The Hepatitis B Virus X Protein Functionally Interacts with CREB

The Hepatitis B Virus X Protein Functionally Interacts withCREB-binding Protein/p300 in the Regulation ofCREB-mediated Transcription*

Received for publication, July 17, 2006, and in revised form, December 5, 2006 Published, JBC Papers in Press, December 11, 2006, DOI 10.1074/jbc.M606774200

Delphine Cougot1, Yuanfei Wu2, Stefano Cairo3, Julie Caramel4, Claire-Angelique Renard, Laurence Levy5,Marie Annick Buendia, and Christine Neuveut6

From the Unite d’Oncogenese et Virologie Moleculaire, Institut Pasteur and INSERM U579, 28 rue du Dr. Roux, 75015 Paris, France

The hepatitis B virus infects more than 350 million peopleworldwide and is a leading cause of liver cancer. The virus encodesa multifunctional regulator, the hepatitis B virus X protein (HBx),that is essential for virus replication.HBx is involved inmodulatingsignal transduction pathways and transcription mediated by vari-ous factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investi-gated the role of HBx and its potential interaction with CBP/p300in regulatingCREBtranscriptional activity.WeshowthatHBxandCBP/p300 synergistically enhanced CREB activity and that CREBphosphorylation by protein kinase A was a prerequisite for thecooperative action of HBx and CBP/p300. We further show thatHBx interacted directly with CBP/p300 in vitro and in vivo. Usingchromatin immunoprecipitation, we provide evidence that HBxphysically occupied the CREB-binding domain of CREB-respon-sive promoters of endogenous cellular genes such as interleukin 8andproliferatingcellnuclearantigen.MoreoverexpressionofHBxincreased the recruitment of p300 to the interleukin 8 and prolif-erating cell nuclear antigen promoters in cells, and this is associ-ated with increased gene expression. As recruitment of CBP/p300is known to represent the limiting event for activatingCREB targetgenes, HBxmay disrupt this cellular regulation, thus predisposingcells to transformation.

Hepatitis B virus (HBV)7 infection is a worldwide healthproblem and one of the major causes of hepatocellular carci-

noma (HCC) (1). Despite epidemiological evidence linkingHBV infection toHCC, themechanisms underlyingHBV-asso-ciated carcinogenesis are poorly understood. Among the pro-teins encoded by HBV, the hepatitis B virus X protein (HBx) isessential for virus replication in vivo (2, 3) and is thought tocontribute to hepatocarcinogenesis. HBx can transform SV40-immortalizedmurine hepatocytes (4) and induce liver cancer insome transgenic mouse models (5). In other mouse models,HBx acts as a cofactor and may accelerate cancer development(6–8). HBx may participate in cell transformation in severalways. On the one hand, it may act indirectly by activating virusreplication (9–12), which in turn activates immune responsesand causes chronic liver inflammation. This leads to continu-ous destruction and regeneration of the hepatocytes, increasingthe chances of genetic alterations (13). On the other hand, byproviding a favorable environment for virus replication, HBxmay directly disrupt cellular functions such as cell signaling inpart throughmodulation of cytoplasmic calcium, transcription,cell proliferation, and apoptosis (14–21). Thus, dysfunctionsand alterations may accumulate, leading ultimately to celltransformation.HBx exerts most of its activities through interaction with a

large array of cellular partners, such as p53, DDB1, and CRM1(18, 22–25). Furthermore HBx acts as a transcriptional co-acti-vator through direct interaction with various proteins, such asthe RPB5 subunit of RNApolymerase II, TF IIB, TF IIH, TATA-binding protein, and the basic domain-leucine zipper (bZIP)family of proteins that include the cyclic AMP-response ele-ment (CRE)-binding protein (CREB) (14).The CREB/bZIP family of proteins plays an essential role in

the liver by regulating gene expression and different processessuch as gluconeogenesis, lipid metabolism, and cell prolifera-tion (26). Recently CREB has also been implicated in leukemo-genesis and, interestingly, in hepatocarcinogenesis (27, 28).Activation of proteins of the CREB/bZIp family is mediatedthrough phosphorylation of a key acceptor site (serine 133 inCREB) induced by a variety of growth factors and stress signalsthat activate various signaling pathways. Among other signals,

* This work was supported in part by Association pour la Recherche sur leCancer (ARC) Grant 3379. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec-tion 1734 solely to indicate this fact.

1 Supported by a fellowship from the Ministere de la recherche et desTechnologies.

2 Supported by an ARC fellowship. Present address: Program in MolecularMedicine, University of Massachusetts Medical School, 373 Plantation St.,Worcester, MA 01605.

3 Supported by GIS-Institut des Maladies Rares.4 Present address: INSERM U509, Laboratoire de Pathologie Moleculaire des

Cancers, Inst. Curie, 26 rue d’Ulm, 75005 Paris, France.5 Supported by an ARC fellowship. Present address: Laboratory of Develop-

mental Signalling, Cancer Research UK London Research Inst., 44 Lincoln’sInn Fields, London WC2A 3PX, UK.

6 To whom correspondence should be addressed: Unite d’Oncogenese etVirologie Moleculaire (INSERM U579), Institut Pasteur, 28 rue du Dr. Roux,75724 Paris Cedex 15, France. Tel.: 33-145-68-88-51; Fax: 33-145-68-89-43;E-mail: [email protected].

7 The abbreviations used are: HBV, hepatitis B virus; HCC, hepatocellular carci-noma; HBx, hepatitis B virus X protein; CREB, cyclic AMP-response element

(CRE)-binding protein; PKA, cyclic AMP-dependent kinase (protein kinase A);CBP, CREB-binding protein; HTLV-I, human T-cell leukemia virus type I; ChIP,chromatin immunoprecipitation; GST, glutathione S-transferase; IL-8, interleu-kin 8; PCNA, proliferating cell nuclear antigen; CCNA2, cyclin A2; bZIP, basicdomain-leucine zipper; HEK, human embryonic kidney; HA, hemagglutinin;RT, reverse transcription; PIASy, protein inhibitor of activated STATy; ATF, acti-vating transcription factor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 7, pp. 4277–4287, February 16, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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cAMP is known to induce CREB phosphorylation through theactivation of cyclic AMP-dependent kinase (PKA). This phos-phorylation allows the CREB-binding protein (CBP) and p300,co-activator paralogs, to be recruited (29). In turn, CBP/p300mediate the transcriptional activation of target genes by allow-ing a multiprotein activation complex to be formed that linksthe transcription factors to the basal transcriptionalmachinery.CBP/p300 also possess intrinsic acetyltransferase activity,which is responsible for the acetylation of histones thought torelieve chromatin-dependent repression (30). Finally CBP/p300 also modify transcriptional activity through the acetyla-tion of non-histone proteins (31). The CREB/bZIP factors canform homo- or heterodimers that bind to a DNA sequenceknown as the CRE. This is found in the regulatory region ofvarious cellular genes and also as a variant in theHBV enhancerI (32).HBx interacts directly in vivo and in vitro with the CREB/

