1998;58:4531-4536. Published online October 1, 1998.Cancer Res Rajani Ravi, Bijoyesh Mookerjee, Yvette van Hensbergen, et al. Transcriptional Integrator p300

B RelA via theκp53-mediated Repression of Nuclear Factor-

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[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. Universityof Pennsylvania School of Medicine, Philadelphia. Pennsylvania 19104 [W. S. E-D.j

Abstract

Thf /)5J tumor suppressor gene plays an instrumental role in transcrip-

tional regulation of target genes involved in cellular stress responses.p53-dependent transactivation and transrepression require its interaction

with p300/CBP, a coactivator that also interacts with the RelA subunit ofnuclear factor-KB. We find that p53 inhibits RelA-dependent transactivation without altering RelA expression or inducible KB-DNA binding.p53-mediated repression of RelA is relieved by p300 overexpression andthe increased RelA activity conferred by p53-deficiency is counteracted byeither transactivation domain-deficient p300 fragments that bind RelA or

a transdominant mutant of lidt«. Our results suggest that p53 canregulate diverse KB-dependent cellular responses.

Introduction

Investigations of how p53 prevents the genesis or progression ofhuman neoplasia have focused on its role in surveillance mechanismsthat regulate cell cycle progression, apoptosis, and angiogenesis (1).Several lines of evidence suggest that p53 may execute its biologicalfunctions by transcriptional regulation of specific target genes. p53 isa multifunctional transcription factor that includes a transcriptionalactivation domain (AA1-42) that is required for interaction with thebasal transcriptional machinery and a sequence-specific DNA-bindingdomain (AA102-292) that binds to a 20-bp consensus binding site (2).In addition to sequence-specific transcriptional activation, p53 alsorepresses genes, the promoters of which do not contain p53-binding

sites (3). The vast majority of missense mutations in human cancersare clustered in the sequence-specific DNA binding domain and result

in loss of its transcriptional regulatory function. Moreover, both EIAand T-antigen oncoproteins disrupt the transcriptional activity of p53by binding to p300/CBP,4 a coactivator that is required for p53-

dependent transactivation and transrepression (4-7). In addition to

serving as a transcriptional adaptor, p300 mediates functional interactions between p53 and other transcriptional factors that interact withp300. For example, the association of p53 with p300 interferes withcoactivation of other p300-dependent factors such as AP-1 or hypoxiainducible factor-1 (6, 8). The amino-terminal of p300 interacts with

Received 7/29/98; accepted 8/31/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Funded by Grant 1 R29CA71660-01A1 from the National Cancer Institute and Grant

R21 CA/ES66204 from the NIH (to A. B.). A. B. is a recipient of a Passano PhysicianScientist award, a Valvano Foundation Scholar award, a Jose Carreras American Societyof Hematology Scholar award, and grants from the American Cancer Society.

2 These authors contributed equally to this work.1To whom requests for reprints should be addressed, at 3-120 Johns Hopkins Oncol

ogy Center. 600 North Wolfe Street. Baltimore, MD 21287-8967. Phone: (410) 614-3844:Fax: (410) 955-1969: E-mail: rbedi@welchlink.welch.jhu.edu.

4 The abbreviations used are: CBP, CREB-binding protein; AP, activator protein;

NF-KB, nuclear factor-KB; iKBa, inhibitor of KB; HIV-CAT. HIV-chloramphenicolacetyltransferase: HPV, human papilloma virus; TNF-a. tumor necrosis factor a: EMSA.electrophoretic mobility shift assays; C/H. cystidine/histidine rich.

the transcriptional activation domain of the RelA (p65) subunit ofNF-KB (9, 10), a family of heterodimeric transcription factors that

regulate immune, inflammatory, and stress responses (11). RelA transcriptional activity is regulated by its interaction with p300 and isrepressed by p300 binding proteins such as cyclinE-cdk2 (9, 10).

These observations prompted us to investigate whether the interactionof p53 with p300 exerts a similar influence on RelA-dependent

transcriptional activity.In this study, we show that p53 interferes with p300-dependent

coactivation of RelA without altering NF-KB DNA binding activity.

