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 Â¡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 lidtÂ«. 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 IÂ«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-Â«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 37Â°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 ÃŸ-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 â€”¿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 ÃŸ-galactosidase Repression of NF-KB RelA-dependent Transcription by p53. activity by cotransfection with ÃŸ-galactosidase plasmids (pON260). To determine whether RelA-dependent transactivation is influenced Â¡nVitro Transcription and Translation. In vitro transcription and trans by p53, mouse embryonic fibroblasts of p53+/+ and p53â€”/â€”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-/â€” 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, scriptional activity of NF-KB RelA, p53â€”/â€”MEFs were cotrans- Bedford, MA), and probed with appropriate dilutions of the following primary fected with the HIV-CAT reporter and a RelA expression vector (pGD antibodies: anti-NF-xB p65 (C-20), ami-NF-KBp65 (A), anti-NF-Â«Bp50 (nu RelA) with or without an expression vector encoding wild-type p53 clear localization sequence), anti-lKBa (C-12), anti-p53 (DO-1), anti-p300 (pC53-SN-3). Although RelA stimulated HIV-CAT activity when (C-20), anti-Actin (C-ll: Santa Cruz Biotechnology, Santa Cruz, CA), and transfected separately in p53â€”/â€”MEFs, RelA-induced transactiva anti-p53 (pAb421; Oncogene Science, Cambridge, MA). Immunoreactive pro tion was inhibited in a dose-dependent fashion by cotransfection of tein complexes were visualized with enhanced chemiluminescence detection the p53-encoding plasmid (Fig. \A). Cotransfection of p53â€”/â€”MEFs (Amersham Corp.). For immunoprecipitations, 300-500 /ig of whole cell with iKBaM, a combined NH2- and COOH-terminal phosphorylation lysates were precleared with protein A (Pharmacia, Piscataway, NJ) and incubated with 1 /Â¿g/ml primary antibody. The immune complexes were mutant IKBÂ«that resists degradation (17), inhibited RelA-mediated immunoprecipitated with protein A-Sepharose beads for at least 1 hour, and the stimulation of HIV-CAT, thereby confirming the dependence of re bound complexes were resolved by SDS-PAGE, transferred, and immuno- porter activity on NF-KB (Fig. 1/4). blotted as described above. The HPV16 E6 oncoprotein induces ubiquitin-dependent conjuga Electromobility Shift Assays. Nuclear extracts were prepared as described tion and degradation of p53 (20). To investigate the effect of endog (19). Double-stranded oligonucleotides containing either a consensus binding enous p53 on RelA-dependent transactivation, the PA-1 cell line was site for NF-KB (5'-GGGGACTTTCCC-3') or a mutant consensus sequence (G to C substitution; Santa Cruz Biotechnology) were 5' end-labeled using stably transfected with an expression vector encoding HPV-16 E6 polynucleotide kinase and [-y-1:!P]dATP. Nuclear extracts (2.5-5 /xg) were (PA-1 E6) or empty vector (PA-1 Neo; Ref. 12). PA-1 Neo or PA-1 incubated with ~1 /nl of labeled oligonucleotide (20,000 cpm) in 20 fi\ E6 cells were cotransfected with the HIV-CAT reporter and either incubation buffer (10 ITIMTris, 40 rtiM NaCl. 1 mM EDTA, l mM ÃŸ-mercap- exposed to ionizing radiation (10 Gy) or left unirradiated. PA-1 neo toethanol. 2% glycerol, and 1-2 ng of poly dl-dC) for 20 minutes at 25Â°C.The cells exhibited only weak stimulation of KB-dependent transcription specificity of NF-Â«B DNA-binding activity was confirmed by competition that was completely silenced in response to irradiation (Fig. IÃŸ).In