bZIP family of proteins via their bZIP domain and increasestheir DNA binding affinity and transcriptional activity (33, 34).It is still unknown whether HBx stabilizes CREB binding toDNA or increases CREB dimerization (35–37). However, stud-ies have clearly shown that an increase in CREB DNA bindingaffinity by HBx is necessary but not sufficient to explain theco-activation of CREB byHBx (38, 39). In particular, it has beensuggested that HBx might recruit CBP/p300 to CREB/CREindependently of CREB phosphorylation at Ser-133 (39). Thismechanism has already been proposed for the Tax protein ofthe human T-cell leukemia virus type I (HTLV-I) (40).In this study, we investigated potential interactions between

HBx andCBP/p300.We first addressed the question ofwhetherHBx cooperates with CBP/p300 to activate CREB-dependenttranscription in human hepatoma cells by reporter assays. Wealso examined the requirement of PKA-induced phosphoryla-tion ofCREB inHBx transactivation capacity. Thendirect bind-ing ofHBx toCBP/p300was assessed in vitro and in vivo.More-over we used chromatin immunoprecipitation (ChIP) assay toanalyze the mechanisms underlying the cooperation betweenHBx and CBP/p300. Our data show that activated transcrip-tion of endogenous HBx target genes is associated withincreased p300 occupancy at CREB binding sites in the pro-moters of these genes. Thus, interaction between HBx andp300 could play a predominant role in HBx-dependent reg-ulation of gene transcription.

EXPERIMENTAL PROCEDURES

Cell Culture, Infection, and Transfection—HeLa, HEK293,and HEK293 T cells were maintained in Dulbecco’s modifiedEagle’s medium with 10% fetal calf serum. HepG2 andHepAD38 cells weremaintained inDulbecco’smodified Eagle’smedium/F-12 complemented with 10% fetal calf serum, 3.5 �10�7 M hydrocortisone, and 5 �g/ml insulin. Cells were trans-fected with different vectors as indicated in the figure legendsusing the Exgen reagent (Euromedex) or Lipofectamine reagent(Invitrogen). Total amounts of transfectedDNAwere kept con-stant by adding corresponding empty vectors. All transfectionexperiments were repeated at least three times in duplicate. Forluciferase assays, cells were lysed and assayed 48 h after trans-fection. HBx was found to activate transcription of the thymi-

dine kinase-�-galactosidase plasmid used to normalize lucifer-ase activity for transfection efficiency. We therefore confirmedthe results by multiple independent assays. Human primaryhepatocytes were prepared from resected normal human liversas described previously (41). Briefly cells were seeded on colla-gen I-coated plates and maintained in William’s medium sup-plemented with 10 nM insulin, 100 mM triiodothyronine, and 1mg/ml bovine serum albumin. All experimental procedureswere in conformity with French laws and regulations and withinformed consent of the patients. For infection, virions wereproduced inHEK293T cells as described previously (42).Hepa-tocytes were incubated for 2 hwith virion preparations normal-ized to 800 ng/ml viral p24. Freshmediumwas then added, andcells were incubated further for 48 h.Antibodies—Mouse monoclonal (sc-7300) and rabbit poly-

clonal antibodies (sc-369) against CBP and rabbit polyclonalantibody (sc-8981) against p300were fromSanta Cruz Biotech-nology. Monoclonal anti-p300 antibody was from Upstate Bio-technology (05-267), mouse monoclonal anti-HBx antibodywas from Affinity Bioreagent (MAI-081), mouse monoclonalanti-actin was from Sigma (A-5441), and mouse monoclonalanti-HA antibody was from Covance (MMS-101R). For West-ern blotting, CBP and p300 antibodies were diluted to 1:500 inblocking solution. HBx and HA antibodies were diluted to1:1000, and actin antibody was diluted to 1:10,000.Plasmids—cDNAs of wild type HBx (adw subtype) and dele-

tionmutants, kindly provided by V. Kumar (43), were amplifiedby PCR and inserted into the pcDNA3 vector to generatecDNAs carrying a C-terminal Myc tag (pcDNAX0 topcDNAX14). N-terminally HA-tagged HBx expression vectorswere constructed similarly. All constructs were verified bysequencing. The expression vector for full-length p300 wasprovided by Y. Nakatami (44). The RSV-PKA expression con-struct was from R. Maurer (45). The pCRE-Luc reporter plas-mid, which carries four consensus CRE sites, was from Strat-agene, andG5-E1B-Luc, which contains five Gal4 binding sites,was provided by J. G. Judde (46). Full-length Gal4-CREB andthe Gal4-CREB-M1 mutant were provided by M. Montminy(47). GST-CBP fusion constructs were kindly provided by T.Kouzarides (48), and the GST-KIX construct (CBP amino acids588–683) by J. Nyborg (49). GST-CBP-(1–495) andGST-CBP-(409–683) were constructed by inserting the correspondingCBP fragments in frame with GST into pGEX5X-1. HA-PIASyplasmid was provided by A. Dejean (50). pTRIP-HBx plasmidwas generated by cloning the BglII-XhoI fragment containingwild type HBx (adw subtype) cDNA in the BamHI-XhoI sites ofthe lentiviral vector pTRIP�U3 (51).Recombinant Proteins and in Vitro Binding Assays—GST-

CBP fusion proteins were expressed in E. coliBL21 and purifiedby glutathione-Sepharose affinity (Sigma) according to stand-ard protocols. HBx proteins were translated in vitro in the pres-ence of [35S]methionine using the TNT coupled reticulocytelysate system (Promega). For in vitro binding assay, 35S-labeledHBx proteins were mixed with Sepharose beads carrying eitherGSTorGST-CBP and incubated for 2 h at 4 °C in binding buffer(20 mM Hepes, pH 7.9, 300 mM NaCl, 1 mM MgCl2, 0.8% Non-idet P-40, 1 mM dithiothreitol, and 0.02% bovine serum albu-min). The beads were then washed three times with binding