We find that RelA interacts with two specific regions of the p300coactivator, one located at the amino terminal and the other at aCOOH-terminal site, which also binds p53. p53-mediated repression

of RelA is overcome by enforced overexpression of p300 and theincreased RelA activity conferred by p53-deficiency is counteractedby a transdominant negative phosphorylation-mutant I«Ba or p300

fragments that incorporate RelA binding sites, but lack the criticalCOOH-terminus transactivation domain. Our results suggest a mechanism by which p53 can regulate diverse NF-«B-dependent cellular

responses.

Materials and Methods

Cells and Cell Culture. PA-1 ovarian teratocarcinoma cells stably trans-

fected with HPV 16 E6 or empty vector, generated as described (12). werecultured in Basal Eagle medium containing 0.5 mg/ml G418. p53+/+ andp53-/- MEFs and RelA+/+ and RelA-/- mouse fibroblasts have been

described (13, 14). Fibroblasts of all genotypes were cultured in DMEM.Thymocytes were isolated from homozygous p53-deficient transgenic mice,6-8 weeks of age (C57BL/6J-Trp53tmITyj: The Jackson Laboratory, BarHarbor, ME), and their age/sex-matched wild-type counterparts (C57BL/6)

and cultured in RPMI medium. All culture media were supplemented with 10%fetal bovine serum, 100 units/ml penicillin, and 100 ng/ml streptomycinsulfate. Cells were maintained in humidified atmosphere containing 5% CO2at 37°C. Ionizing radiation was delivered with a 137Cs dual source y-cell

irradiator (Atomic Energy Commission. Canada). Human recombinant TNF-a

was purchased from Genzyme (Cambridge. MA).Expression Vectors. CMVp53 (pC53-SN-3: Bert Vogelstein, The Johns

Hopkins University. Baltimore. MD) has been described (15). The vectorsencoding full-length p300 (CMVp300) or the p300-deletion mutants [p300( 1-742), p300( 1514-1922). p300(964-1922)) have been described (6). The plas-

mid encoding RelA (pGD RelA) and the backbone plasmid (pGD) have beendescribed (16). The plasmid expressing UBaM (pLIxBaMSN) and the emptycontrol vector (pLXSN: Dr. Douglas Green. La Jolla Institute of Allergy andImmunology. La Jolla, CA). have been described (17). The HIV-CAT reporter

plasmid (Dr. Mira Jung, Georgetown University. Washington. DC) has beendescribed (18). The ß-galactosidase plasmid. pON260. was obtained from

Promega (Madison, WI).Transfections and Reporter Assays. Cells were plated at —¿�30%conflu

ence 16-24 hours before serum-free transfection (12-16 hours) with lipo-

fectamine (Life Technologies, Inc., Gaithersburg, MD). After the addition offresh medium supplemented with 10% fetal bovine serum, cells were cultured

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p53-MEDlATED REPRESSION OF NF-*B RELA

for 24-48 hours before harvest. Transfections for reporter assays were carried

out using a reporter activator DNA ratio of 1:2, using the quantities indicatedin the figure legends. When necessary, total amount of transfected DNA wasequalized using the appropriate control backbone plasmid (CMVO, LXSN. orpGD). CAT assays were performed using thin layer chromatography, and theresults were quantitated as percentage conversion using a Phosphorlmager(Molecular Dynamics). All reporter assays were normalized to ß-galactosidaseactivity by cotransfection with ß-galactosidase plasmids (pON260).

¡nVitro Transcription and Translation. In vitro transcription and translations were performed using a TNT coupled reticulocyte lysate system (Pro-

mega). The p300 deletion mutants cloned into the Gal4 vector were transcribedusing T7 RNA polymerase. Translation of the mRNAs was performed in rabbitreticulocyte lysates using [<5S]methionine (Amersham Corp.) according to themanufacturer's protocol.

