p63 and p73 are required for p53- dependent apoptosis in ... - Nature

letters to nature experiments shown in Fig. 4a, b were exactly as described5, except that the binding and washing buffer for the GST – LHP1 results shown in Fig. 4a consisted of 50 mM sodium phosphate and 25 mM NaCl at pH 6.0. For Fig. 4a, bound proteins were separated by 4 – 20% gradient SDS – PAGE, blotted, and detected with an anti-GST monoclonal antibody (Pierce). For Fig. 4b, the bound biotinylated peptides (Upstate Biotechnology) were separated by 18% SDS – PAGE, blotted, and detected with streptavidin HRP conjugate (Upstate Biotechnology).

Competing interests statement

Interaction of CMT3 with LHP1

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A six-histidine fusion construct was made by cloning full-length LHP1 into the XhoI and PstI sites of pRSETB, and expressed in E. coli strain BL21. Proteins were purified on NiNTA-agarose (Qiagen) and eluted with 100 mM imidazole before mixing with glutathione-agarose-bound GST fusion proteins. Binding and wash buffers were the same as in the peptide binding assays5. Bound proteins were separated by 4– 20% gradient SDS – PAGE, blotted, and detected with INDIA HisProbe-HRP (Pierce). Received 15 January; accepted 4 March 2002. Published online 17 March 2002, DOI 10.1038/nature731. 1. Rea, S. et al. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406, 593–599 (2000). 2. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D. & Grewal, S. I. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110– 113 (2001). 3. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41 –45 (2000). 4. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001). 5. Lachner, M., O’Carroll, D., Rea, S., Mechtler, K. & Jenuwein, T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116– 120 (2001). 6. Bannister, A. J. et al. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120–124 (2001). 7. Jacobs, S. A. et al. Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J. 20, 5232 –5241 (2001). 8. Tamaru, H. & Selker, E. U. A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414, 277–283 (2001). 9. Lindroth, A. M. et al. Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation. Science 292, 2077– 2080 (2001). 10. Tschiersch, B. et al. The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13, 3822– 3831 (1994). 11. Allshire, R. C., Nimmo, E. R., Ekwall, K., Javerzat, J. P. & Cranston, G. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 9, 218–233 (1995). 12. Ivanova, A. V., Bonaduce, M. J., Ivanov, S. V. & Klar, A. J. The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nature Genet. 19, 192–195 (1998). 13. Peters, A. H. et al. Loss of the suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323–337 (2001). 14. Jacobsen, S. E. & Meyerowitz, E. M. Hypermethylated SUPERMAN epigenetic alleles in Arabidopsis. Science 277, 1100–1103 (1997). 15. Bartee, L., Malagnac, F. & Bender, J. Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev. 15, 1753 –1758 (2001). 16. Baumbusch, L. O. et al. The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes. Nucleic Acids Res. 29, 4319–4333 (2001). 17. Tachibana, M., Sugimoto, K., Fukushima, T. & Shinkai, Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J. Biol. Chem. 276, 25309–25317 (2001). 18. Finnegan, E. J., Peacock, W. J. & Dennis, E. S. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development. Proc. Natl Acad. Sci. USA 93, 8449–8454 (1996). 19. Ronemus, M. J., Galbiati, M., Ticknor, C., Chen, J. & Dellaporta, S. L. Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273, 654–657 (1996). 20. Kishimoto, N. et al. Site specificity of the Arabidopsis METI DNA methyltransferase demonstrated through hypermethylation of the superman locus. Plant Mol. Biol. 46, 171– 183 (2001). 21. Soppe, W. J. et al. The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene. Mol. Cell 6, 791–802 (2000). 22. Vongs, A., Kakutani, T., Martienssen, R. A. & Richards, E. J. Arabidopsis thaliana DNA methylation mutants. Science 260, 1926– 1928 (1993). 23. Steimer, A. Endogenous targets of transcriptional gene silencing in Arabidopsis. Plant Cell 12, 1165– 1178 (2000). 24. Henikoff, S. & Comai, L. A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149, 307–318 (1998). 25. Akhtar, A., Zink, D. & Becker, P. B. Chromodomains are protein–RNA interaction modules. Nature 407, 405–409 (2000). 26. Gaudin, V. et al. Mutations in LIKE HETEROCHROMATIN PROTEIN 1 affect flowering time and plant architecture in Arabidopsis. Development 128, 4847–4858 (2001). 27. Peters, A. H. F. M. et al. Histone H3 lysine 9 methylation is an epigenetic imprint of facultative heterochromatin. Nature Genet. 30, 77–80 (2002).