Functional Interaction between HBx and CBP/p300

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buffer, and bound proteins were analyzed by SDS-PAGE andautoradiography.Immunoprecipitation and Western Blotting—Cells in 10-cm

dishes were lysed in 800 �l of lysis buffer (150mMNaCl, 50 mMTris/HCl (pH 7.4), 0.5% (v/v)Nonidet P-40, and protease inhib-itor mixture (Roche Applied Science)) and quickly frozen ondry ice. Lysates were thawed and centrifuged at 14,000 rpm for10 min, and 700 �l of the supernatant were cleared by incuba-tion with 30 �l of protein A/protein Gmixture. Antibodies (2.5�g) were initially incubated with 50 �l of 50% (v/v) proteinA/protein G-Sepharose for 2 h and then added to the lysates.After overnight incubation, the beads were extensively washedwith the same buffer, and bound proteins were resolved bySDS-PAGE.ForWestern blot analysis, protein lysates were transferred to

nitrocellulose membrane and incubated with the indicatedantibodies for 1 h. Reactive proteins were developed with sec-ondary antibodies conjugated to alkaline phosphatase and visu-alized using chemiluminescence according to the manufactur-er’s protocol (Tropix).Small Interfering RNAs—RNA oligonucleotides specifically

directed against CBP (forward, 5�-UUGAGGAAUCAACAGC-CGCTT-3�; reverse, 5�-GCGGCUGUUGAUUCCUCAATT-3�) and p300 (forward, 5�-CAGAGCAGUCCUGGAUUAGTT-3�; reverse, 5�-CUAAUCCAGGACUGCUCUGTT-3�) weresynthesized (Eurogentec). Small interfering RNA (siRNA) neg-ative control duplex was purchased from Eurogentec. HeLacells were transfected with either p300 and CBP siRNA (20 nMeach) or control siRNA (40 nM) using Oligofectamine (Invitro-gen). Protein extractswere prepared 48 h after transfection, andthe expression of CBP and p300 was analyzed byWestern blot-ting. Alternatively 24 h after siRNA transfection, HeLa cellswere co-transfected with the G5-E1B-Luc reporter plasmid,Gal4-CREB fusion protein, and PKA with or without HBxexpression vectors using Exgen (Euromedex). The cells werelysed 48 h later and assayed for luciferase activity.ChIP Assay—HeLa cell extracts were prepared 24 h after

transfection, andChIP assayswere carried out as described pre-viously (52) withminormodifications. In brief, cells were cross-linked by incubation with 1% formaldehyde for 5 min at 37 °C,and the nuclear extracts were prepared and sonicated. Thebound protein-DNA complexes were incubated overnight at4 °C with or without 2 �g of rabbit polyclonal anti-p300 anti-bodies. Immune complexes were incubated (2 h at 4 °C withrotation) with 50 �l of a protein A/protein G-agarose mixture.The immunoprecipitates were washed five times in radioim-munoprecipitation assay buffer, once in LiCl buffer, and twicein Tris-EDTA buffer and then eluted in elution buffer (1% SDS,0.1% NaHCO3, 0.5 mM Pefabloc, protease inhibitors (SigmaP8340; dilution, 1:1000), and phosphatase inhibitors (SigmaP5726; dilution, 1:1000). After purification of the immunopre-cipitatedDNA, a 180-bp region encompassing theGal4 site wasamplified using semiquantitative stepdown PCR (see below)using the following primers: pG5gal4-5�, 5�-TACCCTCTAG-AGTCGACGGAT-3�, and pG5gal4-3�, 5�-AAGCTAATTCC-CGGGATCCGC-3� (Tm � 67 °C).

As negative control, a sequence within the �-lactamase genewas amplified with the following primers: Ampr-c5�, 5�-CGG-

CATCAGAGCAGATTGTA-3�, and Ampr-c3�, 5�-CTGGCG-TAATAGCGAAGAGG-3�. For analysis of endogenous pro-moters, 6 � 106 HepG2 or HepAD38 cells were seeded in75-cm2 flasks and treated or not with 0.3 �g/ml tetracycline.After 5 days, the cells were treated for 1 h with forskolin (10�M), and ChIP assays were carried out using 2 �g of anti-p300antibody or 5�g ofmousemonoclonal anti-HBx for the immu-noprecipitation step.Semiquantitative stepdown PCR was performed as follows:

95 °C for 5 min � 1 cycle; 95 °C for 1 min, 70 °C for 1 min, and72 °C for 1 min � 4 cycles; 95° for 1 min, 68 °C for 1 min, and72 °C for 1 min � 4 cycles; 95 °C for 1 min, 66 °C for 1 min,and 72 °C for 1min� 4 cycles, and finally 95° for 1min, primer-specific Tm for 1 min, and 72 °C for 1 min � 20–25 cycles. Thefollowing primerswere used for PCR-mediated amplification ofthe IL-8, cyclin A2, and PCNA promoters at the indicated Tm:hIL-8-CHIP5�, 5�-AAACTTTCGTCATACTCCGTATTTG-3�, and hIL8-CHIP3�, 5�-GCTCCGGTGGTTTTTATATC-3�(Tm � 60 °C); hCCNA2-CHIP5�, 5�-CCTGCTCAGTTTCCT-TTGGT-3� and hCCNA2-CHIP3�, 5�-AGACGCCCAGAG-ATGCAG-3� (Tm � 64 °C); hPCNA-CHIP5�, 5�-GGCTCACA-GTTCCCTTAGCA-3� and hPCNA-CHIP3�, 5�-ATATTCCG-GACCGTGATGG-3� (Tm � 64 °C). The relative intensities ofthe PCR fragments were quantified using Gene Snap/GeneTools from SynGene. Intensities were corrected by subtractingthe intensity of the corresponding background and then nor-malized with the input.RT and Quantitative PCR—Total RNA was extracted from

primary human hepatocyte cultures or HepAD38 cells usingthe RNeasy kit (Qiagen) according to the manufacturer’sinstructions. RNA (1�g) was reverse transcribed using randomprimers and the Superscript reverse transcriptase (Invitrogen).Real time quantitative PCRwas carried out on the ABI PRISM�7900HT Sequence Detection System (Applied Biosystems)using the standard PCR protocol (denaturation at 95 °C andannealing/extension at 60 °C) with the addition of a final disso-ciation step to ensure amplicon-specific detection by SYBRGreen. Samples were prepared by adding cDNA to SYBRGreenPCRMaster Mix (Applied) using the following primers: IL-8.s,5�-GCCTTCCTGATTTCTGCAGC-3�, and IL-8.a, 5�-CGCA-GTGTGGTCCACTCTCA-3�; PCNA.s, 5�-GGTCAGCCTTC-CCTAGCC-3�, and PCNA.a, 5�-CGCCTCTCGACTCTG-CTC-3�; CCNA2.s, 5�-TTGCTGGAGCTGCCTTTCAT-3�,and CCNA2.a, 5�-GCATGCTGTGGTGCTTTGAG-3�. Wechose PNN as a reference gene because it has a very low varia-tion coefficient in arrays of human liver tumors and in liver celllines.8 The primers were as follows: PNN.s, 5�-CCTTTCTGG-TCCTGGTGGAG-3�, and PNN.a, 5�-TGATTCTCTTCTGG-TCCGACG-3�.