Immunoblotting and Immunoprecipitations. Cell lysates were preparedas described (19), and 50-100 ng of protein were resolved by SDS-PAGE,transferred onto lmmobilon-P polyvinylidene diflouride membrane (Millipore,

Bedford, MA), and probed with appropriate dilutions of the following primaryantibodies: anti-NF-xB p65 (C-20), ami-NF-KBp65 (A), anti-NF-«Bp50 (nuclear localization sequence), anti-lKBa (C-12), anti-p53 (DO-1), anti-p300(C-20), anti-Actin (C-ll: Santa Cruz Biotechnology, Santa Cruz, CA), andanti-p53 (pAb421; Oncogene Science, Cambridge, MA). Immunoreactive pro

tein complexes were visualized with enhanced chemiluminescence detection(Amersham Corp.). For immunoprecipitations, 300-500 /ig of whole cell

lysates were precleared with protein A (Pharmacia, Piscataway, NJ) andincubated with 1 /¿g/ml primary antibody. The immune complexes wereimmunoprecipitated with protein A-Sepharose beads for at least 1 hour, and thebound complexes were resolved by SDS-PAGE, transferred, and immuno-

blotted as described above.Electromobility Shift Assays. Nuclear extracts were prepared as described

(19). Double-stranded oligonucleotides containing either a consensus bindingsite for NF-KB (5'-GGGGACTTTCCC-3') or a mutant consensus sequence (Gto C substitution; Santa Cruz Biotechnology) were 5' end-labeled usingpolynucleotide kinase and [-y-1:!P]dATP. Nuclear extracts (2.5-5 /xg) were

incubated with ~1 /nl of labeled oligonucleotide (20,000 cpm) in 20 fi\incubation buffer (10 ITIMTris, 40 rtiM NaCl. 1 mM EDTA, l mM ß-mercap-toethanol. 2% glycerol, and 1-2 ng of poly dl-dC) for 20 minutes at 25°C.The

specificity of NF-«B DNA-binding activity was confirmed by competition

with excess cold wild-type or mutant oligonucleotide. Samples were loaded on

a 5% polyacrylamide gel, electrophoresed, and analyzed by autoradiography ofthe dried gels.

Results

Repression of NF-KB RelA-dependent Transcription by p53.To determine whether RelA-dependent transactivation is influencedby p53, mouse embryonic fibroblasts of p53+/+ and p53—/—gen

otypes were transfected with a HIV-CAT reporter, which is driven by

two KB sites contained in the long terminal repeat (18). Assessment ofCAT activity in cellular extracts prepared 2 days after transfection,showed low HIV gene expression in p53+/+ MEFs, whereasp53-/— MEFs demonstrated comparatively strong activation of HIV-

CAT (Fig. 1A). To directly examine the effect of p53 on the tran-scriptional activity of NF-KB RelA, p53—/—MEFs were cotrans-

fected with the HIV-CAT reporter and a RelA expression vector (pGDRelA) with or without an expression vector encoding wild-type p53(pC53-SN-3). Although RelA stimulated HIV-CAT activity whentransfected separately in p53—/—MEFs, RelA-induced transactiva

tion was inhibited in a dose-dependent fashion by cotransfection ofthe p53-encoding plasmid (Fig. \A). Cotransfection of p53—/—MEFs

with iKBaM, a combined NH2- and COOH-terminal phosphorylationmutant IKB«that resists degradation (17), inhibited RelA-mediatedstimulation of HIV-CAT, thereby confirming the dependence of reporter activity on NF-KB (Fig. 1/4).

The HPV16 E6 oncoprotein induces ubiquitin-dependent conjuga

tion and degradation of p53 (20). To investigate the effect of endogenous p53 on RelA-dependent transactivation, the PA-1 cell line wasstably transfected with an expression vector encoding HPV-16 E6(PA-1 E6) or empty vector (PA-1 Neo; Ref. 12). PA-1 Neo or PA-1E6 cells were cotransfected with the HIV-CAT reporter and eitherexposed to ionizing radiation (10 Gy) or left unirradiated. PA-1 neocells exhibited only weak stimulation of KB-dependent transcription

that was completely silenced in response to irradiation (Fig. Iß).In

Fig. I. Repression of NF-KB RelA-dependent transcriptional activity by p53. A, effect of p53 on KB-dependent HIV-CAT geneexpression. p53-/- or p53-t-/+ MEFs were transfected with theHIV-CAT reporter and the poN260 ß-galactosidase expression vector. p53-/- MEFs were cotransfected with HIV-CAT and a RelA-

