Figure 1.
Venn diagram illustrating genes down regulated in the absence of the p53 family members.
Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple).
Figure 2.
Heatmap showing unsupervised hierarchical clustering analysis of p53, p63, and p73−/− E1A MEFs after doxorubicin treatment.
Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73-deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap.
Table 1.
p53 family response elements assayed by ChIP.
Figure 3.
Genes involved in DNA repair are differentially expressed in MEFs deficient for p63 and/or p73.
Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/−, p63−/−, p73−/− and p63−/−;p73−/−) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001.
Figure 4.
Low expression of BRCA2 and Rad51 in cells and mammary tumors deficient for p63 and p73.
(A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody.
Figure 5.
p63 and/or p73 bind to intronic regions within genes involved in DNA repair.
Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members (p53−/−, p63−/− and p73−/−) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate.
Figure 6.
ΔN isoforms of p63 and p73 transactivate Rad51 and BRCA2 luciferase reporter genes.
Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM.
Figure 7.
Cells deficient for p63, p73, or both p63 and p73 have an impaired ability to repair damaged DNA and exhibit increased sensitivity to ionizing radiation.
(A–D) DNA damage (tail moment) detected by the Comet assay at 0 (untreated), 1, and 16 hours in E1A MEFs treated with (A) 5 Gy γ radiation, (B) 0.34 µM doxorubicin or primary MEFs treated with (C) 5 Gy γ radiation, and (D) 0.34 µM doxorubicin. Genotypes are indicated on the x-axis and tail moment is shown on the Y-axis. Three independent MEF lines were assayed for each genotype in triplicate. Asterisks indicate statistical significance compared to wild-type (p <0.0001). (E,F) Clonogenic survival of E1A MEFs of the indicated genotypes following (E) gamma radiation and (F) doxorubicin. (G,H) Clonogenic survival of primary MEFs of the indicated genotypes following (G) gamma radiation and (H) doxorubicin. Percent (%) survival is indicated on the Y-axis for each dose of gamma-irradiation (0, 1, 2, 3 Gy) or doxorubicin (0.34, 0.5, 1 µM) on the x-axis. Three independent MEF lines were assayed for each genotype in triplicate. Data are represented as the mean ± SEM.