Supplementary Information accompanies the paper on Nature’s website (http://www.nature.com).

Acknowledgements We thank T. Jenuwein for the GST – Suv constructs and H3 N-terminal peptides, Y. Shinkai for the H3 N-terminal GST fusion constructs, A. Kouzarides for an HP1 construct, and S. Peyvandi for technical assistance. This work was supported by grants from the National Institutes of Health, the Beckman Young Investigator programme, and the Searle Scholars Foundation to S.E.J. J.P.J. was supported by an NIH training grant and A.M.L. by a postdoctoral fellowship from the Damon Runyon Walter Winchel Foundation.

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The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to S.E.J. (e-mail: [email protected]).

p63 and p73 are required for p53dependent apoptosis in response to DNA damage Elsa R. Flores*, Kenneth Y. Tsai*†, Denise Crowley*§, Shomit Sengupta*§, Annie Yang‡, Frank McKeon‡ & Tyler Jacks*§ * Massachusetts Institute of Technology, Department of Biology and Center for Cancer Research, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA † Harvard–Massachusetts Institute of Technology Division of Health Sciences and Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA ‡ Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA § Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20185, USA .............................................................................................................................................................................

The tumour-suppressor gene p53 is frequently mutated in human cancers and is important in the cellular response to DNA damage1,2. Although the p53 family members p63 and p73 are structurally related to p53, they have not been directly linked to tumour suppression, although they have been implicated in apoptosis3 – 9. Given the similarity between this family of genes and the ability of p63 and p73 to transactivate p53 target genes10,11, we explore here their role in DNA damage-induced apoptosis. Mouse embryo fibroblasts deficient for one or a combination of p53 family members were sensitized to undergo apoptosis through the expression of the adenovirus E1A oncogene12 – 14. While using the E1A system facilitated our ability to perform biochemical analyses, we also examined the functions of p63 and p73 using an in vivo system in which apoptosis has been shown to be dependent on p53. Using both systems, we show here that the combined loss of p63 and p73 results in the failure of cells containing functional p53 to undergo apoptosis in response to DNA damage. Previous work has shown that p53-deficient E1A mouse embryo fibroblasts (MEFs) treated with DNA-damaging agents are highly resistant to apoptosis12 – 14. To determine whether p63 and/or p73 are involved in apoptosis induced by DNA damage, p63-deficient (p632/2) and p73-deficient (p732/2) E1A-expressing MEFs were generated and treated with doxorubicin for 0, 6, 12, 24 and 48 h (Fig. 1A; Supplementary Information, Fig. S1a), stained with annexin V coupled to fluorescein isothiocyanate, and analysed by flow cytometry. E1A MEFs lacking p63 or p73 exhibited a partial resistance to apoptosis in response to DNA damage; 70% of the p632/2 E1A MEFs and 80% of the p732/2 E1A MEFs were viable, compared with 42% of the wild-type E1A MEFs at 12 h. At 24 h, 50% of the p632/2 E1A MEFs and 65% of the p732/2 E1A MEFs were viable, compared with 5% of the wild-type E1A MEFs (Fig. 1A). Because p63-deficient and p73-deficient E1A MEFs exhibited a partial resistance to apoptosis, we hypothesized that they might cooperate with p53 or with each other in the DNA damage response. Therefore, double-homozygous E1A MEFs deficient for all pairs of combinations (p532/2; p632/2, p532/2; p732/2, p632/2; p732/2) were generated and treated with doxorubicin. As shown in Fig. 1A, all the double-knockout cells, including the p632/2; p732/2 E1A MEFs, were resistant to apoptosis in response to DNA damage. The p532/2; p632/2 and p532/2; p732/2 E1A MEFs were more resistant

