Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 ...

MOLECULAR AND CELLULAR BIOLOGY, Sept. 2006, p. 6859–6869 0270-7306/06/$08.00⫹0 doi:10.1128/MCB.00062-06 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 26, No. 18

Acetylation of Mouse p53 at Lysine 317 Negatively Regulates p53 Apoptotic Activities after DNA Damage† Connie Chao,1 Zhiqun Wu,1 Sharlyn J. Mazur,2 Helena Borges,1 Matteo Rossi,2 Tongxiang Lin,1 Jean Y. J. Wang,1 Carl W. Anderson,3 Ettore Appella,2 and Yang Xu1* Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-03221; Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 208922; and Biology Department, Brookhaven National Laboratory, Upton, New York 119733 Received 11 January 2006/Returned for modification 7 February 2006/Accepted 3 July 2006

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53K317R mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53K317R MEFs after various types of DNA damage, increased p53dependent apoptosis was observed in p53K317R thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53K317R thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage. ent sites, primarily through the phosphorylation of serine or threonine residues and the acetylation of lysines. Most sites of posttranslational modification reside in the amino- or carboxyterminal segments of the p53 polypeptide, both of which are predicted to be largely unstructured when not associated with other proteins (3). Both regions of p53 interact with a large number of cellular proteins that may modulate p53 stability and activity, thereby differentially regulating the expression of p53 target genes that control cell cycle progression, apoptosis, or senescence (28). In response to DNA damage and other cellular stresses, the protein levels and the activity of p53 as a transcription factor are greatly increased (29). The interaction between p53 and MDM2, Pirh2, or COP1, each of which is a transcriptional target of p53, leads to the ubiquitination of p53 and its rapid degradation (5). In this context, MDM2, Pirh2, and COP1 each act as a p53-specific E3 ubiquitin ligase, thus functioning in feedback systems that negatively regulate p53 stability (5). In addition, the interaction between Mdm2 or MdmX and the N terminus of p53 inhibits p53 transcriptional activities (39). Human p53 becomes acetylated at multiple lysine residues in its C terminus in response to various stresses. Six of these, Lys305, -370, -372, -373, -381, and -382, can be acetylated in vitro by the histone acetyltransferase p300 or CBP, which are closely related enzymes that function as coactivators for a number of transcription factors (19, 50). These studies suggest that these acetylation events activate p53 transcriptional activities. Some or all of these C-terminal lysine residues can also be ubiquitinated by Mdm2 and modified by other mechanisms, such as neddylation and sumoylation (5, 13, 54). Cell line transfection experiments have shown that the Mdm2-mediated ubiquitination of the C-terminal lysine residues is important for p53 destabilization (33, 44, 49). However, by mutating the

The p53 gene is the most commonly mutated tumor suppressor gene in human cancers (22). p53 plays multiple roles in cellular responses to various stresses, including the induction of cell cycle arrest, senescence, differentiation, and apoptosis (29). p53 functions as a transcription factor and has a sequence-specific DNA-binding domain in its central region, a transcriptional activation domain at its amino (N) terminus, and a tetramerization domain at its carboxyl (C) terminus (29). p53 can directly activate the expression of a large panel of genes, including Cdkn1a (p21), which is a universal inhibitor of cyclin-dependent kinases and plays a major role in mediating p53-dependent cell cycle G1 arrest (6, 14, 15). In addition, p53 also activates the expression of a number of proapoptotic genes, including the Puma, Noxa, Perp, Pidd, and Killer 5 genes (45). p53 can also directly suppress the expression of genes, including those encoding MAP4, Survivin, and Nanog, contributing to apoptosis and cellular differentiation (34, 43). The importance of p53-dependent transcription in p53-dependent apoptosis has been underscored by the findings that Puma is critical for mediating p53-dependent apoptosis after DNA damage (25, 52). In addition, a transcription-inactive p53 mutant fails to induce p53-dependent apoptosis after DNA damage (11, 26, 27). Recent studies also indicate that p53 might induce apoptosis in a transcription-independent manner (41). Posttranslational modifications of p53 play important roles in regulating p53 stability and activity (1, 55). The p53 protein can be posttranslationally modified at approximately 30 differ* Corresponding author. Mailing address: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322. Phone: (858) 822-1084. Fax: (858) 534-0053. E-mail: [email protected]. † Supplemental material for this article may be found at http://mcb .asm.org/. 6859