RESULTS

HBx Cooperates with CBP/p300 in CREB/ATF Family-de-pendent Transcriptional Activation—We used reporter assaysto investigate whether HBx cooperates with CBP/p300 in theactivation of CREB/ATF-dependent transcription. HeLa cellswere transfectedwith pCRE-Luc, a synthetic luciferase reporter

8 S. Cairo, unpublished data.




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containing four CRE sites either alone or together with HBx,PKA, and p300. Although HBx alone could not efficiently acti-vate the pCRE-Luc reporter, we observed a 15.4-fold increase inpCRE-Luc activity when HBx was co-transfected with PKA(Fig. 1). Consistent with previous reports (29), p300 increasedpCRE-Luc reporter activation about 16-fold after the inductionof CREB phosphorylation by PKA. CRE reporter activity was53.7 times higher after co-transfection withHBx and p300 thanwith the reporter alone, suggesting cooperative action of thetwo factors (Fig. 1A). We also observed a similar cooperationbetween HBx and CBP/p300 in activating CREB/ATF-depend-ent transcription in HEK293 cells (data not shown).We then investigated whether HBx cooperates with p300 in

activating CREB/ATF-dependent transcription in hepatomacells. HepG2 cells were transfected with pCRE-Luc togetherwith different combinations of HBx, PKA, and p300 expressionvectors. In HepG2 cells transfected with PKA, co-transfectionwith HBx and p300 led to a 28-fold induction, whereas co-transfection with HBx or p300 alone led to 3.5- and 15-foldinduction (Fig. 1B). In cells that were not transfected with PKA,HBx did not cooperate with p300. Our results show that HBx

and p300 cooperatively activateCREB/ATF-mediated transcriptionwhen the cAMP pathway isactivated.Interaction of HBx with CBP/

p300—We next investigated whe-ther HBx directly interacts withCBP and p300. HeLa cells weretransfected with a plasmid express-ing HA-tagged HBx, and cell lysateswere immunoprecipitated withanti-CBP or anti-p300 antibodiesfollowed by Western blotting withanti-HA antibodies. HBx co-immu-noprecipitated with endogenousCBP and p300 (Fig. 2), whereas nosignal was detected after controlimmunoprecipitation with irrele-

vant anti-FLAG antibody. To confirm the specificity of theHBx/CBP and HBx/p300 interactions, we used cells expressingHA-tagged PIASy. Indeed it has been shown previously thatPIASy does not interact with CBP/p300 (53). In our conditions,HA-PIASy did not co-immunoprecipitate with p300 or CBP(Fig. 2).We then investigated the CBP/p300 domains involved in

HBx binding using pulldown assays. Different CBP fragmentsexpressed as GST fusion proteins (depicted in Fig. 3A, upperpanel, left) were incubated with in vitro translated, radiolabeledfull-length HBx. HBx bound to the N-terminal domain of CBPas shown by the interaction with CBP-(1–1099) and CBP-(1–495), both of which encompass the CH1 domain (Fig. 3A, upperpanel, right). We also observed HBx binding to CBP-(1099–1877) and CBP-(1620–1877), both of which contain the CH3domain, and a weaker interaction with CBP-(1099–1758),which may reflect that HBx binds to the small part of the CH3domain present in this fragment. We observed no interactionbetween HBx and CBP-(409–683), which contains the KIXdomain (amino acids 588–683).We repeated the binding assays using GST-CBP-(1–495)

and GST-CBP-(1099–1877) fusion proteins together with dif-ferent in vitro translated, in-frame deletion mutants of HBx toidentify the domain that interacts with CBP in HBx. Weobserved no binding using the X7 mutant, indicating that theHBx region spanning the amino acids 58–84 was needed forbinding toCBP-(1–495) andCBP-(1099–1877) (Fig. 3B). How-ever, amino acids 120–140 might also have been involvedbecause deletion of this region (X10 mutant) resulted in veryweak binding to CBP-(1–495) and CBP-(1099–1877) (Fig. 3B).Finally the X9mutant, which contains a deletion of amino acids85–119, had a minimal residual binding with both CBP-(1–495) andCBP-(1099–1877). Thus, the central part of HBx fromamino acids 61 to 140 is necessary for maximal binding to CBP.It has been reported that the minimum HBx domain requiredfor a direct CREB interaction is the region from amino acids 49to 115 (38), and thus it overlaps with the domains needed forCBP binding.We confirmed that these domains are required for HBx and

CBP cooperation in the co-activation of CREB/ATF-mediated

FIGURE 1. Cooperation of HBx and CBP/p300 in CREB/ATF-dependent transcription. A, HeLa cells weretransfected with 0.5 �g of pCRE-Luc reporter plasmid together with different combinations of PKA (0.1 �g),HBx (0.5 �g), or p300 plasmid (0.5 �g). The basal activity of cells co-transfected with the pCRE-Luc reporter andempty vectors was set at 1. B, HepG2 cells were transfected as in A except that cells were transfected with 0.2 �gof HBx vector and 0.25 �g of p300. In both experiments, luciferase activities were determined 48 h aftertransfection, and the results are the average of three independent experiments carried out in duplicate.

FIGURE 2. HBx interacts with endogenous CBP/p300 in vivo. After transfec-tion of HeLa cells with 2 �g of HA-tagged HBx, HA-tagged PIASy, or emptyvector, cellular extracts were co-immunoprecipitated with either anti-CBP oranti-p300 antibodies or with irrelevant anti-FLAG antibodies, and proteinswere resolved by SDS-PAGE. HBx and PIASy proteins were detected by immu-noblotting using anti-HA antibodies. The amounts of CBP and p300 in theprecipitate and of HBx or PIASy in the total lysate were determined by West-ern blotting with anti-CBP, anti-p300, and anti-HA antibodies, respectively,after loading 1:10 of the precipitate and 1:32 of the total lysate. WB, Westernblot; IP, immunoprecipitate.