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letters to nature than the p532/2 E1A MEFs, whereas the p632/2; p732/2 E1A MEFs were as resistant as p532/2 E1A MEFs (Fig. 1A). Cisplatin and girradiation were also tested on E1A MEFs of the different genotypes with similar results (Supplementary Information, Fig. S1). These findings indicate that even in the presence of p53, the E1A MEFs deficient for both p63 and p73 are resistant to apoptosis, suggesting that these genes might act together with p53 or in an obligatory parallel pathway to induce apoptosis subsequent to DNA damage. Several isoforms have been identified for p63 and p73, including some containing (TAp63 and TAp73) and lacking (DNp63 and DNp73) an amino-terminal transactivation domain4. The p632/2; p732/2 E1A MEFs carry mutations in the central DNA-binding domains and are therefore deficient for all of these isoforms. To determine whether the transactivation domain of these proteins is important in the response to DNA damage, wild-type, p532/2 and p632/2; p732/2 E1A MEFs were transfected with expression vectors for TAp63a, TAp73a, DNp63a or DNp73a. After 24 h the cells were treated with 0.34 mM doxorubicin for 12 h, and apoptosis was detected by staining with annexin V. Protein expression of the transfected plasmids was detected by western blotting (Supplementary Information, Fig. S1d). Wild-type E1A MEFs transfected with any of the expression vectors underwent apoptosis in response to doxorubicin, whereas p532/2 E1A MEFs transfected with these constructs and treated with doxorubicin did not. Transfection of TAp63a or TAp73a into the p632/2; p732/2 E1A MEFs caused a small but consistent increase in apoptosis after treatment with doxorubicin, whereas transfection with DNp63a or DNp73a had no effect in this system (Supplementary Information, Fig. S1e). These data suggest that the transactivation domains of p63 and p73

are important for DNA-damage induced apoptosis. p53 has been shown to be required for irradiation-induced apoptosis in the developing nervous system15 – 17. To examine the requirements for p63 and p73 in this setting, control and p632/2; p732/2 day-13.5 embryos were analysed. Wild-type, p532/2, p632/2, p732/2 and p632/2; p73 2/2 embryos were treated with girradiation (5 Gy) in utero, harvested 5 h later and analysed for apoptosis by end labelling in situ. Figure 1B, C shows that there was a striking difference between the level of apoptosis detected in the girradiated central nervous system (CNS) of the wild-type embryos (29 ^ 8%) compared with the p532/2 (0.18 ^ 0.1%) and the p632/2; p732/2 embryos (0.2 ^ 0.1%) (Fig. 1Bb, Bd, Bf, C). The level of apoptosis in the p632/2; p732/2 embryos was similar to that found in p532/2 embryos, indicating that p63 and p73 are required for p53-dependent apoptosis in this system as well. Both the p632/2 and p732/2 embryos displayed an intermediate phenotype: 7.5 ^ 3.0% and 8.0 ^ 3.0% apoptotic cells, respectively (Fig. 1Bg, Bh, C). No apoptosis was detected in untreated wild-type, p532/2, and p632/2, p732/2 embryos (Fig. 1Ba, Bc, Be). The number of apoptotic cells was divided by the number of total cells to derive the percentage of apoptotic cells in each sample. These results indicate that both p63 and p73 are required for p53dependent apoptosis in vivo and support the results previously derived from E1A expressing MEFs. Given that the p632/2; p732/2 MEFs expressing E1A were as resistant to apoptosis as cells lacking p53 itself, experiments were performed to determine the function of p53 in the double-mutant cells. In all cells containing functional p53, the p53 protein was expressed at similar levels and was induced on treatment with

Figure 1 E1A MEFs and the CNS from 13.5-day embryos mutant for p53-family proteins are resistant to apoptosis in response to DNA damage. A, Histogram representing the percentage of viable cells scored by annexin 12 and 24 h after treatment with 0.34 mM doxorubicin. B, CNS of embryos assayed for apoptosis by using FRAGEL. a, c, e, Untreated embryos: wild type (a), p532/2 (c) and p632/2; p732/2 (e).

b, d, f, g, h Embryos treated in utero with g-irradiation (5 Gy) for 5 h: wild type (b), p532/2 (d), p632/2; p732/2 (f), p632/2 (g) and p732/2 (h). Positive cells are indicated by arrows. p632/2; p732/2 embryos were histologically indistinguishable from wild-type embryos (see description in Supplementary Information). C, Histogram showing the percentages of apoptotic cells after g-irradiation for 5 h from the analysis in B.