Fig. I. Repression of NF-KB RelA-dependent transcriptional ac tivity by p53. A, effect of p53 on KB-dependent HIV-CAT gene expression. p53-/- or p53-t-/+ MEFs were transfected with the HIV-CAT reporter and the poN260 ÃŸ-galactosidase expression vec tor. p53-/- MEFs were cotransfected with HIV-CAT and a RelA- expression vector (pGD RelA). together with plasmids encoding either p53 (pC53-SN-3; 0.5 Â¿igand l /ig) or IKBaM (pLlKBaMSN). 1 2345 Transfections were carried out using reporter and activator DNA in % Conv.: 5 37 51 4 1 a 1:2 ratio, and a control plasmid (pLXSN) was included to equalize Mouse Embryonic Fibroblasts the total amounts of expression vectors contained in each condition. HIV-CAT activity was quantitated as percentage conversion and represented as values normali/ed relative to ÃŸ-galactosidase. B. siimulalion of KB-dependent HIV-l gene expression by HPV-E6. PA-1 Neo and PA-1 E6 cells were transfected with the HIV-CAT P53+/+ reporter construct and poN260 ÃŸ-galactosidase expression vector. p53-/- Twenty-four hours after transfection, cells were irradiated ( 10 Gy) or left untreated, followed by assessment of HIV-CAT expression 12 hours later. HIV-CAT activity was normalized relative to ÃŸ-galac tosidase, and values were expressed as percentage conversion.

4532

MEF B PA-1 Neo PA-1 E6

MEFs Thymocytes Â«Â»teto

â€¢¿â€¢â€¢â€¢Â§â€¢â€¢â€¢ Irradiation ++++ --- + + + + -+- + - + TNF-a -++ ++ -++ + + Time(min)o459045904590 o 459045904590 o 90o so o 90 100 C PA-1 Neo PA-1 E6

O PA-1 Neo â€¢¿PA-1E6

IR TNF-a HIV-CAT

Fig. 2. p53 does not alter RelA expression or inducible NF-KB-DNA binding. A, RelA-dependent expression of endogenous 1KBÂ«isrepressed by p53. Immunoblot analyses of RelA und iKBa expression in RelAâ€”/â€”orReIA+/+ mouse fibroblasts, p53â€”/â€”orp53+/+ mouse thymocytes, and PA-1 Neo or PA-1 E6 cells. B, effect of p53 on inducible NF-*B DNA binding activity. Nuclear extracts of PA-1 Neo and PA-1 E6 cells after treatment with 10 Gy of irradiation in the presence or absence of 50 ng/ml of TNF-a were analyzed by EMSA. EMSA of nuclear extracts was prepared from irradiated (90 minutes after 10 Gy) and unirradiated p53+/+ or p53-/â€” MEF or from MEFs stably transfected with iKBaM. C, effect of HPV16-E6 on TNF-a-induced KB-dependent gene expression. PA-1 Neo and PA-t E6 cells transfected with the poN260 ÃŸ-galactosidase expression vector with or without the HIV-CAT reponer gene construct were treated 24 hours after transfection with either ionizing radiation ( 10 Gy) with or without TNF-a (50 ng/ml) and analyzed for HIV-CAT activity 12 hours later. HIV-CAT activity was normalized relative to 0-galactosidase, and values were expressed as percentage conversion. contrast, PA-1 E6 cells displayed a higher constitutive HIV-CAT their p53-deficient counterparts (Fig. 2B). In contrast, the repression expression that was not diminished by irradiation. These data confirm of HIV-CAT in MEFs transfected with IxBaM was associated with a that endogenous p53 represses RelA-dependent transactivation. reduction in KB DNA binding activity in the same experiments. These The IxBa gene promoter contains Â«Bmotifs that are stimulated by data suggest that unlike IKBa, p53 inhibits RelA transactivating RelA (21). In addition to transcriptional activation, RelA also in ability through a mechanism independent of NF-KB DNA binding creases IÂ«Baexpression by protein stabilization, thereby participating activity. in an inducible autoregulatory pathway (22). Compared with Association of p300 with p53 and RelA. The transcriptional ac RelA+/+ fibroblasts, their counterparts derived from RelAâ€”Iâ€” tivity of both p53 and RelA depend upon their respective interactions mouse embryos demonstrate a reduction in IxBa expression (Fig. 2A). with the p300 coactivator. We first defined the specific regions of Because IÂ«Baexpression reflects endogenous RelA activity, we next p300 that interact with p53 and RelA. 35S-methionine-labeled in examined the expression of IKBa in PA-1 Neo and PA-1 E6 cells. vf/ro-translated proteins were generated from p300 deletion mutants Consistent with the repression of basal Â«B-reporter activity by p53, using a transcription/translation reticulocyte-based system. The p300 IKBa expression was reduced in PA-1 Neo cells compared with PA-1 deletion mutants were chosen to define the avidity of the interaction E6 cells. Analogous results were obtained in thymocytes isolated from of p53 or RelA with each of the specific functional cysteine/histidine- p53+/+ and p53â€”/â€”mice. Immunoblot analyses showed a compar rich domains of p300 (Fig. 3/1 and fi). Endogenous p53 was immu- atively reduced basal expression of IÂ«Ba in p53+/+ thymocytes noprecipitated from cellular extracts of wild-type mouse embryonic compared with their p53-deficient counterparts (Fig. 2A). These data fibroblasts (p53+/+) or their p53â€”/â€”counterparts mixed with each demonstrate that p53 represses RelA-dependent expression of IxBa in w'rro-translated p300 deletion protein. p53 immune complexes were and indicate that IÂ«Ba does not mediate p53-induced repression of found to contain p300(1514-1922), but not p300(l-742)(Fig. 3D). RelA. These data confirm that the p53 binding site on p300 resides between p53 Does Not Alter RelA Protein Levels or Inducible NF-icB- amino acids 1514 and 1922, a region immediately upstream of and DNA Binding Activity. p53 did not alter the levels of expression of overlapping the C/H3 domain. To identify the RelA-interacting re RelA in either PA-1 cells or thymocytes (Fig. 2A). Unlike TNF-a, gions, in i'Ã/ro-translated fragments of p300 were added to cell extracts stimulation of RelA-dependent transactivation by expression of E6 in prepared from fibroblasts derived from RelA-deficient mouse em PA-1 cells was not attended with any significant increase in Â«B-DNA bryos (RelA-/-) or wild-type mouse fibroblasts (RelA+/+). and binding activity in EMSA (Fig. 2B). In addition, PA-1 Neo and PA-1 immunoprecipitated with an anti-RelA antibody. Anti-RelA antibody- E6 cells demonstrated no difference in TNF-a-induced stimulation of precipitated complexes were found to avidly bind the amino terminal Â«B-DNAbinding. However, PA-1 Neo cells stimulated with TNF-a p300( 1-742) fragment, and to a lesser extent, the deletion mutants demonstrated a significantly lower activation of HIV-CAT compared p300 (1514-1922) and p300 (964-1922; Fig. 3Q. These results with PA-1 E6 cells (Fig. 2Q. Similarly, EMSA demonstrated no demonstrate that RelA interacts with two regions of p300, one incor differences in KB DNA-binding activity between wild-type MEFs and porating the amino terminal containing the C/HI domain, and the 4533

