p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage

Abstract

p53-mediated transcription activity is essential for cell cycle arrest, but its importance for apoptosis remains controversial. To address this question, we employed homologous recombination and LoxP/Cre-mediated deletion to produce mutant murine embryonic stem (ES) cells that express p53 with Gln and Ser in place of Leu25 and Trp26, respectively. p53(Gln25Ser26) was stable but did not accumulate after DNA damage; the expression of p21/Waf1 and PERP was not induced, and p53-dependent repression of MAP4 expression was abolished. Therefore, p53(Gln25Ser26) is completely deficient in transcriptional activation and repression activities. After DNA damage by UV radiation, p53(Gln25Ser26) was phosphorylated at Ser18 but was not acetylated at C-terminal sites, and its DNA binding activity did not increase, further supporting a role for p53 acetylation in the activation of sequence-specific DNA binding activity. Most importantly, p53(Gln25Ser26) mouse thymocytes and ES cells, like p53(-/-) cells, did not undergo DNA damage-induced apoptosis. We conclude that the transcriptional activities of p53 are required for p53-dependent apoptosis.

Fig. 1. Construction of p53^Gln25Ser26 ES cells. (A) The mouse germline p53 locus. Blank boxes represent the p53 exons and the two filled bars represent the two probes (A and B) used to detect the wild-type and mutant p53 alleles by Southern blot analysis. The germline 14 kb EcoRI and 5.6 kb BamHI fragments are indicated. (B) The targeting construct. The position of the mutations encoding Gln25 and Ser26 in place of Leu25 and Trp26 in exon 2 of the p53 gene is indicated by an asterisk. The PGK-Neo^r gene flanked by LoxP sites was inserted into an engineered SalI site within intron 4. (C) Targeted p53 locus. The sizes of the mutant EcoRI and BamHI fragments are indicated. The positions of the PCR primer sites that were used to screen for deletion of the PGK-Neo^r segment are indicated by arrowheads. (D) Mutant p53 allele with the PGK-Neo^r gene segment deleted. The size of the mutant BamHI fragment after deletion of the PGK-Neo^r gene is indicated; arrows show the new positions of the PCR primer sites. (E) Southern blot analysis of genomic DNA derived from wild-type (lane 1), heterozygous p53^Gln25Ser26 mutant (lanes 2 and 3) and homozygous mutant (lanes 4 and 5) ES cells with the PGK-Neo^r gene inserted. Genomic DNA was digested with EcoRI and hybridized with probe A. The positions of the EcoRI restriction fragments from the germline alleles are indicated by an arrow. (F) Southern blotting analysis of genomic DNA derived from wild-type (lane 1), homozygous mutant ES cells with the PGK-Neo^r gene inserted (lane 2), and p53^Gln25Ser26 ES cells with the PGK-Neo^r gene deleted (lanes 3 and 4). Genomic DNA was digested with BamHI and hybridized with probe B. The positions of the BamHI fragments derived from wild-type and mutant alleles after deletion of the PGK-Neo^r gene and of the mutant allele with the PGK-Neo^r gene inserted are indicated with arrows.

Fig. 2. Induction of p53 and p53-dependent gene expression in wild-type and p53^Gln25Ser26 cells following DNA damage. Cell extracts were prepared from wild-type and p53^Gln25Ser26 ES cells at the time points indicated after exposure to (A) 5 Gy γ-irradiation or (B) 60 J/m² UV-C light, and the samples were processed for western immunoblot analysis as described in Materials and methods. The genotypes and time points are labeled on the top of the lane. p53 and actin are indicated on the right. (C) p21 protein induction in wild-type, p53^Gln25Ser26 and p53^–/– differentiated ES cells following 60 J/m² UV treatment. The positions of p21 and actin are indicated by arrows. (D) PERP mRNA induction in wild-type, p53^Gln25Ser26 and p53^–/– differentiated ES cells following 60 J/m² UV treatment. The positions of PERP and GAPDH mRNA are indicated by arrows. (E) Repression of MAP4 expression in wild-type, p53^Gln25Ser26 and p53^–/– ES cells following 60 J/m² UV radiation. The positions of MAP4 protein and actin are indicated with arrows. The genotypes and times after DNA damage are given at the top of each panel.