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letters to nature doxorubicin for 12 h, as shown by western blot analysis (Fig. 2a). We also determined that p63 is induced on treatment with doxorubicin. By western blot analysis, wild-type, p532/2 and p532/2; p732/2 E1A-expressing cells exhibited a striking increase in the amount of p63 after treatment with doxorubicin for 12 h (Fig. 2b). Antibodies against mouse p73 were not of sufficient quality to determine p73 induction, although endogenous human p73 has been shown to be induced by E1A and cisplatin6,7,9,7,18. Therefore, p63 and p73 can be induced after forms of stress, including DNA damage and oncogenic stimuli.

Upon DNA damage, p53 is stabilized and accumulates in the nucleus. Indirect immunofluorescence for p53 was performed on wild-type and p632/2; p732/2 E1A MEFs after treatment with doxorubicin, cisplatin or g-irradiation. The extent and pattern of p53 nuclear staining in the p632/2; p732/2 E1A MEFs were comparable to those of wild-type E1A cells (data not shown). Additionally, the localization of p63 was tested in wild-type, p532/2 and p632/2; p732/2 E1A MEFs after treatment with doxorubicin for 6 h (Supplementary Information, Fig. S2). Similarly to p53, p63 nuclear staining was increased under these conditions, consistent

Figure 2 p53 and p63 are induced in E1A MEFs after treatment with DNA-damaging agents. Western blots are shown containing whole cell extracts from MEFs expressing E1A. a, p53 is induced in wild-type, p632/2; p732/2, p632/2 and p732/2 E1A MEFs.

b, p63 is induced in wild-type, p532/2 and p532/2; p732/2 E1A MEFs 12 h after treatment with 0.34 mM doxorubicin (Dox.). Actin was used as a loading control. U, untreated.

Figure 3 p53 target genes are induced differently in p632/2; p732/2 E1A MEFs and the brain of 13.5-day embryos. a, Northern blot analysis of total RNA extracted from E1A MEFs treated with 0.34 mM doxorubicin (Dox.) for 12 h and probed with mdm2, p21, PERP, bax or ARPP P0. b, Histogram indicating fold induction of the p53 target genes

analysed in a calculated with the use of ARPP P0 and the PhosphorImager. c, Western blot analysis of E1A MEFs treated for 12 h with 0.34 mM doxorubicin and probed with antip21 and anti-Bax. d, Northern blot analysis of total RNA extracted from brains of 13.5-day embryos 5 h after treatment with g-irradiation (5 Gy). U, untreated.

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letters to nature with its having a function in the transcriptional activation of target genes involved in the DNA damage response. For the further characterization of MEFs expressing E1A and deficient for either p63 or p73 or both, the expression of known p53 target genes was examined. Specifically, the p21, mdm2, bax and PERP genes were analysed by northern blotting both before and 12 h after treatment with 0.34 mM doxorubicin19 – 21. Figure 3a, b shows that the induction of p21 and mdm2 was comparable in wild-type, p632/2, p732/2 and p632/2; p732/2 E1A MEFs. Interestingly, whereas bax and PERP were also induced to similar extents in wildtype, p632/2 and p732/2 E1A MEFs, these genes were not induced in the p632/2; p732/2 E1A MEFs (see Fig. 3b). Western blot analysis was performed for p21 and bax to confirm these results at the level of protein expression (Fig. 3c). These data show that some p53 targets are regulated normally in p632/2; p732/2 E1A MEFs, whereas genes linked to the apoptotic response were specifically affected. To determine whether p53 target genes were differentially expressed in the CNS of developing p632/2; p732/2 day-13.5 embryos, the brain was microdissected from control wild-type, p532/2 and p632/2; p732/2 embryos or embryos treated in utero with g-irradiation (5 Gy). At 5 h after treatment, total RNA was isolated from each brain for northern blot analysis. Again, p21, mdm2, PERP and bax were found to be induced in wild-type embryos, whereas only p21 and mdm2 were induced in p632=2 ;p732=2 embryos, confirming the finding in the E1A MEFs (Fig. 3d). Given that p53 and p63 are present, induced and enriched in the nuclei of E1A MEFs after DNA damage, we assayed for p53 and p63 occupancy of specific promoter elements in vivo by chromatin immunoprecipitation (ChIP) analysis for p53 target genes. We

