transcriptionalregulationofthebovineleukemiavirus ... · a modulation of the level of tax blv...

Transcriptional Regulation of the Bovine Leukemia VirusPromoter by the Cyclic AMP-response ElementModulator � Isoform*□S

Received for publication, April 11, 2007, and in revised form, May 23, 2007 Published, JBC Papers in Press, May 25, 2007, DOI 10.1074/jbc.M703060200

Thi Lien-Anh Nguyen‡1,2, Stephane de Walque‡2,3, Emmanuelle Veithen‡, Ann Dekoninck‡4, Valerie Martinelli‡5,Yvan de Launoit§, Arsene Burny‡, Robert Harrod¶, and Carine Van Lint‡4,6

From the ‡Institut de Biologie et de Medecine Moleculaires, Laboratoire de Virologie Moleculaire, Universite Libre de Bruxelles, Ruedes Profs Jeener et Brachet 12, 6041 Gosselies, Belgium, the §Institut de Biologie de Lille, Institut Pasteur de Lille, Universite de Lille 1,UMR 8117 CNRS, BP 447, 1 Rue Calmette, 59021 Lille Cedex, France, and the ¶Department of Biological Sciences, Laboratory ofMolecular Virology, Southern Methodist University, Dallas, Texas 75275-0376

Bovine leukemia virus (BLV) expression is controlled at thetranscriptional level through three TaxBLV-responsive elements(TxREs) responsive to the viral transactivator TaxBLV. ThecAMP-responsive element (CRE)-binding protein (CREB) hasbeen shown to interact with CRE-like sequences present in themiddle of eachof theseTxREs and toplay critical transcriptionalroles in both basal and TaxBLV-transactivated BLV promoteractivity. In this study,wehave investigated thepotential involve-ment of the cAMP-response elementmodulator (CREM) inBLVtranscriptional regulation, and we have demonstrated thatCREM proteins were expressed in BLV-infected cells andbound to the three BLV TxREs in vitro. Chromatin immuno-precipitation assays using BLV-infected cell lines demon-strated in the context of chromatin that CREM proteins wererecruited to the BLV promoter TxRE region in vivo. Func-tional studies, in the absence of TaxBLV, indicated that ectopicCREM� protein had a CRE-dependent stimulatory effect onBLV promoter transcriptional activity. Cross-link of the B-cellreceptor potentiated CREM� transactivation of the viral pro-

moter. Further experiments supported the notion that thispotentiation involved CREM� Ser-117 phosphorylation andrecruitment of CBP/p300 to the BLVpromoter. AlthoughCREBand TaxBLV synergistically transactivated the BLV promoter,CREM� repressed this TaxBLV/CREB synergism, suggesting thata modulation of the level of TaxBLV transactivation throughopposite actions of CREB and CREM� could facilitate immuneescape and allow tumor development.

Bovine leukemia virus (BLV)7 infection is characterized byviral latency in the large majority of infected cells and by theabsence of viremia. These features are thought to be due to thetranscriptional repression of viral expression in vivo (1, 2). BLVtranscription initiates at the unique promoter located in the5�-long terminal repeat (5�-LTR) of the BLV genome. The5�-LTR is composed of theU3, R, andU5 regions and transcrip-tion initiates at the U3-R junction. BLV exhibits two distinctfunctional transcriptional states as follows: a low basal level oftranscription ensured by host cellular transcription factors, anda high level of transcription directed by the virus-encoded tran-scriptional activator TaxBLV (3, 4). In the early stages of BLVtranscription, before TaxBLV expression and transactivation,the basal transcriptional promoter activity is ensured by severalcis-acting elements located in the 5�-LTR. In the U3 region arepresent the promoter CAAT and TATA boxes (5, 6), and three21-bp enhancers, each containing an imperfectly conserved8-bp cyclic AMP-responsive element (CRE), which binds atleast three proteins: CRE-binding protein (CREB) and acti-vating transcription factors 1 and 2 (ATF-1 and ATF-2)(7–10). Importantly, the 21-bp enhancers are also calledTaxBLV-responsive elements (TxREs) because transactiva-tion of the BLV LTR by TaxBLV requires these enhancers. Ithas been proposed that TaxBLV activation of transcription