220-i" 65 Nuclear 45 - Receptor STATIa Genotype RelA, + - + - + â€”¿ FVW c-Jun 'c-Fos P/CAF ' STATto c Myti Input STATIÂ« TFW and-RelA (1-742) MyoO RNA polymerase II RNA helicase A pA.I PA-1 PA-1 PA-1 C/HI (864.1922) Neo E6 Neo Ea IR - IR P300 )â€¢â€¢â€¢

IP ita Western anti-p300 l P anti-p300 l P anti-RelA L Western anti-p300 Western anti-RelA Fig. 3. Association of p300 with p53 and NF-KB RelA. A. schematic representation of full-length p30() depicting the three C/H domains (C/Hl, C/H2. and C/H3) and their interactions. The p53-binding region is included between AA 1514 and 1922. The three deletion mutants used (p300 (1-742). p300 (964-1922). and p300 (1514-1922)] and the interaction domains included in these fragments are indicated. B. in ri/ro-translated proteins derived from p300 deletion mutants shown in A. C. regions of p300 that interact wilh RelA. In firm-translated t5S-labeled proteins shown in B were added to protein extracts derived from RelAâ€”/- ( â€”¿)orRelA+/+ ( + ) mouse fibroblasts. and anti-RelA immunoprecipitates were resolved by 89ÃSDS-PAGE. To assess the avidity of interaction between RelA and each deletion fragment, equivalent input quantities of each labeled deletion protein shown in fl were used. 0. inleraction of p53 with the region of p300 between AA 1514-1922. In riVro-translated "S-labeled proteins p300( 1-742) and p300( 1514-1922) were added to protein extracts derived from p53-/- (-) or p53+/+ (+ ) MEFs. and anti-p53 immunoprecipitates were resolved by 10% SDS-PAGE. Â£.effect of HPV E6 on the association between p300 and RelA in vi\'i>.Protein extracts prepared from PA-1 Neo or PA-1 E6 cells, before and 1 hour after ioni/ing radiation (10 Gy). were incubated with either anti-RelA or control (C) antibodies, and RclA-associated immune complexes were analy/cd by immunoblotting with anti-p300. F, effect of HPV E6 on endogenous complexes of RelA and p300. p3(K)-immunoprecipilales from protein extracts prepared as in Â£were immunohlotted with anti-RelA antibody. C. immunoblot analysis of immune complexes precipitated by a control antibody uintirahhit IgG). G. detection of Re!A-p300 complexes in p53+/ + and p53â€”/â€”mouse thymocytes. Tap, immunoblot analyses of extracts prepared from thymocytes before and I hour after irradiation using an anti-p3(M) antibody. Bollimi. anti-RelA immune complexes precipitated from thymocyte extracts as in E and immunoblotted with anti-p3(X) antibody.