expression vector (pGD RelA). together with plasmids encodingeither p53 (pC53-SN-3; 0.5 ¿igand l /ig) or IKBaM (pLlKBaMSN).Transfections were carried out using reporter and activator DNA ina 1:2 ratio, and a control plasmid (pLXSN) was included to equalizethe total amounts of expression vectors contained in each condition.HIV-CAT activity was quantitated as percentage conversion andrepresented as values normali/ed relative to ß-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-CATreporter construct and poN260 ß-galactosidase expression vector.Twenty-four hours after transfection, cells were irradiated ( 10 Gy) orleft untreated, followed by assessment of HIV-CAT expression 12hours later. HIV-CAT activity was normalized relative to ß-galactosidase, and values were expressed as percentage conversion.

1% Conv.: 5

2345

37 51 4 1

Mouse Embryonic Fibroblasts

P53+/+p53-/-

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p53-MEDlATED REPRESSION OF NF-KB RELA

BMEF

PA-1 Neo PA-1 E6

MEFs Thymocytes

«»teto

100

•¿�•••§•••Irradiation ++++ --- + + + + -+- + - +

TNF-a -++ ++ -++ + +

Time(min)o 459045904590 o 459045904590 o 90o so o 90

C PA-1 Neo PA-1 E6

O PA-1 Neo

•¿�PA-1 E6

IRTNF-a

HIV-CAT

Fig. 2. p53 does not alter RelA expression or inducible NF-KB-DNA binding. A, RelA-dependent expression of endogenous 1KB«is repressed by p53. Immunoblot analyses of RelAund iKBa expression in RelA—/—or ReIA+/+ mouse fibroblasts, p53—/—or p53+/+ mouse thymocytes, and PA-1 Neo or PA-1 E6 cells. B, effect of p53 on inducible NF-*B DNAbinding 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-/— MEF or from MEFs stably transfected with iKBaM. C, effectof HPV16-E6 on TNF-a-induced KB-dependent gene expression. PA-1 Neo and PA-t E6 cells transfected with the poN260 ß-galactosidase expression vector with or without theHIV-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 activity12 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

expression that was not diminished by irradiation. These data confirmthat endogenous p53 represses RelA-dependent transactivation.

The IxBa gene promoter contains «Bmotifs that are stimulated byRelA (21). In addition to transcriptional activation, RelA also increases I«Baexpression by protein stabilization, thereby participatingin an inducible autoregulatory pathway (22). Compared withRelA+/+ fibroblasts, their counterparts derived from RelA—I—

mouse embryos demonstrate a reduction in IxBa expression (Fig. 2A).Because I«Baexpression reflects endogenous RelA activity, we nextexamined the expression of IKBa in PA-1 Neo and PA-1 E6 cells.Consistent with the repression of basal «B-reporter activity by p53,IKBa expression was reduced in PA-1 Neo cells compared with PA-1

E6 cells. Analogous results were obtained in thymocytes isolated fromp53+/+ and p53—/—mice. Immunoblot analyses showed a compar

atively reduced basal expression of I«Ba in p53+/+ thymocytescompared with their p53-deficient counterparts (Fig. 2A). These datademonstrate that p53 represses RelA-dependent expression of IxBaand indicate that I«Ba does not mediate p53-induced repression of

RelA.p53 Does Not Alter RelA Protein Levels or Inducible NF-icB-

DNA Binding Activity. p53 did not alter the levels of expression ofRelA in either PA-1 cells or thymocytes (Fig. 2A). Unlike TNF-a,stimulation of RelA-dependent transactivation by expression of E6 inPA-1 cells was not attended with any significant increase in «B-DNAbinding activity in EMSA (Fig. 2B). In addition, PA-1 Neo and PA-1E6 cells demonstrated no difference in TNF-a-induced stimulation of«B-DNAbinding. However, PA-1 Neo cells stimulated with TNF-ademonstrated a significantly lower activation of HIV-CAT comparedwith PA-1 E6 cells (Fig. 2Q. Similarly, EMSA demonstrated nodifferences in KB DNA-binding activity between wild-type MEFs and

their p53-deficient counterparts (Fig. 2B). In contrast, the repressionof HIV-CAT in MEFs transfected with IxBaM was associated with a