examined the mdm2 and p21 promoters, which had been shown to be induced in both wild-type and p632/2; p732/2 E1A MEFs by northern analysis, and bax and PERP, which were induced in wildtype but not p632/2; p732/2 E1A MEFs (Fig. 3a–c). Additionally, the NOXA promoter was used as another example of a p53 target gene believed to be important for p53-dependent apoptosis22. Figure 4a, b shows that after DNA damage there was increased association of p53 and p63 with the p21, mdm2, bax, PERP and NOXA promoters in wild-type MEFs expressing E1A. Similarly, increased association of p53 with the p21 and mdm2 promoters was evident in p632/2; p732/2 E1A MEFs under these conditions, although at somewhat lower levels than in wild-type E1A MEFs. In striking contrast, we observed no association of p53 with the PERP, NOXA and bax promoters after DNA damage in p632/2; p732/2 E1A cells. These results correlate with the differential induction of p53 target genes obtained in the northern blot analyses (Fig. 3a–d), and indicate that after DNA damage p63 and p73 might regulate the ability of p53 to bind at certain selected promoters. Interestingly, p63 was also found at the bax, PERP and NOXA promoters in p532/2 E1A MEFs after treatment with doxorubicin for 12 h (Fig. 4b), whereas p63 was not apparent at the mdm2 and p21 promoters in these cells. Notably, p63 was found to bind both the 5 0 and 3 0 p53-binding sites on the PERP promoter21 and was more strongly associated with the 5 0 site, whereas p53 was detected only at the 3 0 site, suggesting a complex regulation of this gene by the p53 family members. These results indicate that p63 is present at p53 target genes after DNA damage and, importantly, that it is present in the absence of p53 at target genes involved in the apoptotic process. (For a complete set of controls, see Supplementary Information, Fig. S4.) p53 has long been recognized as central to the induction of apoptosis in response to DNA damage, a function thought to be critical for tumour suppression and the response of tumour cells to chemotherapeutic agents. Although our data do not diminish the significance of p53 in this process, they do establish a role for p63 and p73. The combined absence of p63 and p73 severely impaired the induction of p53-dependent apoptosis in response to DNA damage in E1A-expressing cells and in the developing mouse CNS. This can be explained by the inability of p53 to bind the promoters of apoptosis-associated target genes and to upregulate their transcription in p632/2; p732/2 E1A cells and the developing CNS. There are two pieces of evidence that p63 and p73 are important players in recruiting p53 to promoters at apoptosis-related genes. First, p63 is present at such promoters, even in the absence of p53. Second, p53 is not present at these promoters in the absence of p63 and p73. Our data support the notion that there might be two classes of p53-family target genes. One class includes genes such as mdm2 and p21, which p53 regulates in the presence or absence of p63 and p73. The other includes genes such as PERP, bax and NOXA, for which genetic and biochemical data indicate a requirement for p63 or p73 for p53 to be recruited and function properly. These data show that p63 and p73 are important components of the response to DNA damage, and may portend a greater role for these proteins in tumour suppression and chemosensitivity. A

Methods Cell culture

Figure 4 Differential binding of p53 and p63 in vivo at various promoters in E1A MEFs. a, ChIP analysis for the presence of p53 at the p21, mdm2, PERP (3 0 site), NOXA and bax promoters in E1A MEFs before (U) and after treatment for 12 h with 0.34 mM doxorubicin (Dox.). b, ChIP analysis for the presence of p63 at the p21, mdm2, PERP (5 0 site), NOXA and bax promoters in E1A MEFs. Input, total input chromatin from untreated wild-type E1A MEFs. For additional controls see Supplementary Information, Fig. S4. NATURE | VOL 416 | 4 APRIL 2002 | www.nature.com