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transcription by investigating the activity of HBx deletionmutants by reporter assays. HeLa cells were transfected withthe pCRE-Luc reporter and PKA expression vector togetherwith wild type or mutated forms of HBx in the presence orabsence of p300. In these experiments, fold activations wereexpressed relative to the activity in cells co-transfected withpCRE-Luc reporter and PKA. The X7 (�58–84) and X9 (�85–119) mutants that bind neither CREB nor CBP/p300 failed tostimulate CREB/ATF transcriptional activity (Fig. 3C, comparelanes 8, 10, and 13). Moreover the X10 (�120–140) mutant,which binds CREB but binds CBP/p300 very weakly, did notactivate CREB or cooperate with CBP/p300 in CREB/ATFco-activation (Fig. 3C, lanes 11 and 12). These results indi-cate that the physical interaction of HBx with CBP/p300 is

required for HBx co-activation activity in CREB/ATF-de-pendent transcription.HBx Cooperates with CBP/p300 in the Activation of Gal4-

CREB-dependent Transcription and Requires the Phosphoryla-tion of CREB—Previous studies have suggested that theincreased affinity of CREBDNAbinding byHBx contributes to,but does not fully explain,HBx activity onCREB (38, 39).More-over our results suggested that an interaction betweenHBx andCBP/p300 is needed for CREB transcriptional activation byHBx. Therefore, we tested whether HBx still cooperates withp300 independently fromCREBDNAbinding.Weused aGal4-CREB chimera containing full-length CREB fused to the Gal4DNA-binding domain (47). It has been reported that HBx doesnot affect the binding of Gal4-CREB to Gal4 response elements

FIGURE 3. Interaction between HBx and CBP/p300 is essential for activation of CREB/ATF transcriptional activity. A, mapping by GST pull-down assay ofCBP domains that interact with HBx. A schematic illustration of full-length CBP with representative domains and the GST-CBP fusion proteins used for the invitro binding studies are shown in the left panel. Right panel, in vitro translated and [35S]methionine-labeled HBx was incubated with GST or GST-CBP fusionproteins. The input reflects 20% of the protein incubated with the beads. Binding was assessed after extensive washing, SDS-PAGE resolution, and autoradiogra-phy. Bottom right panel, expression of the GST and the different GST-CBP constructs was analyzed by SDS-PAGE and staining with Coomassie Blue. B, determinationof the CBP-binding region of HBx protein. Left panel, schematic representation of full-length HBx protein with the in-frame HBx deletion mutants used for thein vitro pulldown assay. The minimal binding domain for CREB is shown. KD, Kunitz domain-like region; PSR, proline-serine-rich domain. Right panel, [35S]me-thionine-labeled HBx and different deletion mutants were incubated with GST, GST-CBP-(1– 495), and GST-CBP-(1099 –1877). The input represents 10% of theprotein incubated with the beads. C, activity of HBx deletion mutants on CREB/ATF-mediated transcription. HeLa cells were co-transfected with 0.5 �g ofpCRE-Luc reporter and RSV-PKA plasmids with different combinations of wild type HBx or deletion mutants (X0 and X5, X6, X7, X9, and X10, respectively;described in Fig. 2C) (0.5 �g) and p300 (0.5 �g). Luciferase activities were determined 48 h after transfection. The basal activity of the cells co-transfected withpCRE-Luc and the PKA expression vector was taken as 1. The results are the average of three independent experiments carried out in duplicate.




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(34). We transfected 293 cells with the G5-E1B-Luc reporter,which contains five Gal4 binding sites, alone or in combinationwith HBx, PKA, and p300. PKA activated Gal4-CREB 2.7-foldcompared with the level in cells co-transfected with G5-E1B-Luc and Gal4-CREB plasmids (Fig. 4, lanes 2 and 7). HBx co-transfected with PKA further increased Gal4-CREB activity4.4-fold (Fig. 4, compare lanes 4 and 7). Moreover G5-E1B-Lucactivity was increased 3.3-fold in cells co-transfected with p300and PKA compared with PKA alone (Fig. 4, compare lanes 6and 7), and co-transfection of HBx and p300 with PKA furtherincreasedCREB activity 2.7-fold (Fig. 4, compare lanes 5 and 6).These results indicate that, in addition to its known effect onCREB DNA binding, HBx cooperates with CBP/p300 in activa-tion of CREB independently of CREB DNA binding affinity.A previous study had suggested that the activation of CREB-

dependent transcription by HBx relies on its ability to recruitCBP/p300 to non-phosphorylated CREB (39). However, ourresults showed that HBx could only activate CREB-dependenttranscription after activation of CREB by PKA, suggesting thatphosphorylation of CREB is required for transcriptional activa-tion by HBx. We tested whether HBx can cooperate with CBP/p300 in the context of non-phosphorylated CREB in a reporterassay using a Gal4-CREB-M1 mutant (S133A) that cannot bephosphorylated by PKA. HBx cannot activate the luciferasereporter when the cAMP signaling pathway is not activated(Fig. 4, compare lanes 10 and 11). However, co-transfectionwith HBx and PKA slightly increased the transcriptional activ-ity of CREB-M1 (2.9-fold), but this activation was lower thanthe activation obtained with the wild type CREB (12-fold) (Fig.4, compare lanes 11 and 12 with lanes 3 and 4). There was nocooperation betweenHBx andp300whenusingCREB-M1 (Fig.4, compare lanes 12, 13, and 14 with lanes 4, 5, and 6). Thesedata show that HBx activates CREB and cooperates with CBP/p300 only when CREB is phosphorylated at Ser-133.