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endogenous mouse p53 gene to change these C-terminal lysines to arginines, recent knock-in studies indicate that the posttranslational modifications at these lysine residues are not important for regulating p53 stability and only fine-tune p53 activity (17, 30). Lys320 of human p53 is acetylated in vitro by PCAF, the p300/CBP-associated factor (36, 50); this site is also acetylated in vivo following DNA damage. The corresponding lysine residue of mouse p53 is also acetylated in vivo after DNA damage (10). PCAF is a component of several large chromatin remodeling complexes, e.g., SAGA, that include proteins important for p53-mediated transcription and apoptosis, such as hADA2 and hADA3 (8, 53). Based on several reports that Lys320 acetylation is correlated with increased sequence-specific DNA binding and transcriptional activation, it has been thought that this acetylation positively regulates p53 responses (2, 50, 51). However, other experiments failed to show a significant requirement for acetylation of the human K320 site for the p53dependent transcriptional activation of reporter genes in transient transfection assays (36). To determine the physiological roles of this acetylation in regulating p53 responses to DNA damage, a missense Lys317 (corresponding to human Lys320)to-Arg mutation was introduced into both alleles of the endogenous p53 gene of mice. Analysis of the p53-dependent responses to DNA damage in p53K317R mice indicated that this acetylation negatively regulates p53 apoptotic responses to DNA damage. MATERIALS AND METHODS Culture and treatment of MEFs and thymocytes. Mouse embryonic fibroblasts (MEFs) were recovered from day E12 embryos and were cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum, penicillin-streptomycin, and ␤-mercaptoethanol as described previously (9). For the analysis of p53-mediated responses after DNA damage, MEFs were treated with UVC light (60 J/m2), ionizing radiation (IR) (20 Gy), or doxorubicin (0.25 ␮M) and harvested at the indicated times after treatment. Thymocytes were recovered from 4- to 6-week-old mice and resuspended in 5% serum and 25 mM HEPES in DMEM prior to being exposed to various dosages of IR. For p53-dependent apoptosis, thymocytes were harvested at 10 h following irradiation, stained with annexin V (BD Biosciences/Pharmingen) to detect apoptotic cells, and analyzed by flow cytometry as previously described (9). Western immunoblot analysis. Cell extracts were prepared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer, and the proteins from 5 ⫻ 105 MEFs or 107 thymocytes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% (for Mdm2), 10% (for p53), or 12% (for p21Waf1) polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were probed with a rabbit polyclonal antibody against p21 (Santa Cruz Biotechnology, Inc.), a polyclonal antibody against p53 (CM-5; Novocastra Laboratories Ltd.), a polyclonal antibody against mouse acetyl-K317 p53 (Trevigen Inc.), or a monoclonal antibody against Mdm2 (2A10; Calbiochem/Oncogene Sciences). Filters were subsequently incubated with horseradish peroxideconjugated secondary antibody and developed with ECL Plus (Amersham Biosciences, Inc.). The anti-acetyl-K317 p53 immunoblot was developed using an IR dye-tagged secondary antibody and scanned using the Odyssey imager (LI-COR Biosciences). To control for loading, filters were stripped and probed with a rabbit polyclonal antibody against actin (Santa Cruz Biotechnology, Inc.). p53-Mdm2 coimmunoprecipitation. MEF cells were lysed in TNEP buffer (20 mM Tris, pH 7.6, 170 mM NaCl, 1 mM EDTA, 0.5% NP-40, 10 mM NaF, 0.2 mM Na3VO4 with protease inhibitors) and centrifuged. Protein lysate (1 mg) was immunoprecipitated with a polyclonal antibody against p53 that was conjugated to agarose beads (FL-393AC; Santa Cruz Biotechnology, Inc.). The immune complexes were rinsed three times with NETN (20 mM Tris-HCl, pH 7.7, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) and analyzed by Western blotting. Cell cycle analysis. MEFs were seeded at 1 million cells/10-cm plate and synchronized at G1/G0 for 96 h in DMEM containing 0.1% fetal bovine serum. The cultures were released from the cell cycle block by transfer to normal

MOL. CELL. BIOL. DMEM supplemented with 10% fetal calf serum. Untreated MEFs or MEFs treated with 45 J/m2 UVC were continuously labeled with BrdU (10 ␮M; SigmaAldrich Co.) for 24 h and harvested by being fixed overnight in 70% ethanol. The S-phase cells were revealed by staining with anti-BrdU antibody, followed by flow cytometric analysis as described previously (9). The G1/G0- and G2/M-phase cells were revealed by staining with propidium iodide. Analysis of p53-dependent apoptosis in mouse tissues. Mice were handled according to guidelines from the UCSD Animal Subjects Committee. Thymocytes were recovered from 4- to 6-week-old mice, and single-cell suspensions were exposed to various dosages of IR. The apoptotic cells were analyzed 10 h following IR by staining with annexin V (BD Biosciences/Pharmingen). To analyze p53-dependent apoptosis in the mouse small intestine, 2-month-old mice were irradiated with 8 Gy of IR, and the irradiated animals were sacrificed 5 h later. Their small intestines were fixed overnight in 10% formalin, embedded in paraffin, and then sectioned. The apoptotic cells in sections were revealed by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining by using a Klenow FragEL kit (Calbiochem/Oncogene Science) as recommended by the manufacturer. Sections were examined by fluorescence microscopy. Retinal apoptosis assays were performed as described previously (4). Briefly, all experiments were performed using postnatal day 3 (P3) littermate pups (day 0 is the day of birth). P3 mice were exposed to 2 Gy whole-body irradiation using a 137Cs irradiator. Mice were kept warm and sacrificed at 6 h following irradiation. The eyes were removed and immersed in fixation buffer (4% paraformaldehyde in phosphate-buffered saline, pH 7.4); the tails were used for DNA extraction and genotyping. Following overnight fixation, the eyes were infiltrated with 20% sucrose in phosphate-buffered saline and embedded in tissue-embedding medium, OCT (Tissue-Tek; Sakura). Transverse frozen sections were processed for the TUNEL assay according to the manufacturer’s protocol (Roche) and then counterstained with 1 ␮g/ml bisbenzimide (Hoechst-332558; SigmaAldrich Co.). Quantification of retinal apoptotic cells was realized in the inner nuclear layer (INL) and neuroblastic layer (NBL) under the light microscope (magnification, ⫻400) using a grid coupled to the eyepiece. TUNEL-positive nuclei were counted in two to three fields (field ⬇ 0.04 mm2) per animal. Histograms show the mean and standard error of the mean from four to five animals per genotype. Statistic analysis was realized using Prisma 4 (GraphPad Software Inc.). Quantitative real-time PCR analysis. Total RNA was purified from MEFs and thymocytes using TRIzol (Invitrogen), followed by a further purification using the RNeasy mini kit (QIAGEN Inc.). RNA samples were treated with RNasefree DNase I (QIAGEN Inc.) and converted to cDNA using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was performed on an ABI 7000 machine (Applied Biosystems) using a 2⫻ SYBR green PCR master mix (Applied Biosystems). PCR conditions were as follows: 10-min hot start at 95°C, followed by 40 cycles of 30 s at 95°C and 1 min at 61°C. Reactions were run in triplicate wells, and the average cycle threshold for each gene was calculated as previously described (9); mRNA levels of each gene were standardized to the mRNA levels of GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Primers for p21, Mdm2, and Noxa have been described previously (9). The primers for Lrdd (Pidd) were 5⬘-CTGGTTCCTGGTCGTTTTACG-3⬘ and 5⬘-GGATGAC CTGAGGAGCAGAGA-3⬘. Promoter luciferase assay. E1A/Ras-expressing MEFs were seeded onto the 24-well plate at a density of 200,000 cells per well. Twenty-four hours after plating, 50 ng of p21-Luc (48) or Pidd-Luc (35) construct was cotransfected with 4 ng of thymidine kinase promoter-Renilla luciferase vector using HyFect transfection reagent according to the manufacturer’s instructions (Denville Scientific Inc.). After transfection, cells were cultured in normal MEF medium with or without 1 ␮M doxorubicin for 6 h. The luciferase activity was determined with the Dual-Luciferase reporter assay system (Promega Corporation) as previously described (34). Microarray analysis of p53-regulated genes. Thymocytes from wild-type or p53K317R mice were cultured as described above and either left untreated or exposed to 5 Gy IR. After 5 h, total RNA was isolated as described above. Total RNA (1 ␮g) was converted to cRNA, linearly amplified, and fluorescently labeled using a low-input RNA linear amplification kit (Agilent Technologies, Inc.) and Cy3- or Cy5-labeled CTP (PerkinElmer) following the manufacturer’s directions. Untreated (Cy3 labeled, 750 ng) and IR-treated (Cy5 labeled, 750 ng) samples were combined and hybridized to a microarray (Agilent mouse oligonucleotide array, ⬃22,000 features) for 17 h at 60°C. Following low-stringency and high-stringency washes, microarray slides were dried and stored under nitrogen in the dark. Hybridization signals (Agilent microarray scanner) were background corrected and Lowess normalized (Agilent Feature Extraction software). Results from duplicate arrays for each of two independent wild-type or