MEFs were derived from 13.5-day embryos from the following mouse crosses: p532/2 embryos were derived from p53þ/- crosses maintained on a 129/SvJae background; p632/2 and p732/2 embryos were similarly derived from p63þ/- (maintained on a mixed C57BL/6 £ 129/SvJae) and p73þ/- crosses (maintained on a 129/SvJae background), respectively. Double-mutant MEFs (p532/2; p732/2, p532/2; p632/2 and p632/2; p732/2) were derived by first generating double-heterozygous mice and crossing to generate doublehomozygous embryos. The genotypes of the MEFs were determined by polymerase chain reaction (PCR) as described previously23 – 25. E1A-expressing MEFs of each of the above genotypes were generated by infection with high-titre ecotropic retroviruses derived by using the Phoenix retrovirus packaging system26. At 48 h after infection, E1A-expressing MEFs were selected by the addition of 2 mg ml21 puromycin for 2 d. For transfection experiments, E1A MEFs were transfected with 0.5 mg mouse TAp63a, DNp63a, DNp73a,


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letters to nature human TAp73a or pcDNA3 with the use of FuGene (Roche). At 24 h after transfection, cells were treated with doxorubicin for 12 h and assayed for apoptosis as described below.

Annexin assay At 24 h after plating, E1A MEFs were treated with 0.34 mM doxorubicin. Floating and adherent cells were collected 0, 6, 12, 24 and 48 h after treatment, stained with annexin V coupled to fluorescein isothiocyanate (Becton Dickinson) and propidium iodide (50 mg ml21) in accordance with the manufacturer’s instructions, and analysed with a Becton Dickinson FACScan with the use of CellQuest software. The percentage of viable cells was calculated by scoring for cells that were not annexin-positive (early apoptotic) or not positive for both annexin and propidium iodide (late apoptotic and necrotic).

End labelling in situ on 13.5-day embryonic CNS Wild-type, p532/2, p632/2, p732/2 and p632/2; p732/2 day-13.5 embryos were derived from the same mouse crosses used to derive the MEFs. Pregnant females at 13 d post coitus (p.c.) were treated with g-irradiation (5 Gy) by using an irradiator with a 137Cs source. At 5 h after treatment, embryos were harvested and fixed in 10% neutral buffered formalin overnight. Embryos were processed, embedded in paraffin and sectioned onto slides. End labelling in situ was performed on slides with the use of the FRAGEL kit from Oncogene Research Products, in accordance with the manufacturer’s protocol. Brown nuclei were scored as positive. To quantify the numbers of apoptotic cells in the CNS of the embryos, brown nuclei in the subventricular zone were counted and divided by the total number of cells in that zone. Four or five embryos per genotype were used in this analysis.

Northern blot analysis At 24 h after plating, E1A MEFs of each genotype were treated for 12 h with 0.34 mM doxorubicin. For embryonic brain analysis, embryos were treated in utero with girradiation (5 Gy) by using an irradiator with a 137Cs source at 13 d post coitus; 5 h after treatment, brains were harvested from the embryos by microdissection. Total RNA was isolated from E1A MEFs or from embryonic brains by using Tri reagent (Sigma) and subjected to electrophoresis on a 1% agarose gel in 1 £ MOPS containing 6% formaldehyde. Blots probed with mdm2, p21 and ARPP P0 were prehybridized and hybridized in ExpressHyb solution at 65 8C (Clontech). Blots probed with bax and PERP were prehybridized in 0.5 M NaPO4 pH 7.2, 1 mM EDTA, 1% BSA, 7% SDS and hybridized in 30% formamide, 0.2 M NaPO4 pH 7.2, 1 mM EDTA, 1% BSA, 7% SDS at 42 8C. Washes were performed at 60 8C in 2 £ SSC, 0.1% SDS for 15 min followed by 0.2 £ SSC, 0.1% SDS for 15 min. Radiolabelled probes from complementary DNAs corresponding to mdm2, p21, bax, PERP and ARPP P0 were prepared by random primer labelling with the Prime-It II kit (Stratagene) and [a-32P]dATP.

