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P53-Mediated Repression of Nuclear Factor-Kbreia Via The [CANCER RESEARCH 58. 4531-4536. October 15. 1998] Advances in Brief p53-mediated Repression of Nuclear Factor- KB ReiA via the Transcriptional Integrator Rajani Ravi,2 Bijoyesh Mookerjee,2 Yvette van Hensbergen, Cauri C. Bedi, Antonio Giordano, Wafik S. El-Deiry, Ephraim J. Fuchs, and Atul Bedi3 Johns Hopkins Oncology Center. The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-8967 ¡R.R.. B. M., Y. \: H.. C. C. B., E. J. F.. A. B.I: Sbarro Institute for Cancer Research and Molecular Medicine. Thomas Jefferson University. Philadelphia. Pennsylvania 19107 [A. C.!: and Howard Hughes Medical Institute. University of Pennsylvania School of Medicine, Philadelphia. Pennsylvania 19104 [W. S. E-D.j Abstract the transcriptional activation domain of the RelA (p65) subunit of NF-KB (9, 10), a family of heterodimeric transcription factors that Thf /)5J tumor suppressor gene plays an instrumental role in transcrip- regulate immune, inflammatory, and stress responses (11). RelA tran tional regulation of target genes involved in cellular stress responses. scriptional activity is regulated by its interaction with p300 and is p53-dependent transactivation and transrepression require its interaction repressed by p300 binding proteins such as cyclinE-cdk2 (9, 10). with p300/CBP, a coactivator that also interacts with the RelA subunit of nuclear factor-KB. We find that p53 inhibits RelA-dependent transacti These observations prompted us to investigate whether the interaction vation without altering RelA expression or inducible KB-DNA binding. of p53 with p300 exerts a similar influence on RelA-dependent p53-mediated repression of RelA is relieved by p300 overexpression and transcriptional activity. the increased RelA activity conferred by p53-deficiency is counteracted by In this study, we show that p53 interferes with p300-dependent either transactivation domain-deficient p300 fragments that bind RelA or coactivation of RelA without altering NF-KB DNA binding activity. a transdominant mutant of lidt«. Our results suggest that p53 can We find that RelA interacts with two specific regions of the p300 regulate diverse KB-dependent cellular responses. coactivator, one located at the amino terminal and the other at a COOH-terminal site, which also binds p53. p53-mediated repression Introduction of RelA is overcome by enforced overexpression of p300 and the increased RelA activity conferred by p53-deficiency is counteracted Investigations of how p53 prevents the genesis or progression of by a transdominant negative phosphorylation-mutant I«Ba or p300 human neoplasia have focused on its role in surveillance mechanisms that regulate cell cycle progression, apoptosis, and angiogenesis (1). fragments that incorporate RelA binding sites, but lack the critical COOH-terminus transactivation domain. Our results suggest a mech Several lines of evidence suggest that p53 may execute its biological anism by which p53 can regulate diverse NF-«B-dependent cellular functions by transcriptional regulation of specific target genes. p53 is a multifunctional transcription factor that includes a transcriptional responses. activation domain (AA1-42) that is required for interaction with the basal transcriptional machinery and a sequence-specific DNA-binding Materials and Methods domain (AA102-292) that binds to a 20-bp consensus binding site (2). Cells and Cell Culture. PA-1 ovarian teratocarcinoma cells stably trans- In addition to sequence-specific transcriptional activation, p53 also fected with HPV 16 E6 or empty vector, generated as described (12). were represses genes, the promoters of which do not contain p53-binding cultured in Basal Eagle medium containing 0.5 mg/ml G418. p53+/+ and sites (3). The vast majority of missense mutations in human cancers p53-/- MEFs and RelA+/+ and RelA-/- mouse fibroblasts have been are clustered in the sequence-specific DNA binding domain and result described (13, 14). Fibroblasts of all genotypes were cultured in DMEM. in loss of its transcriptional regulatory function. Moreover, both EIA Thymocytes were isolated from homozygous p53-deficient transgenic mice, and T-antigen oncoproteins disrupt the transcriptional activity of p53 6-8 weeks of age (C57BL/6J-Trp53tmITyj: The Jackson Laboratory, Bar by binding to p300/CBP,4 a coactivator that is required for p53- Harbor, ME), and their age/sex-matched wild-type counterparts (C57BL/6) dependent transactivation and transrepression (4-7). In addition to and cultured in RPMI medium. All culture media were supplemented with 10% serving as a transcriptional adaptor, p300 mediates functional inter fetal bovine serum, 100 units/ml penicillin, and 100 ng/ml streptomycin sulfate. Cells were maintained in humidified atmosphere