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CHARACTERIZATION OF p53 K317R KNOCK-IN MOUSE MUTANT

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FIG. 1. Construction of p53K317R mice. (A) The endogenous mouse p53 locus. Open boxes represent the p53 exons, and filled boxes represent the two probes used to detect the wild-type and p53K317R alleles by Southern blot analysis. The sizes of the germ line EcoRI and HindIII fragments are indicated. (B) Targeting construct. The PGK-neor gene flanked by two LoxP sites was inserted into an engineered SalI site within intron 4. The asterisk represents the K317R mutation. (C) Targeted p53 locus. The positions of the PCR primers used to screen for deletion of the PGK-neor gene are indicated by arrowheads. The size of the mutant EcoRI fragment is indicated. (D) Knock-in allele after Cre-mediated deletion of the PGK-neor gene. The size of the mutant HindIII fragment after deletion of the PGK-neor gene is indicated. (E) Southern blot analysis of genomic DNA derived from the tails of a wild-type (lane 1) and a heterozygous mutant p53K317R (lane 2) mouse with the PGK-neor gene inserted. Genomic DNA was digested with EcoRI and hybridized with probe A; the wild-type (Wt) and mutant bands are indicated at the right. (F) Genomic DNA derived from the tail of wild-type (lane 1), heterozygous mutant p53K317R (lane 2) and homozygous mutant p53K317R (lane 3) mice with the PGK-neor gene deleted. Genomic DNA was digested with HindIII and hybridized with probe B; the fragments derived from the wild-type allele and the knock-in allele are indicated at the right. (G) Acetylation of mouse p53 at K317 in E1A/Ras-expressing wild-type and p53K317R MEFs 6 h after doxorubicin treatment (125 ng/ml). The MEFs were treated with trichostatin A for 4 hours prior to harvest as previously described (9).

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FIG. 2. p53 stability and activity in p53K317R MEFs in response to DNA damage. p53 protein levels at the indicated times following UV irradiation with 60 J/m2 (A), exposure to 5 Gy IR (B), or treatment with 0.25 ␮M doxorubicin (C) are shown. Genotypes and time points after treatment are indicated at the top; p53 and actin are indicated at the right. (D) Interaction of Mdm2 and p53 in wild-type (Wt) and p53K317R MEFs by coimmunoprecipitation. MEFs were harvested at different times after exposure to 60 J/m2 UV light. Unirradiated samples were treated with the proteasome inhibitor ALLN at 50 ␮M for 4 h prior to harvesting to ensure detectable levels of p53. The whole-cell extract was immunoprecipitated with a polyclonal antibody against p53 conjugated to agarose beads, and the amount of p53 and Mdm2 in the immunoprecipitate was determined by Western blotting. Genotypes and time points are indicated at the top; p53 and Mdm2 are indicated at the right.

K317R thymocyte preparations for a total of eight arrays were analyzed collectively. Signals with a geometric mean normalized intensity of greater than 0.15 were analyzed further. Genes responsive to IR were identified by applying the Student t test and selecting those with a P value of less than 0.001. Genes for which the IR-induced level was significantly affected by the K317R mutation were identified by applying the Student t test and selecting those with P values of less than 0.05.