Western blot analysis E1A MEFs treated for 12 h with 0.34 mM doxorubicin were lysed on ice in lysis buffer (100 mM Tris, 100 mM NaCl, 1% Nonidet P40, protease inhibitor cocktail (Roche)), 100 mg of protein was loaded on a SDS/10% polyacrylamide gel and transferred to a poly(vinylidene difluoride) membrane (Millipore). Blots were blocked in TBST (10 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% Tween) containing 5% milk and probed with the following primary antibodies: anti-p21 (clone F5; Santa Cruz), anti-p53 (Ab-3; Oncogene Research Products), anti-p63 (clone 4A4; F. McKeon), anti-Bax (clone N-20; Santa Cruz), anti-c-Myc (clone 9E-10; Santa Cruz) and anti-haemagglutinin (clone Y11, Santa Cruz). After incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Jackson Immunochemicals), detection was performed with the ECL Plus kit (Amersham). To ensure even loading of protein samples, each blot was stripped with strip buffer (125 mM Tris-HCl pH 6.8, 2% SDS, 0.68% 2-mercaptoethanol) at 50 8C for 30 min followed by a 30-min wash in TBST at 25 8C and probed with actin (Santa Cruz).

p63 immunofluorescence At 24 h after plating, E1A MEFs were treated for 6 h with 0.34 mM doxorubicin and fixed on coverslips in 1:1 methanol/acetone for 1 min. Cells were rehydrated in 1 £ PBS, blocked in 1% normal goat serum and incubated with a mouse monoclonal anti-p63 antibody (clone 4A4, F. McKeon) at 25 8C for 2 h. Detection was performed with an anti-mouse Texas Red secondary antibody (Molecular Probes) at 25 8C for 2 h.

annealing temperature 59 8C. PERP 3 0 site: 5 0 end, 5 0 -AGCCACTGGGACCTGCTGGTCA CCC-3 0 ; 3 0 end, 5 0 -CTACCTGCTGGGTCACCCGG GCGGC-3 0 , at site -218; annealing temperature 68 8C. NOXA: 5 0 end, 5 0 -CCTGGCCC CACCCCACCCCACCCCC-3 0 ; 3 0 end, 5 0 -TCAGGGCTATTTTACGCTCTCGGCC-3 0 , at site -174; annealing temperature 65 8C. Received 9 November 2001; accepted 25 February 2002. 1. Levine, A. J. p53, the cellular gatekeeper for growth and division. Cell 88, 323–331 (1997). 2. Evan, G. I. & Vousden, K. H. Proliferation, cell cycle and apoptosis in cancer. Nature 411, 342– 348 (2001). 3. Yang, A. et al. p63, a p53 homolog at 3q27 –29, encodes multiple products with transactivating, deathinducing, and dominant-negative activities. Mol. Cell 2, 305–316 (1998). 4. Irwin, M. S. & Kaelin, W. G.Jr Role of the newer p53 family proteins in malignancy. Apoptosis 6, 17–29 (2001). 5. Stiewe, T. & Putzer, B. M. Role of the p53-homologue p73 in E2F1-induced apoptosis. Nature Genet. 26, 464–469 (2000). 6. Agami, R., Blandino, G., Oren, M. & Shaul, Y. Interaction of c-Abl and p73a and their collaboration to induce apoptosis. Nature 399, 809– 813 (1999). 7. Gong, J. G. et al. The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage. Nature 399, 806–809 (1999). 8. Jost, C. A., Marin, M. C. & Kaelin, W. G.Jr p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature 389, 191–194 (1997). 9. Yuan, Z. M. et al. p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage. Nature 399, 814–817 (1999). 10. Di Como, C. J., Gaiddon, C. & Prives, C. p73 function is inhibited by tumor-derived p53 mutants in mammalian cells. Mol. Cell. Biol. 19, 1438–1449 (1999). 11. Gaiddon, C., Lokshin, M., Ahn, J., Zhang, T. & Prives, C. A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain. Mol. Cell. Biol. 21, 1874 –1887 (2001). 12. Lowe, S. W., Ruley, H. E., Jacks, T. & Housman, D. E. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 74, 957–967 (1993). 13. Lowe, S. W. & Ruley, H. E. Stabilization of the p53 tumor suppressor is induced by adenovirus 5 E1A and accompanies apoptosis. Genes Dev. 7, 535–545 (1993). 14. Lowe, S. W., Jacks, T., Housman, D. E. & Ruley, H. E. Abrogation of oncogene-associated apoptosis allows transformation of p53-deficient cells. Proc. Natl Acad. Sci. USA 91, 2026–2030 (1994). 15. Lee, Y., Chong, M. J. & McKinnon, P. J. Ataxia telangiectasia mutated-dependent apoptosis after genotoxic stress in the developing nervous system is determined by cellular differentiation status. J. Neurosci. 21, 6687– 6693 (2001). 16. Herzog, K. H., Chong, M. J., Kapsetaki, M., Morgan, J. I. & McKinnon, P. J. Requirement for Atm in ionizing radiation-induced cell death in the developing central nervous system. Science 280, 1089–1091 (1998). 17. Chong, M. J. et al. Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system. Proc. Natl Acad. Sci. USA 97, 889–894 (2000). 18. Zaika, A., Irwin, M., Sansome, C. & Moll, U. M. Oncogenes induce and activate endogenous p73 protein. J. Biol. Chem. 276, 11310– 11316 (2001). 19. Miyashita, T. et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9, 1799–1805 (1994). 20. Yin, C., Knudson, C. M., Korsmeyer, S. J. & Van Dyke, T. Bax suppresses tumorigenesis and stimulates apoptosis in vivo. Nature 385, 637–640 (1997). 21. Attardi, L. D. et al. PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. Genes Dev. 14, 704–718 (2000). 22. Oda, E. et al. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288, 1053 –1058 (2000). 23. Jacks, T. et al. Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4, 1– 7 (1994). 24. Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718 (1999). 25. Yang, A. et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404, 99– 103 (2000). 26. Kinsella, T. M. & Nolan, G. P. Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7, 1405–1413 (1996). 27. Wells, J., Boyd, K. E., Fry, C. J., Bartley, S. M. & Farnham, P. J. Target gene specificity of E2F and pocket protein family members in living cells. Mol. Cell. Biol. 20, 5797 –5807 (2000).

