ABSTRACTCellular DNA damage causes stabilization and activation of the tumor suppressor and transcription factor p53, in part by promoting multiple covalent modifica-tions of the p53 protein, including acetylation. We investigated the importance of acetylation in p53 func-tion and the mechanism by which acetylation influences p53 activity. Acetylation site substitutions reduced p53-dependent transcriptional induction and G1 cell cycle arrest. Chromatin immunoprecipitation analysis of the endogenous p21 promoter showed increased association of p53, coactivators (СВР and TRRAP), and acetylated histones following cell irradiation. Results with acetylation-defective p53 demonstrate that the critical function of acetylation is not to increase the DNA binding affinity of p53 but rather to promote coactivator recruitment and histone acetylation. Therefore, we propose that an acetylation cascade consisting of p53 acetylation-dependent recruitment of coactivators/HATs is crucial for p53 function.
Attardi, L.D., Reczek, E.E., Cosmas, C., Demicco, E.G., McCurrach, M.E., Lowe, S.W., and Jacks, T. (2000). PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family. Genes Dev. 14, 704-718.
Banin, S., Moyal, L., Shieh, S., Taya, Y., Anderson, C.W., Chessa, L., Smorodinsky, N.I., Prives, C., Reiss, Y., Shiloh, Y., and Ziv, Y. (1998). Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 281, 1674-1677.
Bannister, A.J., and Kouzarides, T. (1996). The СВР co-activator is a hist one acetyltransf erase. Nature 384, 641-643. Brown, C.E., Lechner, Т., Howe, L., and Workman, J.L. (2000). The many HATs of transcription coactivators. Trends Biochem. Sci. 25, 15-19.
Canman, C.E., Lim, D.S., Cimprich, K.A., Taya, Y., Tamai, K., Saka-guchi,K., Appella, E., Kastan, M.B.,and Siliciano, J.D. (1998). Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 281, 1677-1679.
Chao, C., Saito, S., Kang, J., Anderson, C.W., Appella, E., and Xu, Y. (2000). p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage. EMBO J. 19, 4967-4975.
Chehab, N.H., Malikzay, A., Appel, М., and Halazonetis, T.D. (2000). Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev. 14, 278-288.
Espinosa, J.М., and Emerson, B.M. (2001). Transcriptional regulation by p53 through intrinsic DNA/chromatin binding and site-directed cofactor recruitment. Mol. Cell 8, 57-69.
Gartel, A.L., and Tyner, A.L. (1999). Transcriptional regulation of the p21 (Waf1/CIP1) gene. Exp. Cell Res. 246, 280-289.
Giaccia, A.J., and Kastan, M.B. (1998). The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev. 12, 2973-2983.
Gu, W., and Roeder, R.G. (1997). Activation of p53 sequence-specific DNA binding by acetylation of the p53 С-terminal domain. Cell 90, 595-606.
Gu, W., Shi, X.L., and Roeder, R.G. (1997). Synergistic activation of transcription by СВР and p53. Nature 387, 819-823.
Halazonetis, T.D., Davis, L.J., and Kandil, A.N. (1993). Wild-type p53 adopts a ‘mutant’-like conformation when bound to DNA. EMBO J. 12, 1021-1028.
Haupt, Y., Maya, R., Kazaz, A., and Oren, M. (1997). Mdm2 promotes the rapid degradation of p53. Nature 387, 296-299.
Hirao, A., Kong, Y.Y., Matsuoka, S., Wakeham, A., Ruland, J., Yo-shida, H., Liu, D., Elledge, S.J., and Mak, T.W. (2000). DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287, 1824-1827.
Hupp, Т., and Lane, D. (1994). Allosteric activation of latent p53 tetramers. Curr. Biol. 4, 865-875.
Hupp, Т., Meek, D., Midgley, C., and Lane, D. (1992). Regulation of the specific DNA binding function of p53. Cell 71, 875-886.
Ikura, Т., Ogryzko, V.V., Grigoriev, М., Groisman, R., Wang, J., Hori-koshi, М., Scully, R,, Qin, J., and Nakatani, Y. (2000). Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis. Cell 102, 463-473.
Ishov, A.М., and Maul, G.G. (1996). The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition. J. Cell Biol. 134, 815-826.
Jimenez, G.S., Nister, М., Stommel, J.M., Beeche, М., Barcarse, E.A., Zhang, X.Q., О’Gorman, S., and Wahl, G.M. (2000). A transactivation-deficient mouse model provides insights into Trp53 regulation and function. Nat. Genet. 26, 37-43.
Ко, L.J., and Prives, C. (1996). p53: puzzle and paradigm. Genes Dev. 10, 1054-1072.
Ко, L.J., Shieh, S.Y., Chen, X., Jayaraman, L,, Tamai, K., Taya, Y., Prives, C., and Pan, Z.Q. (1997). p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner. Mol. Cell. Biol. 17,7220-7229.
Kouzarides, T. (1999). Histone acetylases and deacetylases in cell proliferation. Curr. Opin. Genet. Dev. 9, 40-48.
Kubbutat, M.H., Jones, S.N., and Vousden, K.H. (1997). Regulation of p53 stability by Mdm2. Nature 387, 299-303.
Kuo, M.H., and Allis, C.D. (1998). Roles of histone acetyltransf erases and deacetyl ases in gene regulation. Bioessays 20, 615-626.
Lakin, N.D., and Jackson, S.P. (1999). Regulation of p53 in response to DNA damage. Oncogene 18, 7644-7655.