RESULTS K317R

Construction of p53 mice. Lys317 is encoded within exon 9 of the mouse p53 gene. The strategy to introduce the K317R mutation (AAA to AGA) was the same as that used previously to introduce phosphorylation site mutations into the endogenous p53 gene in mice (10). Briefly, site-directed mutagenesis was performed to introduce the K317R mutation into a mouse p53 genomic DNA fragment harboring exons 2 through 10. The targeting vector was constructed by inserting the LoxP-flanked PGK-neor gene into an engineered, unique SalI site within intron 4. A HindIII site within intron 9 was mutated to assist in screening for the replacement of the wildtype exon 9 by the targeting construct. Homologous recombination events between the wild-type p53 allele and the targeting vector were detected by Southern blot analysis after EcoRI digestion of the genomic DNA and hybridization with probe A, giving a 15-kb germ line band and a 6-kbp mutant band (Fig. 1A, B, and E). The heterozygous mutant embryonic stem cells were used to generate chimeric mice that transmitted the targeted allele into the mouse germ line. To delete the PGK-neor gene from the targeted allele, germ line mutant mice were bred with cytomegalovirus-Cre transgenic mice. Cre expression caused excision of the PGK-neor gene from the targeted allele, and the resulting mice with the neor gene deletion were identified by PCR and confirmed by Southern blot analysis after HindIII digestion and hybridization with probe B, which revealed a 3-kbp fragment from the wild-type allele and a 10-kbp fragment from the PGK-neor cassette-deleted allele (Fig. 1C, D, and F). The heterozygous mutant mice were intercrossed to generate homozygous mutant mice that are denoted p53K317R hereinafter. Sequence analysis of the entire coding sequence of the p53 mRNA expressed in p53K317R mouse embryonic fibro-

blasts and thymocytes confirmed that the K317R mutation but no other mutation was introduced into the p53 gene in p53K317R mice. In addition, DNA damage-induced acetylation of mouse p53 at K317 could not be detected in p53K317R MEFs after DNA damage using an antibody specific for p53 acetylated at K317 (Fig. 1G). There is no apparent difference in the life span between the wild-type and p53K317R mice. p53 stability and activity in MEFs after DNA damage. To investigate the effects of lysine 317 acetylation on p53 stabilization after DNA damage, we analyzed p53 protein levels at different times in p53K317R and control wild-type MEFs following DNA damage induced by UVC radiation, IR, or doxorubicin treatment. Our analyses indicated that p53 accumulated to normal levels in MEFs after each of these treatments (Fig. 2A to C). Consistent with these findings, the p53-Mdm2 interactions were disrupted similarly in wild-type and p53K317R MEFs after UV radiation (Fig. 2D). To test whether the K317R mutation affected p53 activity, we analyzed the p53-dependent induction of p21 and activation of the G1/S cell cycle checkpoint in p53K317R and control wildtype MEFs after UV radiation. Normal p21 induction and G1/S cell cycle arrest were observed in p53K317R MEFs, indicating that lysine 317 acetylation was not required to activate p53 in MEFs after UV radiation (Fig. 3A and B). In further support of this conclusion, the induced mRNA levels of several p53 target genes, including Cdnk1 (p21), Pidd, Noxa, and Mdm2, were similar in wild-type and p53K317R MEFs after UV radiation, IR, or doxorubicin treatment (Fig. 3C to E). p53-dependent apoptosis in thymocytes. To determine the role of lysine 317 acetylation in regulating p53 apoptotic function, p53-dependent apoptosis was analyzed in p53⫹/⫹ and p53K317R thymocytes after IR. Consistent with the findings for MEFs, p53 protein accumulated to similar levels in p53⫹/⫹ and p53K317R thymocytes after IR (Fig. 4A). However, compared with that of p53⫹/⫹ thymocytes, p53-dependent apoptosis was increased about twofold in p53K317R thymocytes, indicating higher p53-dependent apoptotic activity in p53K317R thymocytes after IR (Fig. 4B). In support of this conclusion, p53dependent expression levels of proapoptotic Noxa and Pidd

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FIG. 3. (A) Western blot showing induction of p21Waf1 in wild-type (Wt) and p53K317R MEFs at different times after exposure to 60 J/m2 UV radiation. (B) Cell cycle profiles of wild-type and p53K317R MEFs without (⫺) or 24 h after (⫹) exposure to 45 J/m2 UV radiation with continuous BrdU labeling. The percentage of cells in each phase of the cell cycle represents the mean value from three independent experiments; error bars are shown. The ratios of the mRNA levels of several p53 target genes in wild-type and p53K317R MEFs 12 h after 60 J/m2 of UV radiation (C) or 20 Gy of IR (D) or 6 h after 0.5 ␮M doxorubicin treatment (E) are shown. The mRNA levels were determined by quantitative real-time PCR and normalized to the mRNA levels of GAPDH as previously described (9). Mean values from three independent experiments are presented with error bars.

were increased in p53K317R thymocytes after IR compared with those in p53⫹/⫹ thymocytes (Fig. 4C). p53-dependent apoptosis in small intestinal epithelial cells and retina after IR. The epithelial cells of the mouse small intestine undergo p53-dependent apoptosis after IR (38). To investigate the effects of lysine 317 acetylation on p53 apoptotic activities in other tissues, we analyzed p53-dependent apoptosis of epithelial cells from the small intestine in p53K317R mice 5 h after whole-body irradiation with 8 Gy IR. Quantitative analysis of apoptosis in small intestinal epithelial cells indicated that the average number of apoptotic cells per crypt in p53K317R mice was twice that in similarly treated wild-type samples (Fig. 5A and B). Therefore, p53-dependent apoptotic activity was increased in epithelial cells of the small intestine in p53K317R mice compared to these cells in wild-type mice. Developing central nervous systems, including retinas, un-

dergo p53-dependent apoptosis after IR (4). Therefore, we also analyzed p53-dependent apoptotic responses to 2 Gy IR in the retinas of neonatal wild-type and p53K317R mice 6 h after exposure. Apoptosis in the INL and NBL of retinas was increased in neonatal p53K317R mice compared with that in the retinas of wild-type mice (Fig. 5C and D). Thus, p53-dependent apoptosis was increased in the thymocytes, small intestinal epithelial cells, and retinas of p53K317R mice compared to that in wild-type mice. p53-dependent apoptosis in E1A/Ras-expressing MEFs after DNA damage. Although MEFs generally undergo cell cycle arrest in response to DNA-damaging treatments, overexpression of the oncoproteins E1A and Ras sensitizes MEFs to p53-dependent apoptosis after DNA damage (37). Therefore, E1A/Ras-expressing p53K317R and control MEFs were generated by infection with a retroviral vector that expresses both E1A and Ras as described previously (7). Significantly more