Endogenous CBP/p300 Are Required for HBx Activation ofCREB-mediated Transcription—We confirmed the role ofCBP/p300 in HBx-induced activation of CREB using siRNAs toinhibit the endogenous expression of CBP and p300 in HeLacells. The transfection of HeLa cells with an siRNA specific forp300 greatly reduced p300 expression without affecting CBPexpression (Fig. 5, upper panel). Similarly transfection with theCBP-specific siRNA greatly reduced CBP expression withoutaffecting p300 expression. By contrast, the control siRNA didnot affect expression of either CBP or p300 as compared withendogenous levels in non-transfected cells (Fig. 5). Therefore,we tested whether HBx could activate CREB-dependent tran-scription in the presence of siRNAs against p300 andCBP usingthe Gal4-CREB chimera and the G5-E1B-Luc reporter. Deple-tion of p300 and CBP by siRNAs greatly reduced PKA-medi-ated stimulation of CREB-dependent transcription consistentwith the crucial role of CBP/p300 recruitment by phospho-CREB for transactivation (Fig. 5, lower panel). Transcriptionalactivation by HBx was abolished in cells transfected with siR-NAs directed against CBP and p300. Taken together, these datasuggest thatHBx cooperates withCBP/p300 in the activation of

FIGURE 4. Cooperation of HBx and CBP/p300 in the activation of Gal4-CREB-mediated transcription. HEK293 cells were transfected with theG5-E1B-Luc plasmid carrying five Gal4 binding sites (0.05 �g) and the Gal4plasmid (1 �g) or the expression vector for the fusion protein Gal4 and wildtype CREB (Gal4-CREB) (1 �g) or a phosphorylation-deficient CREB mutant(Gal4-CREB-M1) (1 �g) together with different combinations of PKA (0.1 �g),HBx (0.5 �g), and p300 (0.5 �g). The basal activity of the cells co-transfectedwith the G5-E1B-Luc reporter, the Gal4 vector, and empty vectors was set at 1.The results are the average of three independent experiments carried out induplicate.

FIGURE 5. Role of endogenous CBP/p300 in CREB-dependent transcrip-tional activity of HBx. Upper panel, HeLa cells were either mock-transfectedor transfected with siRNAs specific for p300 or CBP or with negative controlsiRNA, and expression of CBP and p300 was followed using Western blotting.Actin was used as loading control. Lower panel, HeLa cells were first trans-fected with the indicated siRNA. After 24 h, the cells were co-transfected withthe G5-E1B-Luc vector and the Gal4-CREB and PKA vectors in the absence orpresence of increasing concentrations of HBx. The activity of the cells co-transfected with the G5-E1B-Luc reporter, the Gal4-CREB fusion protein, andPKA was taken as 1. The results shown are the average of three independentexperiments carried out in duplicate. WB, Western blot.




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CREB-mediated transcription and that endogenous CBP/p300is necessary for HBx transcriptional activity.HBx Increases the Binding of CBP/p300 onDNA-boundGal4-

CREB in Vivo—As HBx interacts and cooperates with CBP/p300 but cannot recruit CBP/p300 to non-phosphorylatedCREB, we wondered whether HBx could increase the recruit-ment of CBP/p300 onto DNA-bound CREB. Therefore, wetransfectedHeLa cells with theG5-E1B-Luc reporter andGal4-CREB construct alone or in combination with PKA and HBxand then assessed the occupancy of CRE sites by endogenousCBP/p300 using a ChIP assay. Equivalent amounts of cross-linked chromatin were immunoprecipitated or not with p300antibodies. The precipitated DNA was then subjected to semi-quantitative PCR amplification using primers encompassingthe Gal4 binding sites. We used primers targeting plasmidsequences outside the Gal4 binding sites in the �-lactamasegene as negative controls. AlthoughPKAefficiently induced therecruitment of p300 toGal4 binding sites (Fig. 6, lane 7), HBx inthe absence of PKA was unable to induce significant recruit-ment of p300 (Fig. 6, lane 9). This observation correlates withour previous finding that HBx was unable to activate CREB-de-pendent transcription in the absence of activation of the cAMPpathway. Importantly the amount of p300 onGal4 binding siteswas higher in the presence of HBx and PKA than in cells trans-fectedwith PKA alone (Fig. 6, compare lanes 11 and 7). Recruit-ment of p300 to Gal4 binding sites was specific because we didnot observe any amplification of the �-lactamase gene (Fig. 6).These data showing that HBx increased CBP/p300 occupancyof a synthetic promoter suggested that HBxmight reinforce therecruitment of p300 on phospho-CREB.

HBx Increases the Recruitment ofCBP/p300 to Endogenous IL-8 andPCNA Promoters in Vivo—We nextinvestigated whether HBx couldalso activate p300 recruitment onendogenous cellular promoters. Forthese studies, we took advantage ofour recent microarray-based screenfor HBx target genes in primaryhuman hepatocytes,9 and weselected three CREB-responsivegenes: IL-8 andPCNA thatwe foundto be activated by HBx (54–56) andthe cyclin A2 gene (CCNA2) forwhich no activation was associatedto HBx expression (57, 58). First theability of HBx to up-regulate PCNAand IL-8 was verified in primaryhuman hepatocytes using real timePCR. Hepatocytes at 24 h postplat-ing were infected with the lentiviralrecombinant vector pTRIP-HBxexpressingHBx under control of thecytomegalovirus promoter. Controlhepatocytes were infected with thevector pTRIP�U3-GFP (51). Asreported previously, the transduced

protein was expressed in 90% of cells (41), and transgeneexpression was ascertained by Northern blotting (data notshown). RNA was extracted at 48 h after infection, and expres-sion of IL-8 and PCNA was analyzed by quantitative RT-PCR.As shown is Fig. 7A, expression of IL-8 was activated by 2-fold,and PCNA expression was activated by 3-fold in hepatocytesexpressing HBx compared with the green fluorescent proteincontrol. By contrast, the CCNA2 gene was not induced by HBxin our experiments (Fig. 7A).We then analyzed the expression of PCNA, IL-8, andCCNA2

in HepAD38, a HepG2-derived cell line that expresses the HBVgenome under tetracycline control (59). This cell line is usefulfor studying the regulation of cellular genes by HBx as HBxexpression is significantly increased by tetracycline removal(Fig. 7B). RNA was extracted from HepAD38 cells grown withor without tetracycline and treated or not with the cAMP ago-nist forskolin that activates PKA. Quantitative RT-PCR wasused to investigate expression of PCNA, IL-8, and cyclin A2genes in these different conditions. We observed a significantincrease in IL-8 and PCNA expression in cells treated with for-skolin and grown in the absence of tetracycline, whereas cyclinA2 expression remained unchanged (Fig. 7C). Our results sug-gest that, in HepAD38 cells, the expression of HBx can up-reg-ulate the expression of IL-8 and PCNA only when the cAMPpathway is activated.Next we studied the recruitment of HBx to PCNA, IL-8, and

CCNA2 promoters in tetracycline-treated or untreatedHepAD38 cells. ChIP assays were carried out in HepAD38 cells

9 L. Levy, C.-A. Renard, M. A. Buendia, and C. Neuveut, unpublished data.

FIGURE 6. HBx increases the recruitment of p300 to a synthetic promoter. HeLa cells were transfectedwith 3 �g of G5-E1B-Luc reporter vector in combination with Gal4-CREB (5 �g), RSV-PKA (2.5 �g), andHA-HBx (5 �g). At 24 h after transfection, ChIP assays were carried out using specific antibodies againstp300 (2 �g). Immunoprecipitated DNA or control DNA was amplified using specific primers for either theGal4 binding site region or the �-lactamase gene. Densitometry of ChIP PCR normalized to input is shownin the bottom panel. The value of amplification products from cells transfected with the G5-E1B-Luc andGal4-CREB plasmids was taken as 1 with other values being calculated accordingly. The graph is theaverage of three separate experiments.