other overlapping with the p53-binding site adjacent to the C/H3 sites of p300 may facilitate its dimerization by overcoming the steric El A-interacting domain. constraint imposed by COOH-terminal domain (24). In either sce We next examined whether p53 influences complex formation nario, binding of RelA by p300 fragments that do not contain both the between RelA and p300. Protein extracts from untreated or irradiated binding sites would be expected to sequester RelA and repress its PA-1 Neo or PA-1 E6 cells were immunoprecipitated with anti-RelA coactivation. Cotransfection of p300( 1514-1922) resulted in strong or control antibodies, and the immune complexes were immuno repression of RelA-stimulated HIV-CAT activity in both PA-1 Neo blotted with an antibody against p300. p300 was detected at equiva and PA-1-E6 cells (Fig. 4). HIV-CAT expression in p53-/- cells lent levels in anti-RelA immune complexes precipitated from both was also inhibited by the p300( 1-742) deletion mutant that binds unirradiated and irradiated cell extracts of either PA-1 Neo or PA-1 RelA but lacks the p53-binding site, the C/H3 domain, and the E6 cells (Fig. 35). To confirm the interaction between RelA and p300, COOH-terminus. The inhibition of RelA activity by coexpression of anti-p3(X) immunoprecipitates from cells extracts were immuno either p300 fragment was similar to that observed with introduction of blotted with antibodies against RelA or the nuclear localization se IÂ«BaM. Conversely, transfection of the vector encoding full length quence region of NF-KB p50. RelA was detected in immune com p300 (pGal4p300) into PA-1 neo cells resulted in a dose-dependent plexes precipitated by anti-p300, but not by the control antibody (Fig. increase in RelA activity, such that high concentrations of p300 3F). In contrast to RelA, NF-Â«Bp50 was not found in p300 immune restored HIV-CAT expression to levels observed in PA-1 cells ex complexes (data not shown). The association between RelA and p300 pressing E6 or PA-1 neo cells transfected with the RelA-expression was not influenced by p53 status or irradiation (Fig. 3Â£and 3>F). vector (Fig. 4). The restoration of RelA activity in PA-1 Neo cells by Analogous to PA-1 cells, equivalent levels of p300 were observed in overexpression of p300, together with the repression of RelA in PA-1 anti-RelA immune complexes precipitated from extracts derived from E6 cells by dominant negative fragments of p300, indicate that p53- either untreated or irradiated p53-/- or p53+/+ thymocytes (Fig. mediated repression of RelA activity is mediated via interference with 3G). Since p53 did not influence expression of p300 (Fig. 3G), these the coactivating function of p300. data confirm that p53 does not prevent complex formation between RelA and p300. Discussion p53 Represses p300-mediated Coactivation of RelA. Since both p53 and RelA are present in complex with p3(X), we next examined p53-mediated transrepression is believed to involve protein-protein whether p53 influences p300-dependent coactivation of RelA. RelA interactions with basal components of the transcriptional apparatus interacts with two discrete regions of p300 (AA1 and 742, and A1514 such as TATA-associated factors (TFIID) and the transcriptional and 1922). Since the COOH-terminal region between amino acids coactivator/integrator p300 (6). p300 and its highly related family 1514 and 1922 also interacts with basal transcription factors such as member CBP contain well conserved regions, such as the C/H do RNA polymerase 11and RNA helicase A (23), it is possible that the mains, that interact with several sequence-specific transcription fac binding of RelA with this region is responsible for its coactivation via tors and nuclear receptors (25). The coactivation of specific signal- recruitment of these cofactors. Alternatively, binding of RelA to both dependent transcriptional events by p300 involves recruitment of 4534

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Rajani Ravi, Bijoyesh Mookerjee, Yvette van Hensbergen, et al.

Cancer Res 1998;58:4531-4536.

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