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FIG. 4. p53 stabilization and activity in wild-type and p53K317R thymocytes after IR. (A) p53 protein levels in wild-type and p53K317R thymocytes at different times after exposure to 5 Gy IR. Genotypes are indicated at the top, and p53 and actin are indicated at the right. (B) p53-dependent apoptosis in wild-type, p53K317R, and p53⫺/⫺ thymocytes 10 h after IR treatment. The mean values from three independent experiments are shown with error bars. (C) mRNA levels of three proapoptotic p53 target genes in wild-type and p53K317R thymocytes before and 5 h after 5 Gy of IR. The mRNA levels were determined by quantitative real-time PCR and normalized to the mRNA levels of GAPDH as previously described (9). The mRNA levels in the wild-type thymocytes without IR were set to 1. The bar graph depicts the mean values from three independent experiments with error bars.

apoptosis was observed in E1A/Ras-expressing p53K317R MEFs than in E1A/Ras-expressing wild-type MEFs after doxorubicin treatment, indicating increased p53 apoptotic activities in E1A/Ras-expressing p53K317R MEFs after DNA damage (Fig. 6A). Consistent with this finding, the mRNA levels of the proapoptotic p53 target genes, the Puma, Noxa, Fas (Killer/ DR5), and Lrdd (Pidd) genes, were higher in E1A/Ras-expressing p53K317R MEFs than in E1A/Ras-expressing p53 wildtype MEFs (Fig. 6B). The effect of the K317R mutation on the ability of endogenous p53 to transactivate expression of the luciferase gene from constructs containing a minimal TATA promoter and the distal p53 response element from the human p21 promoter (48) or the p53 response element from the mouse Lrdd (Pidd) promoter (35) were tested in E1A/Ras-expressing MEFs. The relative luciferase activity expressed by the p21-Luc construct did not differ significantly between the p53 wild-type and p53K317R MEFs, either without the addition of a DNA-dam-

aging agent or in the presence of doxorubicin (Fig. 6C). The relative expression of luciferase from the Pidd-Luc construct was significantly greater (P ⬍ 0.005) in the p53K317R E1A/Rasexpressing MEFs than in the corresponding p53 wild-type cells in the absence of a DNA-damaging agent. In the presence of doxorubicin, however, expression from the Pidd-Luc construct did not differ significantly between the p53⫹/⫹ and p53K317R cells (Fig. 6C). In general, DNA transfection processes activate p53 in mammalian cells (47). The approximately twofoldhigher luciferase expression resulting from the Pidd-Luc vector observed in the p53K317R compared to p53⫹/⫹ MEFs in the absence of additional DNA-damaging treatment may indicate that p53K317R is more easily activated by the transfectionderived stress signals than is wild-type p53. Microarray analysis. To investigate the global effects of the K317R mutation on p53-dependent transcriptional responses to IR, differentially labeled RNA samples from untreated thymocytes and thymocytes harvested 5 h after exposure to 5 Gy

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FIG. 5. p53-dependent apoptosis in the small intestine and retina of wild-type and p53K317R mice. (A) Representative TUNEL staining of the small intestine at 5 h after whole-body irradiation with 8 Gy. (B) Quantitative analysis of p53-dependent apoptosis in the small intestines of wild-type and p53K317R mice. For each irradiated sample, the average number of apoptotic cells per crypt was derived from the total number of apoptotic cells in at least 40 crypts. Mean values from three sets of wild-type and p53K317R samples are shown with error bars. Genotypes are indicated on the bottom. (C) IR-induced, p53-dependent apoptosis in the developing retina of wild-type and p53K317R mice. Photomicrographs are shown of retinal sections of wild-type and p53K317R mice processed for a TUNEL assay (red labeling) and counterstained with Hoechst (blue labeling) 6 h after exposure to 2 Gy IR. Retinal layers: NBL, INL, inner plexiform layer (IPL), and ganglion cell layer (GCL). (D) Quantification of TUNEL-positive nuclei in the developing retina after IR of wild-type and p53K317R mice (P ⫽ 0.0237, Student’s t test).

IR were hybridized to spotted microarrays corresponding to approximately 20,000 genes. Normalized and aggregated hybridization intensities were filtered to select approximately 1,400 genes whose expression was significantly affected (Student’s t test P values of less than 0.001) in wild-type or mutant thymocytes after exposure to IR. The distribution of the effects of IR and the K317R mutation on the expression of these genes is shown in Fig. 7. Approximately 35% of the IR-responsive genes exhibited increased expression in response to IR, while 65% were repressed. The proportion of genes induced or repressed by IR was similar to that reported previously (40). Approximately 150 of these genes are significantly affected by the K317R mutation (see Table S1 in the supplemental material). The selection was based on a univariant test (P ⬍ 0.05) applied successively to each gene. Assuming equal variance, we expect that approximately eight of the genes in Table S1 may be “false positives” in which the appearance of an effect of the K317R mutation has arisen by chance. The fraction of the 150

genes induced or repressed was not significantly different from those that responded to IR; 39 percent were induced while 61 percent were repressed. This list includes previously identified direct and indirect targets of p53 and may include additional p53 target genes that have not yet been identified. However, 25 of the genes significantly affected by the K317R mutation are direct targets of p53, as determined from literature sources (Table 1). The distribution of the effects of IR and the K317R mutation on the p53-responsive gene subset is also shown in Fig. 7. Of the 25 mutation-affected, p53-responsive genes, 12 were induced following exposure to IR and 13 were repressed. All of the induced genes exhibited greater induction in thymocytes from p53K317R mice than in thymocytes from wild-type mice. Six of these, namely, Bbc3 (Puma), Phlda3, Pmaip1 (Noxa), Apaf1, Lrdd (Pidd), and Fas (Tnfrsf6), are strongly proapoptotic. Of the mutant-affected genes that were repressed following IR, 10 exhibited greater repression in the mutant. Only three genes, DNA polymerase (Pold1), calmod-