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treated with forskolin using primers designed to amplifyregions containing the CRE sites. In the PCNA promoter, theCRE site has been located from �52 to �45 from the start site.For the IL-8 gene, we used the rVISTA algorithm and compar-ative analysis of human and mouse promoters to define a puta-tive CRE sequence located from�76 to�56 from the start site,consistentwith previous studies (55, 60). TheCCNA2promotercontains CRE sites located from �67 to �44. We found that intetracycline-treated HepAD38 cells, which expressed very lowlevels of HBx, the PCNA and IL-8 promoters were occupied byan almost undetectable level of HBx (Fig. 8A, lane 3). By con-trast, HBx recruitment greatly increased in the absence of tet-racycline, which correlateswith activatedHBx expression (Figs.8A, lane 5, and 7B). As a negative control, we observed no PCRamplification of IL-8 and PCNA in forskolin-treated HepG2cells. HBx occupancy at the CCNA2 promoter could not bedetected in forskolin-treated HepAD38 cells in the absence oftetracycline.

We then investigated the role of HBx on the recruitment ofCBP/p300 to endogenous promoters in HepAD38 cells treatedwith forskolin. High levels of HBx increased p300 recruitmentto the IL-8 promoter 3.1-fold and to the PCNA promoter 17.3-fold versus cells treated with forskolin and tetracycline (Fig. 8B,compare lane 3with lane 1). HBx did not affect the recruitmentof p300 to the CCNA2 promoter. These data showed a goodcorrelation between the increase in CBP/p300 recruitment byHBx to the CRE binding site of the IL-8 and PCNA promotersand the induction of the corresponding genes by HBx (Figs. 8Band 7C). Taken together, our results show that HBx activatesCREB transcriptional activity by increasing the recruitment ofCBP/p300 to the promoter of CREB endogenous cellular targetgenes.

DISCUSSION

HBx is described as a multifunctional protein essential forvirus replication. It can subvert many cellular processes such assignal transduction, transcription, and apoptosis through pro-tein-protein interaction (14). In this study, we showed thatHBxinteracts in vitro and in vivo with the co-activators CBP/p300.We studied this interaction for CREB transcriptional activityand found that HBx cooperates with CBP/p300 in the CREB-mediated activation of a synthetic promoter containing fourCRE sites.We used a CREBmutant that cannot be phosphoryl-ated on Ser-133 to show that HBx cannot recruit CBP/p300 tonon-phosphorylated CREB. Moreover we used ChIP experi-ments to show clearly that HBx favors the recruitment of p300to phosphorylated CREB bound to DNA. The ChIP experi-ments showed thatHBx can also increase the recruitment of theCBP/p300 co-activators to endogenous cellular target genes.The favoring of CBP/p300 recruitment to particular genes byHBx may be very important in vivo as Zhang et al. (60) showedthat tissue-specific activation of selected genes by cAMP is notdue to differences in phospho-CREB occupancy but rather tothe selective recruitment of CBP/p300.In this study, we analyzed the recruitment of p300 to the

promoters of two HBx target genes, IL-8 and PCNA, both ofwhich contain a CRE sequence and are known to be CREB tar-get genes (54, 55). Although we observed p300 recruitment tothe PCNA and IL-8 promoters after treatment with forskolin inHepAD38 cells, HBx overexpression strongly increased p300occupancy levels. The induction of CREB target genesmay playan important role in the development of HCC associated withHBV infection. Indeed CREB is known to participate in manyfunctions, such as cell survival, proliferation, and glucosemetabolism, and is now considered an important actor in can-cer development. A translocation that fuses the bZIP domain ofATF1, a CREB family member, to the oncogene product ofEwing sarcoma is found in clear cell sarcoma of soft tissues, andthis translocation is responsible for the constitutive activationof CREB target genes (61). Recently it has been shown thatCREB plays an important role in hepatocellular carcinomadevelopment by modulating tumor growth, angiogenesis, andapoptosis (27). Shankar et al. (28) revealed that CREB plays arole in leukemogenesis by showing that CREB overexpressioncauses the abnormal proliferation and survival of myeloid cells;this correlates with the up-regulation of cyclin A. Finally virally

FIGURE 7. HBx increases the expression of IL-8 and PCNA genes. A, expres-sion profiles of IL-8, PCNA, and CCNA2 in primary human hepatocytes express-ing or not expressing HBx were analyzed by quantitative RT-PCR in threeindependent pools of HBx-expressing hepatocytes. The results are the aver-age of three independent experiments. B, the expression of HBx in HepAD38cells grown in the presence or absence of tetracycline was analyzed on 50�g of protein extract by Western blotting using anti-HBx antibodies.HepG2 cells were used as a negative control. Actin was used as a loadingcontrol. C, expression profiles of IL-8, PCNA, and CCNA2 in HepAD38 cellsgrown in the presence or absence of tetracycline and treated or not withforskolin (10 �M) for 1 h. RNA levels were analyzed by quantitative RT-PCR.Tet, tetracycline; WB, Western blot.




