6.5 embryos (+/+) and (−/−). Only the magnified embryonic portions are shown.

Figure 2

Jab1^−/− blastocysts failed in outgrowth and display apoptotic activity. (a) Wild-type (Jab1^+/+) and Jab1^−/− E3.5 blastocysts were cultured in vitro and photographed daily, starting from day 1 to day 5 after isolation. (b) TUNEL of Jab1^+/+ and Jab1^−/− cultured blastocysts at 96 hours. (c) Bromodeoxyuridine (BrdU) incorporation during blastocyst outgrowth was detected by immunohistochemical staining (green); nuclei were counterstained with DAPI.

Figure 3

Loss of Jab1 expression results in spontaneous DNA breaks. (a) Immunostaining of MEFs for the specific phosphohistone H2AX (γ-H2AX) before (Control) and after 30 minutes of IR (10 Gy) exposure. Quantification of the number of γ-H2AX foci in 100 cells is indicated. (b) U2OS cells were transfected with Jab1 or control siRNA before IR exposure and immunostained for γ-H2AX foci 24 hours after IR exposure. Nuclei were visualized using DAPI staining. (c) Cell extracts were prepared from Jab1^+/+ and Jab1^+/− MEFs and U2OS cells transfected with siRNA. Left, The levels of phospho-p53 (Ser15), total p53, p21, and JAB1 expression were analyzed by Western blotting. Right, Quantification. The protein levels were quantified using Image software.

Figure 4

Jab1-depletion impairs DNA DSB repair and increases sensitivity of cells to DNA damage stimuli. (a) U2OS cells were transfected with siRNA before IR exposure (control), collected at the indicated time points after IR treatment, and analyzed for the presence of unrepaired DNA lesions using the comet assay. Left, SYBR staining of DNA showed comet tails migrating out of the nuclei of U2OS cells. Right, Quantification of the fluorescence intensity of the cells is shown as the percentage of cells with intact DNA. (b) HR repair assay. DR-GFP cells were treated with siRNA for 24 hours and then transfected with I-SceI-expression plasmid (I) or empty vector (E). Top, DSB repair was indicated by the percentage of cells expressing GFP by flow cytometric analysis. Data were represented of three independent experiments, mean ± SD. Bottom, Western blot analyses to demonstrate the effective Jab1 knockdown levels. (c) U2OS cells were treated with siRNA before IR exposure; the colonies were stained with crystal violet. (d) Jab1^+/+ (WT) and Jab1^+/− (Mut) MEFs were exposed to IR or UV radiation and assessed for the extent of annexin V staining 24 hours later. * (p<0.05), ** (p<0.01).

Figure 5

Knockdown Jab1 decreases expression of the DNA repair gene Rad51. (a) U2OS cells were transfected with Jab1 or control siRNA 48 hours before IR exposure. Cell lysates were prepared at 5 and 24 hours after IR exposure and then blotted with the indicated antibodies related to DNA repair. (b) U2OS cells were transfected as described in (a) and stained with anti-Rad51 antibodies 5 hours after IR exposure. (c) Rad51 and Jab1 mRNA expression in U2OS cells was detected with use of RT-PCR before (control) and 24 hours after IR exposure. (d) DR-GFP cells were treated with siRNA for 24 hours and then co-transfected with I-SceI-expression plasmid or empty vector, with or without HA-Rad51 plasmid DNA. Left, DSB repair was indicated by the percentage of cells expressing GFP. Right, Western blot analyses to demonstrate the effective Jab1 knockdown and ectopic Rad51 expression. (e) U2OS cells were transfected with siRNA with or without HA-Rad51 plasmid DNA; the colonies were stained with crystal violet after IR exposure 12 days later.

Figure 6

Inhibition of Jab1 suppresses Rad51 promoter activities in a p53-dependent manner. (a) U2OS and Saos-2 cells were treated with siRNA 48 hours before transfection with luciferase reporter plasmid - 403pRad-Luc or 50pRad51-Luc. Luciferase activity assays were performed, and normalized according to Renilla luciferase activity. (b) Saos-2 cells were co-transfected with −403pRad51-Luc plasmids and increasing amounts of pcDNA-p53 or pcDNA vector plasmids (0, 4, 20, or 100 ng). The histograms show relative luciferase activity (RLU) normalized to the siRNA control. Error bars from the data (a and b) of the three independent experiments are shown. (c) U2OS and Saos-2 cells were transfected with siRNA 48 hours before IR exposure. Cell lysates were prepared before and 5 hours after IR, and the levels of Jab1, Rad51, and the phosphorylation of p53 (Ser-15) were determined by Western blotting. (d) Luciferase assays with PG13-luc, MG15-luc reporter in U2OS cells that were transfected with control, Jab1, or p53 siRNAs, respectively. (e) Cell lysates were analyzed by Western blotting (top) and RT-PCR (bottom) for p53, Jab1, Rad51, and β-actin or GAPDH. (f) ChIP assay was carried out after IR; PCR products from Rad51 promoter regions containing p53-binding element were shown on top. The bottom represented quantifications of the data with Image software.