Fig. 3. Cellular localization and DNA binding of p53^Gln25Ser26. Indirect immunofluorescence: wild-type (A) and p53^Gln25Ser26 (B) differentiated ES cells were fixed and stained with PAb421 for p53 (left panels) and DAPI for DNA (right panels) as described in Materials and methods. (C) EMSA: nuclear extracts were prepared from wild-type, p53^Gln25Ser26 and p53^–/– differentiated ES cells before or 4 h after exposure to 60 J/m² UV light. EMSA was performed using a ³²P-labeled, double-stranded p53 consensus binding sequence as described in Materials and methods. Shown is an autoradiogram of the polyacrylamide gel. The lanes correspond to the nuclear p53-specific DNA binding activity from: 1, p53^–/– differentiated ES cells; 2, wild-type differentiated ES cells with no treatment; 3, wild-type differentiated ES cells 4 h after exposure to UV; 4, differentiated p53^Gln25Ser26 ES cells with no UV treatment; 5, differentiated p53^Gln25Ser26 ES cells 4 h after exposure to UV. The positions of supershifted (SS) as well as specific (S) p53-specific DNA complexes are indicated. PAb421 antibody against p53 was used to supershift the p53 complexes.

Fig. 4. UV-induced phosphorylation and acetylation of p53 in wild-type and p53^Gln25Ser26 cells. (A) Western immunoblot analysis showing the phosphorylation of mouse p53 at Ser18 in wild-type and p53^Gln25Ser26 ES cells before and 2 or 4 h after exposure to 60 J/m² UV light. The immunoblot was probed with affinity purified antibodies specific for murine p53 phosphorylated at Ser18 (p53-Ser18P, top strip); the blot was then stripped and probed with PAb240 to detect the total p53 signal (bottom strip). (B) Western immunoblot showing the acetylation of mouse p53 at Lys317 and Lys379 (corresponding to human Lys320 and Lys382) in differentiated wild-type and p53^Gln25Ser26 ES cells before and 18 or 24 h after exposure to 60 J/m² UV light. The blot was probed with affinity purified antibodies specific for mouse p53 acetylated at Lys317 (corresponding to Lys320 of human p53; top strip), stripped and reprobed with antibodies specific for mouse p53 acetylated at Lys379 (corresponding to Lys382 of human p53; central strip), and finally stripped and reprobed with PAb240. The genotypes are given above the panels, the positions of p53 are indicated with arrows.

Fig. 5. Induction of apoptosis in wild-type, p53^–/– and p53^Gln25Ser26 ES cells by UV treatment. (A) Flow cytometric analysis of wild-type, p53^–/– and p53^Gln25Ser26 ES cells harvested 12 h after exposure to 60 J/m² UV irradiation. Cell number is plotted as a function of the intensity of staining for annexin V; cells stained positive with annexin V antibodies are apoptotic. The percentages of non-apoptotic cells are indicated. (B) The percentile ratio of non-apoptotic cells in irradiated wild-type, p53^Gln25Ser26 and p53^–/– ES cells relative to non-apoptotic cells in unirradiated controls. The mean and standard deviation from three independent experiments is given.

Fig. 6. Induction of apoptosis in wild-type, p53^Gln25Ser26 and p53^–/– thymocytes by ionizing radiation. (A) Thymocytes were harvested from wild-type, p53^–/– mice and p53^Gln25Ser26–RAG2^–/– chimeric mice, stained for CD4 and CD8, and analyzed by flow cytometry as described in Materials and methods. Cells residing in the lymphocyte gate were analyzed and the percentage of total cells in a particular gate is indicated. (B) The mean value of the percentile ratio of non-apoptotic CD4⁺ thymocyte number in wild-type, p53^Gln25Ser26 and p53^–/– thymocytes treated with 5, 10 and 20 Gy of IR to the non-apoptotic thymocyte number from untreated controls from three independent experiments is given. Error bars show the standard deviation.