containing 5% CO2 actions between p53 and other transcriptional factors that interact with at 37°C. Ionizing radiation was delivered with a 137Cs dual source y-cell p300. For example, the association of p53 with p300 interferes with irradiator (Atomic Energy Commission. Canada). Human recombinant TNF-a coactivation of other p300-dependent factors such as AP-1 or hypoxia was purchased from Genzyme (Cambridge. MA). inducible factor-1 (6, 8). The amino-terminal of p300 interacts with Expression Vectors. CMVp53 (pC53-SN-3: Bert Vogelstein, The Johns Hopkins University. Baltimore. MD) has been described (15). The vectors Received 7/29/98; accepted 8/31/98. encoding full-length p300 (CMVp300) or the p300-deletion mutants [p300( 1- The costs of publication of this article were defrayed in part by the payment of page 742), p300( 1514-1922). p300(964-1922)) have been described (6). The plas- charges. This article must therefore be hereby marked advertisement in accordance with mid encoding RelA (pGD RelA) and the backbone plasmid (pGD) have been 18 U.S.C. Section 1734 solely to indicate this fact. 1Funded by Grant 1 R29CA71660-01A1 from the National Cancer Institute and Grant described (16). The plasmid expressing UBaM (pLIxBaMSN) and the empty R21 CA/ES66204 from the NIH (to A. B.). A. B. is a recipient of a Passano Physician control vector (pLXSN: Dr. Douglas Green. La Jolla Institute of Allergy and Scientist award, a Valvano Foundation Scholar award, a Jose Carreras American Society Immunology. La Jolla, CA). have been described (17). The HIV-CAT reporter of Hematology Scholar award, and grants from the American Cancer Society. 2 These authors contributed equally to this work. plasmid (Dr. Mira Jung, Georgetown University. Washington. DC) has been 1To whom requests for reprints should be addressed, at 3-120 Johns Hopkins Oncol described (18). The ß-galactosidase plasmid. pON260. was obtained from ogy Center. 600 North Wolfe Street. Baltimore, MD 21287-8967. Phone: (410) 614-3844: Promega (Madison, WI). Fax: (410) 955-1969: E-mail: [email protected]. Transfections and Reporter Assays. Cells were plated at —¿30%conflu 4 The abbreviations used are: CBP, CREB-binding protein; AP, activator protein; ence 16-24 hours before serum-free transfection (12-16 hours) with lipo- NF-KB, nuclear factor-KB; iKBa, inhibitor of KB; HIV-CAT. HIV-chloramphenicol acetyltransferase: HPV, human papilloma virus; TNF-a. tumor necrosis factor a: EMSA. fectamine (Life Technologies, Inc., Gaithersburg, MD). After the addition of electrophoretic mobility shift assays; C/H. cystidine/histidine rich. fresh medium supplemented with 10% fetal bovine serum, cells were cultured 4531 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1998 American Association for Cancer Research. p53-MEDlATED REPRESSION OF NF-*B RELA for 24-48 hours before harvest. Transfections for reporter assays were carried with excess cold wild-type or mutant oligonucleotide. Samples were loaded on out using a reporter activator DNA ratio of 1:2, using the quantities indicated a 5% polyacrylamide gel, electrophoresed, and analyzed by autoradiography of in the figure legends. When necessary, total amount of transfected DNA was the dried gels. equalized using the appropriate control backbone plasmid (CMVO, LXSN. or pGD). CAT assays were performed using thin layer chromatography, and the Results results were quantitated as percentage conversion using a Phosphorlmager (Molecular Dynamics). All reporter assays were normalized to ß-galactosidase Repression of NF-KB RelA-dependent Transcription by p53. activity by cotransfection with ß-galactosidase plasmids (pON260). To determine whether RelA-dependent transactivation is influenced ¡nVitro Transcription and Translation. In vitro transcription and trans by p53, mouse embryonic fibroblasts of p53+/+ and p53—/—gen lations were performed using a TNT coupled reticulocyte lysate system (Pro- otypes were transfected with a HIV-CAT reporter, which is driven by mega). The p300 deletion mutants cloned into the Gal4 vector were transcribed two KB sites contained in the long terminal repeat (18). Assessment of using T7 RNA polymerase. Translation of the mRNAs was performed in rabbit reticulocyte lysates using [<5S]methionine (Amersham Corp.) according to the CAT activity in cellular extracts prepared 2 days after transfection, manufacturer's protocol. showed low HIV gene expression in p53+/+ MEFs, whereas p53-/— MEFs demonstrated comparatively strong activation of HIV- Immunoblotting and Immunoprecipitations. Cell lysates were prepared as described (19), and 50-100 ng of protein were resolved by SDS-PAGE, CAT (Fig. 1A). To directly examine the effect of p53 on the tran- transferred onto lmmobilon-P polyvinylidene diflouride membrane (Millipore,
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