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FIG. 7. Distribution of mRNA levels for IR-responsive genes in thymocytes from wild-type and p53K317R mice after exposure to IR. Thymocytes isolated from p53⫹/⫹ and p53K317R mutant mice were untreated or exposed to 5 Gy IR, and RNA was harvested 5 h after treatment. Differentially labeled and amplified cRNA was hybridized to spotted oligonucleotide microarrays representing ⬃20,000 genes. The expression levels of approximately 1,400 genes were significantly affected after exposure to IR (small gray boxes), the K317R mutation significantly affected the levels of 25 p53-responsive genes after exposure to IR (filled diamonds), and the relative levels of an additional 20 IR-responsive p53 target genes were not significantly affected by the K317R mutation (open inverted triangles). For most p53-regulated genes affected by the K317R mutation, the effect of the mutation was to enhance the level of repression or induction.

FIG. 6. p53-dependent apoptosis in E1A/Ras-expressing wild-type and p53K317R MEFs after DNA damage. (A) p53-dependent apoptosis in E1A/Ras-expressing wild-type and p53K317R MEFs 12 h after doxorubicin treatment (125 ng/ml). The mean values from three independent experiments are shown with error bars. (B) Ratio of the mRNA levels of p53 target genes in E1A/Ras-expressing p53K317R MEFs versus those in wild-type controls 12 h after doxorubicin treatment. The mean values from three independent experiments are shown with error bars. (C) Relative transcription activity of p53 response elements from the p21 and Pidd promoters in E1A/Ras-expressing MEFs. E1A/Rasexpressing wild-type and p53K317R MEFs were transfected with p21-luc or Pidd-luc construct together with a thymidine kinase promoter-Renilla luc construct (control for transfection efficiency) as described previously (34); the cells were subsequently treated with doxorubicin (1 ␮M) or left untreated. Six hours after treatment, the cells were harvested for analysis of luciferase activity. The double asterisks indicate a significant difference (P ⬍ 0.005).

ulin 3 (Calm3), and Pycard, exhibit reduced repression in K317R compared with that in wild-type thymocytes. Interestingly, the list of significantly repressed genes does not contain genes whose mechanisms of repression by p53 are well established (21). In summary, for 88% of the p53-responsive genes significantly affected by the K317R mutation, its effect was to increase the magnitude of the response, whether it was induction or repression. This finding suggests that in thymocytes, the effect of acetylation of lysine 317 is to negatively modulate the activity of p53 as a transcriptional activator or repressor for a subset of target genes. For comparison, we selected a set of 20 p53 target genes that were not significantly affected by the K317R mutation (Table 2); the corresponding distribution is plotted in Fig. 7. Expression of 10 of these 20 genes was increased after IR, while 10 were repressed; however, for none of these did expression differ between wild-type and K317R mutant thymocytes by more than 20 percent. Among these 20 genes are the wellstudied p53 targets p21 (Cdkn1a), Bax, and Mdm2; mRNAs for these genes increased to approximately the same level in wildtype and K317R thymocytes. Although both Mtap4 and Birc5 (Survivin) were shown to be repressed by p53 through direct binding to promoter DNA sequences in murine embryonic

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TABLE 1. p53-responsive genes significantly affected in response to IR by the K317R mutationa

Gene

Alias

mRNA level in IRtreated wt cells/mRNA level in untreated wt cells

Description

Ratio

Bbc3 Phlda3 Pmaip1 Wig1 Ei24 Slc19a2 Gtse1 Apaf1 Glipr1 Notch1 Fas Lrdd Aldh4a1 Mad2l2 Clic4 Mcmd6 Hspa9a Pold1 Calm3 Xrcc5 H2afx Hmbs Odc1 Pycard Egr1 a

Puma Noxa Pig8 Thtr1 B99 RTVP-1 Tnfrsf6 Pidd P5cd Mad2b Mortalin Ku80 H2ax Asc

Bcl-2 binding component 3 Pleckstrin homology-like domain, family A, member 3 Phorbol-12-myristate-13-acetate-induced protein 1 Wild-type p53-induced gene 1 Etoposide-induced 2.4 mRNA Solute carrier family 19 (thiamine transporter), member 2 G2/M phase-expressed protein 1 Apoptotic protease-activating factor 1 GLI pathogenesis-related 1 (glioma) Notch gene homolog 1 (Drosophila) Fas (tumor necrosis factor receptor superfamily member) Leucine rich and death domain containing Aldehyde dehydrogenase 4 family, member A1 MAD2 mitotic arrest deficient-like 2 (yeast) Chloride intracellular channel 4 (mitochondrial) Mini chromosome maintenance-deficient 6 (Saccharomyces cerevisiae) Heat shock protein 9A Polymerase (DNA directed), delta 1, catalytic subunit Calmodulin 3 X-ray repair complementing defective repair 5 H2A histone family, member X Hydroxymethylbilane synthase Ornithine decarboxylase, structural 1 PYD and CARD domain containing Early growth response 1

15.25 14.21 5.66 5.25 4.78 4.18 3.42 3.32 2.61 2.18 2.07 2.05 1.71 0.88 0.87 0.67 0.66 0.63 0.55 0.52 0.50 0.49 0.48 0.43 0.24