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encoded oncoproteins act also through CREB signaling to pro-mote cell transformation. The Tax oncoprotein of HTLV-I andE1A of adenovirus (Ads), both of which affect cellular eventssuch as cell proliferation, apoptosis, and oncogenic transforma-tion, can interact with CREB and CBP/p300 (19, 62, 63). Thisinteraction is thought to activate virus replication and alsomodulate the expression of cellular genes. Both proteins caneither activate or repress cellular genes via theCREB/CBP/p300interaction. In this study, we analyzed two HBx target genes con-taining aCRE sequence: IL-8 andPCNA. Those twogenes are verylikelymembers of a large group of HBx target genes thatmay playan important role inHBV-associated cancer development. IndeedIL-8 is described as a leukocyte chemotactic molecule and isthought to be responsible for maintaining the inflammatoryenvironment associated with HBV infection that may play arole in the development of cancer. Moreover IL-8 has beendescribed as amitogenic, motogenic, and angiogenic factor andis up-regulated in a variety of human cancers, implying that itplays an important role in tumorigenesis (41). PCNA is a com-ponent of cellular complexes involved in eukaryotic DNA rep-lication and repair. The dysregulation of these two functions isa key step in neoplastic transformation. Moreover the modula-tion of PCNA expression has been described previously forHTLV-I and adenovirus types 2 and 5 (62, 63). Finally the lack ofactivation byHBx of someCRE-containing genes such as cyclinA2 suggests that HBxmay act via specific combinations of gen-eral transcription and regulatory factors (14, 64).In this study, we showed that HBx binds two separate

domains of the CBP/p300 protein: an N-terminal domain fromamino acids 1 to 495 that contains the nuclear receptor-bindingdomain and the CH1 domain and a region from amino acids

1620 to 1877 that contains the CH3domain. CH1 and CH3 are two cys-teine- and histidine-rich domainsthat bind Zn2� (also termed TAZ1and TAZ2) as well as many cellularand viral proteins, including tran-scription factors or factors of thegeneral transcriptional machinery(30). Among others, CH1 binds p53,mdm2, p65, HTLV-I Tax, and E1A,and CH3 binds p300/CBP-associ-ated factor, p53, RNA helicase A,c-Fos, Myo-D, E1A, and humanimmunodeficiency virus type 1 Tat.We did not investigate whether thebinding of HBx to different CBP/p300 domains affected the differentHBx activities such as transcrip-tional activation or p53 transcrip-tional repression (65). Neverthelessother viral proteins, such asHTLV-ITax or adenovirus E1A, also bind tomultiple p300 and CBP subdo-mains; this modulates their tran-scriptional and transforming activi-ties. For example, Tax is known toactivate CREB transcriptional activ-

ity partly through the recruitment of CBP/p300 via the KIXdomain and to block the activity of transcription factors such asp53 and p73� by competing for the KIX andCH1 domains. Taxalso inhibits nuclear receptor signaling possibly by interactingwith the same SRC-1 domain on CBP/p300 (40). The E1A andCBP/p300 interaction also seems to be very complex as differ-ent E1A domains contact CBP/p300, and E1A binds differentdomains of CBP/p300. The functional consequences of theseinteractions are also very complex: E1A acts as either a co-activator or a co-repressor of transcription factors such asCREB, c-Fos, MyoD, and Sat1 (62, 66).We also used a phosphorylation-deficient CREB mutant in

reporter assays to show that HBx cannot recruit CBP/p300 to anon-phosphorylated CREB bound to target DNA. A study byPflum et al. (39) using in vitro transcription assays with eitherphosphorylated or non-phosphorylated CREB proteinshowed that HBx activates only the non-phosphorylatedform of CREB. However, using only the wild type CREB pro-tein, it cannot be completely excluded that a few CREB mol-ecules may be phosphorylated.HBx can induce the recruitment of CREB and ATF2 to the

CRE-like sequence present in the HBV enhancer I (33). Pol-licino et al. (67) showed that the recruitment of CBP/p300 tothe HBV minichromosome correlates with virus replication.Further studies are needed to determine whether CREB isinvolved in virus replication and whether HBx participates inthe recruitment of CBP/p300 to the minichromosome. More-over CBP/p300 binds a large variety of cellular transcriptionfactors activated by HBx, such as c-Jun, c-Fos, and p65-NF-�B.It will be interesting to test whether HBx is involved in therecruitment of CBP/p300 to these factors. This mechanism has

FIGURE 8. HBx increases the recruitment of p300 to endogenous target gene promoters. A, HBx isrecruited to the IL-8 and PCNA promoters. HepG2 or HepAD38 cells were grown in the presence or in theabsence of tetracycline and treated with forskolin. ChIP assays were carried out using 5 �g of anti-HBx anti-bodies and specific primers to amplify the CRE sites in the IL-8 and PCNA promoters. The CCNA2 promoter wasused as negative control. PCR products were quantified, and values are expressed relative to PCR fragmentamplified from HepG2 cells that was taken as 1. The result shown is the average of three independent experi-ments. B, HBx increases the recruitment of p300 to the IL-8 and PCNA promoters. ChIP assays in HepAD38 cellswere carried out with 2 �g of anti-p300 antibodies. DNA fragments containing the CRE sites in IL-8, PCNA, orCCNA2 promoters were amplified by semiquantitative PCR. PCR products were quantified as in A except thatthe value from HepAD38 cells treated with tetracycline was taken as 1. Results are the average of three inde-pendent experiments. Tet, tetracycline.




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already been proposed for the transcriptional activation of thehypoxia-inducible factor-1� and the early growth response fac-tor by HBx (68, 69).In conclusion, we demonstrated that HBx co-activates CREB

partly through the recruitment ofCBP/p300. Thus,HBxmay beconsidered as a potentiator of the signalmediated byCREB, andthis mechanism may be involved in HBV-mediated oncogene-sis. Indeed HBV infection is associated with continuous liverregeneration; an important feature of liver regeneration is theincrease in intracellular cAMP, which in turn meditates thefinely tuned regulation of gene expression (26). In this cellularenvironment, HBx may facilitate the recruitment of CBP/p300to phospho-CREB, leading to the deregulation of gene expres-sion, and thus predispose cells to tumorigenesis. Finally as HBxhas been described as a pleiotropic co-activator, the stabiliza-tion of CBP/p300 on transcription factors may be a more gen-eral mechanism of HBx activity.

Acknowledgments—We thank Yu Wei and Muge Ogrunc for criticalreading of the manuscript. We are grateful to P. Tiollais and A.Dejean for constant interests in this work. We thank Drs. A. Dejean,J. G. Judde, T. Kouzarides, V. Kumar, J. Seeler, M. Montminy, Y.Nakatani, and J. Nyborg for kindly providing constructs used in thisstudy. We also thank Dr. C. Seeger for the kind gift of HepAD38 cellline.

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Laurence Lévy, Marie Annick Buendia and Christine NeuveutDelphine Cougot, Yuanfei Wu, Stefano Cairo, Julie Caramel, Claire-Angélique Renard,

Protein/p300 in the Regulation of CREB-mediated TranscriptionThe Hepatitis B Virus X Protein Functionally Interacts with CREB-binding

doi: 10.1074/jbc.M606774200 originally published online December 11, 20062007, 282:4277-4287.J. Biol. Chem.

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The Hepatitis B Virus X Protein Functionally Interacts with CREB