Supplementary Information accompanies the paper on Nature’s website (http://www.nature.com).

ChIP assay ChIP analysis was performed as described previously27. In brief, adherent E1A-expressing MEFs untreated or treated with doxorubicin (0.34 mM) for 12 h were removed from the dish with trypsin, which was subsequently inactivated with DMEM containing fetal bovine serum. Cellular proteins were crosslinked to chromatin with 1% formaldehyde for 10 min at 25 8C. p53 –DNA complexes or p63 – DNA complexes were immunoprecipitated from nuclear extracts by using antibodies against mouse p53 (Ab-1 and Ab-4; Oncogene Research Products) or p63 (clone 4A4; F. McKeon), recovered with Staphylococcus A cells and treated with proteinase K to purify DNA. PCR was performed by amplifying ,250base-pair fragments of the promoter sequence. The following primers were used. mdm2: 5 0 end, 5 0 -GGTGCCTGGTCCCGGACTCG CCGGG-3 0 ; 3 0 end, 5 0 -CCGAGAGGGTCCCCCAGGGGTGTCC-3 0 , site located in intron 1; annealing temperature 75 8C. p21: 5 0 end, 5 0 -CCTTTCTATCAGCCCCAGAGGATACC3 0 ; 3 0 end, 5 0 -GGGACGTCCTTAATTATCTGG GGTC-3 0 , at site -2853; annealing temperature 58 8C. bax: 5 0 end, 5 0 -GATGTTGTAGCC ACCGCGTACAGCC-3 0 ; 3 0 end, 5 0 -TTCATGGTAGAGAGCACTAAGGAGG-3 0 , encompassing sites -518 and -324; annealing temperature 65 8C. PERP 5 0 site: 5 0 end, 5 0 -CACACAATCAGAAGGCCTTGG AGGG-3 0 ; 3 0 end, 5 0 -TAATACTCCGAGGTTGAGGC AGGAAG-3 0 , at site -2097;

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Acknowledgements We thank J. Sage for critical reading of the manuscript, W. G. Kaelin and M. S. Irwin for human TAp73a mammalian expression vector, L. Attardi for PERP cDNA, D. MacPherson for helpful discussions, and K. Olive for p53þ/2 mice. This work was supported in part by the NIH and Howard Hughes Medical Institute. E.R.F. is a postdoctoral fellow of the Leukemia and Lymphoma Society of America. K.Y.T. is supported by the Medical Scientist Training Program and a Koch graduate fellowship. T.J. is an Associate Investigator of the Howard Hughes Medical Institute.

Competing interests statement The authors declare that they have no competing financial interests. Correspondence and requests for materials should be addressed to T.J. (e-mail: [email protected]).