P value ⫺5

3.3 ⫻ 10 1.9 ⫻ 10⫺5 1.1 ⫻ 10⫺5 2.7 ⫻ 10⫺5 7.2 ⫻ 10⫺5 1.6 ⫻ 10⫺3 8.6 ⫻ 10⫺5 8.0 ⫻ 10⫺5 2.6 ⫻ 10⫺4 2.0 ⫻ 10⫺4 9.5 ⫻ 10⫺5 3.0 ⫻ 10⫺4 1.7 ⫻ 10⫺3 1.4 ⫻ 10⫺1 1.1 ⫻ 10⫺4 3.2 ⫻ 10⫺3 4.0 ⫻ 10⫺4 3.3 ⫻ 10⫺4 6.4 ⫻ 10⫺4 1.3 ⫻ 10⫺3 5.4 ⫻ 10⫺4 5.2 ⫻ 10⫺4 5.5 ⫻ 10⫺6 1.1 ⫻ 10⫺4 2.2 ⫻ 10⫺5

mRNA level in IRtreated p53K317R cells/ mRNA level in IRtreated wt cells Ratio

P value

1.22 1.44 1.76 1.46 1.23 1.27 1.28 1.12 1.23 1.23 1.21 1.44 1.17 0.64 0.43 0.86 0.80 1.18 1.09 0.81 0.73 0.76 0.81 1.66 0.75

1.3 ⫻ 10⫺2 9.4 ⫻ 10⫺3 3.7 ⫻ 10⫺4 4.6 ⫻ 10⫺3 1.1 ⫻ 10⫺2 3.3 ⫻ 10⫺2 1.6 ⫻ 10⫺2 1.4 ⫻ 10⫺2 3.9 ⫻ 10⫺2 2.3 ⫻ 10⫺2 1.6 ⫻ 10⫺2 1.4 ⫻ 10⫺3 7.2 ⫻ 10⫺3 2.9 ⫻ 10⫺3 3.5 ⫻ 10⫺3 1.8 ⫻ 10⫺2 9.6 ⫻ 10⫺3 2.7 ⫻ 10⫺2 3.7 ⫻ 10⫺2 1.5 ⫻ 10⫺3 1.1 ⫻ 10⫺2 2.8 ⫻ 10⫺2 4.8 ⫻ 10⫺2 1.1 ⫻ 10⫺2 3.5 ⫻ 10⫺2

wt, wild-type.

fibroblast-derived cell lines (42), in thymocytes the responses of these two genes to IR differed. Mtap4 was moderately induced while Birc5 was moderately repressed; however, neither gene’s response was significantly affected by the K317R muta-

tion (Table 2). The cell cycle-controlled genes Cdc2a, Cdc25c, and Ccnb2 were also equally repressed in wild-type and K317R thymocytes. p53-dependent repression of these and certain other cell cycle-controlled genes has been proposed to occur

TABLE 2. Selected p53-responsive genes not significantly affected by the K317R mutationa

Gene

Alias

Description

mRNA level in IRtreated wt cells/mRNA level in untreated wt cells Ratio

Ccng1 Bax Trp53inp1 Ppm1d Cdkn1a Top2b Btg1 Mtap4 Tnfrsf10b Mdm2 Pcna Gadd45a Xpc Birc5 Plk1 Cdc25c Gsn Cdc2a Ccnb2 Myc a

wt, wild-type.

Cyclin G Teap Wip1 p21

Killer

Survivin

Cyclin B2

Cyclin G Bcl2-associated X protein Trp53-inducible nuclear protein 1 Protein phosphatase 1D, Mg dependent, delta Cyclin-dependent kinase inhibitor 1A Topoisomerase (DNA) II beta B-cell translocation gene 1, antiproliferative Microtubule-associated protein 4 Tumor necrosis factor receptor superfamily member Transformed mouse 3T3 cell double-minute 2 Proliferating cell nuclear antigen Growth arrest and DNA damage-inducible 45 alpha Xeroderma pigmentosum, complement, group C Baculoviral IAP repeat-containing 5 Polo-like kinase homolog (Drosophila) Cell division cycle 25 homolog C (S. cerevisiae) Gelsolin Cell division cycle 2 homolog A (Schizosaccharomyces pombe) Cyclin B2 Myelocytomatosis oncogene

4.68 3.70 2.78 2.08 1.91 1.46 1.46 1.38 1.30 1.27 0.98 0.89 0.85 0.83 0.63 0.61 0.51 0.51 0.49 0.19

P value ⫺4

2.0 ⫻ 10 5.2 ⫻ 10⫺5 1.2 ⫻ 10⫺5 8.3 ⫻ 10⫺4 2.8 ⫻ 10⫺2 2.3 ⫻ 10⫺3 3.4 ⫻ 10⫺3 6.4 ⫻ 10⫺4 3.8 ⫻ 10⫺2 4.5 ⫻ 10⫺2 6.1 ⫻ 10⫺1 1.2 ⫻ 10⫺2 3.6 ⫻ 10⫺2 7.2 ⫻ 10⫺2 5.4 ⫻ 10⫺4 1.5 ⫻ 10⫺3 1.4 ⫻ 10⫺5 4.8 ⫻ 10⫺4 5.0 ⫻ 10⫺4 6.5 ⫻ 10⫺5

mRNA level in IRtreated p53K317R cells/mRNA level in IR-treated wt cells Ratio

P value

1.11 1.13 1.09 1.08 1.11 0.98 1.08 0.93 1.00 1.05 0.86 1.13 0.99 0.90 1.20 0.98 1.01 0.89 1.09 0.83

0.15 0.24 0.36 0.54 0.30 0.84 0.36 0.28 0.99 0.41 0.30 0.22 0.90 0.09 0.15 0.86 0.90 0.11 0.21 0.09