* This work was supported in part by grants (to C. V. L.) from the “FondsNational de la Recherche Scientifique” (Belgium), the Televie Program ofthe Fonds National de la Recherche Scientifique, the “Action de RechercheConcertee du Ministere de la Communaute Francaise,” ULB, ARC Program04/09-309, the Internationale Brachet Stiftung, the Interreg III Program(“Intergenes” Project), the Theyskens-Mineur Foundation, the “FortisBanque Assurance,” and the “Federation Belge Contre le Cancer.” The costsof publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact. Thenucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) EF507802.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1 and S2.

1 Fellow of the Belgian Fonds pour la Recherche dans l’Industrie etl’Agriculture (FRIA) and of the FNRS Televie Program. Present address:Terry Fox Molecular Oncology Group, Lady Davis Institute for MedicalResearch, McGill University, 3755 Cote Ste. Catherine, Montreal, QuebecH3T 1E2, Canada.

2 Both authors contributed equally to this work.3 Supported by a postdoctoral fellowship from the Region Wallonne Program

Waleo2 616295.4 Charge de Recherches of the Fonds National de la Recherche Scientifique.5 Fellow of the Fonds National de la Recherche Scientifique Televie Program.6 Directeur de Recherches of the Fonds National de la Recherche Scientifique.

To whom correspondence should be addressed: Universite Libre de Brux-elles, Institut de Biologie et de Medecine Moleculaires, Laboratoire deVirologie Moleculaire, Rue des Profs Jeener et Brachet 12, 6041 Gosselies,Belgium. Tel.: 32-2-650-9807; Fax: 32-2-650-9800; E-mail: [email protected].

7 The abbreviations used are: BLV, bovine leukemia virus; EMSA, electro-phoretic mobility shift assay; ChIP, chromatin immunoprecipitation; CRE,cAMP-responsive element; CREB, CRE-binding protein; ATF-1, ATF-2, andATF-4, activating transcription factor-1, -2, and -4; CREM, cAMP-responsiveelement modulator; BCR, B-cell receptor; LTR, long terminal repeat;HTLV-1, human T-lymphotropic virus, type 1; TxRE, Tax-responsive ele-ment; CBP, CREB-binding protein; p300, protein 300; KID, kinase inducibledomain; RT, reverse transcription; PBMC, peripheral blood mononuclearcells; nt, nucleotide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 29, pp. 20854 –20867, July 20, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

20854 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 29 • JULY 20, 2007

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could be mediated, as reported for human T-cell leukemiavirus-1, through increased binding of the cellular proteinsCREB, ATF-1, and ATF-2 (and possibly other factors yet tobe identified) to the TxREs (7, 9, 11, 12).Among the CREB/CREM/ATF family of transcription fac-

tors, the cAMP-response element modulator (CREM) hasemerged as a unique member because CREM expression isfinely regulated, both transcriptionally and post-transcription-ally, leading to the production of various activator and repres-sor isoforms (13–15). In contrast to CREB and ATF, the CREMisoforms are expressed in a cell- and tissue-specificmanner (13,14, 16–19). Production of the different CREM isoformsdepends uponRNAprocessing and upon selection of transcrip-tion initiation sites or translation initiation sites. CREM iso-forms are highly homologous toCREB, especially in theirDNA-binding domain and kinase-inducible domain (KID). However,with the exception ofCREM�, CREM�1, andCREM�2, all otherCREM isoforms lack a glutamine-rich transcriptional activat-ing domain and are therefore considered to function as repres-sors of cAMP-regulated genes, either by competing with CREBproteins for binding to CREmotifs or by heterodimerizing withCREB proteins and abolishing their transcription activatingpotential.Expression of the CREM isoforms in BLV-infected cells and

recruitment of these CREM isoforms to the BLV promoterthrough the viral CREs have so far not been investigated. In thisstudy, we have examined the potential expression of CREMproteins inBLV-infected cells and the potential specific bindingof CREM proteins to the CRE-like motifs of the U3 BLV LTRregion, and we have investigated by transient cotransfectionexperiments the functional role of the CREM� isoform in basaland TaxBLV-transactivated BLV promoter activity.