reduction in KB DNA binding activity in the same experiments. Thesedata suggest that unlike IKBa, p53 inhibits RelA transactivatingability through a mechanism independent of NF-KB DNA binding

activity.Association of p300 with p53 and RelA. The transcriptional ac

tivity of both p53 and RelA depend upon their respective interactionswith the p300 coactivator. We first defined the specific regions ofp300 that interact with p53 and RelA. 35S-methionine-labeled in

vf/ro-translated proteins were generated from p300 deletion mutantsusing a transcription/translation reticulocyte-based system. The p300

deletion mutants were chosen to define the avidity of the interactionof p53 or RelA with each of the specific functional cysteine/histidine-rich domains of p300 (Fig. 3/1 and fi). Endogenous p53 was immu-noprecipitated from cellular extracts of wild-type mouse embryonicfibroblasts (p53+/+) or their p53—/—counterparts mixed with eachin w'rro-translated p300 deletion protein. p53 immune complexes were

found to contain p300(1514-1922), but not p300(l-742)(Fig. 3D).

These data confirm that the p53 binding site on p300 resides betweenamino acids 1514 and 1922, a region immediately upstream of andoverlapping the C/H3 domain. To identify the RelA-interacting regions, in i'Ã-/ro-translated fragments of p300 were added to cell extractsprepared from fibroblasts derived from RelA-deficient mouse embryos (RelA-/-) or wild-type mouse fibroblasts (RelA+/+). andimmunoprecipitated with an anti-RelA antibody. Anti-RelA antibody-

precipitated complexes were found to avidly bind the amino terminalp300( 1-742) fragment, and to a lesser extent, the deletion mutantsp300 (1514-1922) and p300 (964-1922; Fig. 3Q. These results

demonstrate that RelA interacts with two regions of p300, one incorporating the amino terminal containing the C/HI domain, and the

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pS.I-MEDIATED REPRESSION OF NF-KB RELA

NuclearReceptorFVWSTATto

STATIac-Jun

c Myti

(1-742)

C/HI

'c-Fos P/CAF '

STATI« TFW

MyoO RNA polymerase IIRNA helicase A

Input

220-i"6545 -

Genotype RelA, + - + - + —¿�

and-RelA

(864.1922)pA.INeo

PA-1

E6

PA-1Neo

PA-1Ea

IR -P300

IR

IP

Western

)•••ita

anti-p300 l PL

Western

anti-p300

anti-RelA

l P anti-RelAWestern anti-p300

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 theirinteractions. 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 theinteraction 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 RelA—/- ( —¿�)or RelA+/+ ( + ) mouse fibroblasts. and anti-RelA immunoprecipitates

were resolved by 89Ã-SDS-PAGE. To assess the avidity of interaction between RelA and each deletion fragment, equivalent input quantities of each labeled deletion protein shownin 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. £.effect of HPV E6 on the association between p300and 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 £were immunohlotted with anti-RelA antibody. C. immunoblot analysis of immune complexes precipitated by a controlantibody uintirahhit IgG). G. detection of Re!A-p300 complexes in p53+/ + and p53—/—mouse thymocytes. Tap, immunoblot analyses of extracts prepared from thymocytes beforeand 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/H3El A-interacting domain.

We next examined whether p53 influences complex formationbetween RelA and p300. Protein extracts from untreated or irradiatedPA-1 Neo or PA-1 E6 cells were immunoprecipitated with anti-RelA

or control antibodies, and the immune complexes were immunoblotted with an antibody against p300. p300 was detected at equivalent levels in anti-RelA immune complexes precipitated from bothunirradiated and irradiated cell extracts of either PA-1 Neo or PA-1

E6 cells (Fig. 35). To confirm the interaction between RelA and p300,anti-p3(X) immunoprecipitates from cells extracts were immuno

blotted with antibodies against RelA or the nuclear localization sequence region of NF-KB p50. RelA was detected in immune complexes precipitated by anti-p300, but not by the control antibody (Fig.3F). In contrast to RelA, NF-«Bp50 was not found in p300 immune

complexes (data not shown). The association between RelA and p300was not influenced by p53 status or irradiation (Fig. 3£and 3>F).Analogous to PA-1 cells, equivalent levels of p300 were observed inanti-RelA immune complexes precipitated from extracts derived fromeither untreated or irradiated p53-/- or p53+/+ thymocytes (Fig.