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through the association of p53 to promoter-bound NF-Y trimers and is accompanied by increased acetylation at human p53 K320 (equivalent to mouse K317) and additional C-terminal sites of the promoter-bound p53 (24). Although acetylation of p53 seems to be important for NF-Y-mediated repression, transient transfection experiments with acetylation site mutants of p53, including K320R, resulted in only minor differences in the repression of reporter constructs (24), consistent with the observations reported here. DISCUSSION Accumulating evidence has indicated that posttranslational modifications of p53 play important roles in regulating p53 stability and activity. The roles of the acetylation of p53 at multiple C-terminal lysine residues, which are mediated by coactivators CBP/p300 and PCAF, have been extensively studied using biochemical and cell line transfection assays. Since some of the C-terminal lysine residues of p53 are also ubiquitination targets of Mdm2, published studies have suggested that acetylation at the C-terminal lysine residues might prevent ubiquitination of p53, leading to p53 stabilization (33). Furthermore, transient transfection experiments with mutant p53 in which the multiple lysine residues were changed to alanine or arginine indicate that these mutations prevent Mdm2-mediated ubiquitination and stabilize p53 (44, 49). However, recent knock-in mutations that change the six (K6R) or seven (7KR) C-terminal lysines to arginine in endogenous murine p53 indicate that the C-terminal lysine modifications are not important for p53 stabilization (17, 30). These studies further underscore the importance of using a physiologically relevant model to study the roles of posttranslational modifications in regulating p53 stability and activity. We report here our studies of p53K317R knock-in mice to address the physiological roles of the p53 acetylation at lysine 317 in regulating p53 responses to DNA damage. Our studies indicate that abrogation of this acetylation enhances p53-mediated apoptosis after DNA damage in all cell types analyzed. This unexpected role of K317 acetylation suggests that some posttranslational modifications of p53 may have evolved to keep p53 apoptotic activities in check to prevent excessive apoptosis and loss of physiologically important cells after DNA damage. In addition, since apoptosis can induce aging (31), the posttranslational modifications that negatively regulate p53 apoptotic activities could also play important roles in balancing p53 activities in tumor suppression and aging of the whole organism. Our studies also indicate that K317 acetylation modulates the ability of p53 to activate the expression of a subclass of p53 target genes, several of which are involved in apoptosis. The K317R mutation affects both the genes whose expression is activated by p53, e.g., Pmaip1 (Noxa), Lrdd (Pidd), and Bbc3 (Puma), as well as the genes whose expression is normally repressed by p53, e.g., Egr1, Xrcc5 (Ku80), and Pold1 (21). Significantly, the expression of p21Waf1 (Cdkn1a), Mdm2, and Gadd45a, three genes that are commonly monitored to access p53 function, was not significantly altered by the K317R substitution (Table 2). Thus, our findings show for the first time that a specific modification at the C terminus of p53 can differentially affect the expression of p53-regulated genes and

thus potentially affect the outcome of the cellular stress response. In this context, our previous studies have shown that phosphorylation at Ser18 in the N-terminal transactivation domain is required to regulate the expression of p53 target genes in a promoter-specific manner (9). It remains to be established how K317 acetylation preferentially affects the proapoptotic p53 target genes. One possibility is that the requirements for the optimal binding of p53 to its response elements found in apoptosis-promoting genes differ from those of other classes of p53 target genes. In this context, the response elements of the proapoptotic genes exhibit greater deviation from the p53 consensus pattern and fail to function in Saccharomyces cerevisiae-based reporter assays, while response elements from cell cycle arrest and DNA repair-associated genes conform better to the consensus pattern and also function in the yeast-based assay (46). Alternatively or additionally, the involvement of K317 acetylation in disrupting the recruitment of coactivators to the promoters of proapoptotic and cell cycle arrest-associated genes may be different. In this context, an extensive study of transcriptional regulation of the human p21Waf1 (Cdnk1a) and FAS (Tnfrsf6) promoters revealed distinct patterns of p53-dependent and -independent behaviors of the basal transcriptional machinery that differed at the two promoters (16, 18). The finding that expression of the oncoproteins E1A and Ras recapitulates the increased apoptosis seen in the p53K317R tissues is also suggestive. The deregulation of the E2F transcription factors through the binding of the adenovirus E1A protein to Rb is fundamental to the process of viral transfection (20). E2F1 binds to p53 and promotes p53-dependent apoptosis (32). Furthermore, E2F1 is a transcriptional activator of several proapoptotic cofactors of p53, including ASPP1 and TP53INP1 (32). A second well-studied aspect of E1A is its disruption of the interaction between PCAF and p300/CBP through the preferential binding of E1A to p300/CBP (12). In response to DNA damage, PCAF also acetylates E2F1, resulting in increased stability and activity as a transcriptional activator (23). Therefore, the intrinsic weakness of p53 binding to its response elements in the promoters of proapoptotic genes, the differences in the regulation of basal transcription of these genes, and the effects of proapoptotic cofactors of p53 all might contribute to the observed differential regulation of p53 target proapoptotic and cell cycle arrest-promoting genes in p53K317R cells after DNA damage. ACKNOWLEDGMENTS This work was supported by the National Institutes of Health grant CA94254 to Y.X. C.W.A. was supported in part by funds from the Laboratory Directed Research and Development Program and by the U.S. Army Breast Cancer Idea Award (DAMD17-00-1-0168) at the Brookhaven National Laboratory under contract with the U.S. Department of Energy. REFERENCES 1. Appella, E., and C. W. Anderson. 2001. Post-translational modifications and activation of p53 by genotoxic stresses. Eur. J. Biochem. 268:2764–2772. 2. Barlev, N. A., L. Liu, N. H. Chehab, K. Mansfield, K. G. Harris, T. D. Halazonetis, and S. L. Berger. 2001. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol. Cell 8:1243–1254. 3. Bell, S., C. Klein, L. Muller, S. Hansen, and J. Buchner. 2002. p53 contains large unstructured regions in its native state. J. Mol. Biol. 322:917–927. 4. Borges, H. L., C. Chao, Y. Xu, R. Linden, and J. Y. Wang. 2004. Radiation-

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