Lambert, P.F., Kashanchi, F., Radonovich, M.F., Shiekhattar, R., and Brady, J.N. (1998). Phosphorylation of p53 serine 15 increases interaction with СВР. J. Biol. Chem. 273, 33048-33053.
Lees-Miller, S.P., Sakaguchi, K., Ullrich, S.J., Appella, E., and Anderson, C.W. (1992). Human DNA-activated protein kinase phosphory-lates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol. Cell. Biol. 12, 5041-5049.
Lill, N.L., Grossman, S.R., Ginsberg, D., DeCaprio, J., and Livingston, D.M. (1997). Binding and modulation of p53 by рЗОО/CBP coactivators. Nature 387, 823-827.
Liu, L., Scolnick, D.M., Trievel, R.C., Zhang, H.B., Marmorstein, R,, Halazonetis, T.D., and Berger, S.L. (1999). p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage. Mol. Cell. Biol. 19, 1202-1209.
Luo, J., Su, F., Chen, D., Shiloh, A., and Gu, W. (2000). Deacetylation of p53 modulates its effect on cell growth and apoptosis. Nature 408, 377-381.
Martinez, E,, Palhan, V.B., Tjernberg, A., Lymar, E.S., Gamper, A.M., Kundu, Т.К., Chait, B.T., and Roeder, R.G. (2001). Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. Mol. Cell. Biol. 21, 6782-6795.
McMahon, S.B., Van Buskirk, H.A., Dugan, K.A., Copeland, T.D., and Cole, M.D. (1998). The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins. Cell 94, 363-374.
Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H., and Nakatani, Y. (1996). The transcriptional coactivators p300 and СВР are histone acetyltransf erases. Cell 87, 953-959.
Orlando, W., Strutt, H., and Paro, R. (1997). Analysis of chromatin structure by in vivo formaldehyde cross-linking. Methods 11, 205-214.
Prives, C., and Hall, P.A. (1999). The p53 pathway. J. Pathol. 187, 112-126.
Rachez, C., Gamble, М., Chang, C.P., Atkins, G.B., Lazar, M.A., and Freedman, L.P. (2000). The DRIP complex and SRC-1/p160 coactivators share similar nuclear receptor binding determinants but constitute functionally distinct complexes. Mol. Cell. Biol. 20, 2718-2726.
Sakaguchi, K., Herrera, J.E., Saito, S., Miki, Т., Bustin, М., Vassilev, A., Anderson, C.W., and Appella, E. (1998). DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev. 12, 2831-2841.
Scolnick, D., Chehab, N., Stavridi, E., Lien, М., Caruso, L., Moran, E., Berger, S., and Halazonetis, T. (1997). CREB-binding protein and рЗОО/CBP-associated factor are transcriptional coactivators of the p53 tumor suppressor protein. Cancer Res. 57, 3693-3696.
Shieh, S.Y., Ikeda, М., Taya, Y., and Prives, C. (1997). DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell 91, 325-334.
Shieh, S.Y., Ahn, J., Tamai, K., Taya, Y., and Prives, C. (2000). The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. Genes Dev. 14, 289-300.
Stavridi, E.S., Chehab, N.H., Caruso, L.C., and Halazonetis, T.D. (1999). Change in oligomerization specificity of the p53 tetrameriza-tion domain by hydrophobic amino acid substitutions. Protein Sci. 8, 1773-1779.
Stavridi, E.S., Chehab, N.H., Malikzay, A., and Halazonetis, T.D. (2001). Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest. Cancer Res. 61, 7030-7033.
Sterner, D.E., and Berger, S.L. (2000). Acetylation of hi stones and transcription-related factors. Microbiol. Mol. Biol. Rev. 64,435-459.
Szak, S.T., Mays, D., and Pietenpol, J.A. (2001). Kinetics of p53 binding to promoter sites in vivo. Mol. Cell. Biol. 21, 3375-3386.
Tibbetts, R.S., Brumbaugh, K.M., Williams, J.M., Sarkaria, J.N., Cliby, W.A., Shieh, S.Y., Taya, Y., Prives, C., and Abraham, R.T. (1999). A role for ATR in the DNA damage-induced phosphorylation of p53. Genes Dev. 13, 152-157.
Vassilev, A., Yamauchi, J., Kotani, Т., Prives, C., Avantaggiati, M.L., Qin, J., and Nakatani, Y. (1998). The 400 kDa subunit of the PCAF hist one acetyl ase complex belongs to the ATM superfamily. Mol. Cell 2, 869-875.
Vogel stein, B., Lane, D., and Levine, A.J. (2000). Surfing the p53 network. Nature 408, 307-310.
Wang, Y., and Prives, C. (1995). Increased and altered DNA binding of human p53 by S and G2/M but not G1 cy cl in-dependent kinases. Nature 376, 88-91.
Waterman, J.L., Shenk, J.L., and Halazonetis, T.D. (1995). The dihedral symmetry of the p53tetramerization domain mandates a conformational switch upon DNA binding. EM BO J. 14, 512-519.
Waterman, M.J., Waterman, J.L., and Halazonetis, T.D. (1996). An engineered four-stranded coiled coil substitutes for the tetrameriza-tion domain of wild-type p53 and alleviates transdominant inhibition by tumor-derived p53 mutants. Cancer Res. 56, 158-163.
Zheng, L., Pan, H., Li, S., Flesken-Nikitin, A., Chen, P.L., Boyer, T.G., and Lee, W.H. (2000). Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1. Mol. Cell 6, 757-768.