3G). Since p53 did not influence expression of p300 (Fig. 3G), thesedata confirm that p53 does not prevent complex formation betweenRelA and p300.

p53 Represses p300-mediated Coactivation of RelA. Since bothp53 and RelA are present in complex with p3(X), we next examinedwhether p53 influences p300-dependent coactivation of RelA. RelA

interacts with two discrete regions of p300 (AA1 and 742, and A1514and 1922). Since the COOH-terminal region between amino acids

1514 and 1922 also interacts with basal transcription factors such asRNA polymerase 11and RNA helicase A (23), it is possible that thebinding of RelA with this region is responsible for its coactivation viarecruitment of these cofactors. Alternatively, binding of RelA to both

sites of p300 may facilitate its dimerization by overcoming the stericconstraint imposed by COOH-terminal domain (24). In either sce

nario, binding of RelA by p300 fragments that do not contain both thebinding sites would be expected to sequester RelA and repress itscoactivation. Cotransfection of p300( 1514-1922) resulted in strongrepression of RelA-stimulated HIV-CAT activity in both PA-1 Neoand PA-1-E6 cells (Fig. 4). HIV-CAT expression in p53-/- cells

was also inhibited by the p300( 1-742) deletion mutant that bindsRelA but lacks the p53-binding site, the C/H3 domain, and theCOOH-terminus. The inhibition of RelA activity by coexpression of

either p300 fragment was similar to that observed with introduction ofI«BaM. Conversely, transfection of the vector encoding full lengthp300 (pGal4p300) into PA-1 neo cells resulted in a dose-dependent

increase in RelA activity, such that high concentrations of p300restored HIV-CAT expression to levels observed in PA-1 cells expressing E6 or PA-1 neo cells transfected with the RelA-expressionvector (Fig. 4). The restoration of RelA activity in PA-1 Neo cells byoverexpression of p300, together with the repression of RelA in PA-1E6 cells by dominant negative fragments of p300, indicate that p53-

mediated repression of RelA activity is mediated via interference withthe coactivating function of p300.

Discussion

p53-mediated transrepression is believed to involve protein-protein

interactions with basal components of the transcriptional apparatussuch as TATA-associated factors (TFIID) and the transcriptional

coactivator/integrator p300 (6). p300 and its highly related familymember CBP contain well conserved regions, such as the C/H domains, that interact with several sequence-specific transcription factors and nuclear receptors (25). The coactivation of specific signal-

dependent transcriptional events by p300 involves recruitment of

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15. El-Deiry. W. S.. Tokino. T.. Velculescu. V. E., Levy, D. B., Parsons, R., Trent, J. M.,

Lin. D.. Mercer, W. E., Kinzler, K. W., and Vogelstein. B. WAFI, a potentialmediator of p53 tumor suppression. Cell. 75: 817-825, 1993.

16. Beg, A. A., and Baltimore, D. An essential role for NF-KB in preventing TNF-a-induced cell death (see comments). Science (Washington DC). 274: 782-784. 1996.

17. Van Antwerp. D. J., Martin. S. J., Kafri, T.. Green, D. R.. and Verma. I. M.Suppression of TNF-a-induced apoptosis by NF-KB (see comments). Science (Washington DC). 274.- 787-789. 1996.

18. Nabel. G., and Baltimore, D. An inducible transcription factor activates expression ofhuman immunodeficiency virus in T cells. Nature (Lond.). 326: 711-713, 1987.

19. Ravi. R.. Bedi. A.. Fuchs. E. J.. and Bedi. A. CD95 (Fast-induced caspase-mediatedproteolysis of NF-KB. Cancer Res.. 58: 882-886. 1998.

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22. Scott, M. L.. Fujita. T., Liou. H. C.. Nolan, G. P.. and Baltimore. D. The p65 subunitof NF-KB regulates I*B by two distinct mechanisms. Genes. Dev.. 7: 1266-1276.

1993.23. Nakajima. T.. Uchida. C.. Anderson. S. F., Lee, C. G., Hurwitz. J., Parvin. J. D.. and

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