EXPERIMENTAL PROCEDURES

Cell Lines andCell Culture—Themouse B-lymphoid cell lineA20 was obtained from the American Type Culture Collection(Manassas, VA). A20 cells are BALB/c B-lymphoma cellsderived from a spontaneous reticulum cell neoplasm found inan old BALB/cAnN mouse. A20 cells were cultured in RPMI1640 media (Invitrogen) supplemented with 5% fetal bovineserum, 50 units of penicillin/ml, and 50 �g of streptomycin/ml.Human HeLa cells were maintained in Dulbecco’s modifiedEagle’s medium (Invitrogen) supplemented with 5% fetalbovine serum, 50 units of penicillin/ml, and 50 �g of strepto-mycin/ml. The YR2 B-cell line is derived from leukemic cells ofa BLV-infected sheep and contains a single, monoclonally inte-grated silent proviruswith twomutations inTaxBLV that impairthe infectious potential of the integrated provirus (20). TheYR2cell line was maintained in OptiMEM medium (Invitrogen)supplemented with 10% fetal bovine serum, 50 units of penicil-lin/ml, and 50 �g of streptomycin/ml. All cells were grown at37 °C in an atmosphere of 5% CO2.Isolation of PBMCs and Cell Culture Conditions—Blood

samples were collected from a healthy sheep by jugular veni-puncture, mixed with EDTA as an anticoagulant, and centri-fuged at 1,750� g for 25min at room temperature. The PBMCswere then isolated by Percoll gradient centrifugation (density,1.129 g/ml; Amersham Biosciences) and washed twice in phos-

phate-buffered saline containing 0.075% EDTA. The cells werethen washed with phosphate-buffered saline until the superna-tant became clear. Cells were resuspended at a concentration of106 cells/ml in RPMI 1640 medium supplemented with 10%fetal calf serum, 50 units of penicillin/ml, and 50 �g of strepto-mycin/ml and cultured at 37 °C with 5% CO2.Plasmid Constructs—The pLTRwt-luc, containing the lucif-

erase gene under the control of the complete 5�-LTR of the 344BLV provirus and its derivative pLTR-mutCRE-luc, mutated inthe three viral CRE-like motifs, were previously described (9,10, 21). The eukaryotic expression vectors pSG-TaxBLV andpSG-CREB2 were gifts from Drs. Luc Willems and RichardKettmann (12, 22). The pSV-CREM�-wt expression vector(kindly provided by Dr. Paolo Sassone-Corsi) was describedpreviously (23). Mutation of CREM� serine 117 to alanine wasperformed by the QuikChange site-directed mutagenesismethod (Stratagene) using the pSV-CREM�-wt as substrateand the following pair ofmutagenic oligonucleotides (mutationis highlighted in boldface and the serine 117 codon is under-lined on the coding strand primer): CV 867/868 5�-CTTTCAC-GAAGACCCGCATATAGAAAAATACTG-3�. The mutatedconstruct was fully resequenced after identification by cyclesequencing using the ThermoSequenase DNA sequencing kit(Amersham Biosciences). The resulting plasmid was desig-nated pSV-CREM�-S117A. The expression vectors for thecytomegalovirus wild-type and deleted E1A 12S proteins(p12S-E1A-wt and p12S-E1A/2-36del, respectively) have beendescribed previously (24). The pRc/RSV-CBP expression vec-tor (kindly provided by Dr. D. Trouche) was described previ-ously (25).Transient Transfection and Luciferase Assays—A20 cells

were transfected by using the DEAE-dextran procedure asdescribed previously (26). At 20 h post-transfection, trans-fected cells were mock-treated or treated with a goat anti-mouse IgG antibody at a concentration of 6.5 �g/ml (reference115-006-006, Jackson ImmunoResearch). At 42 h post-trans-fection, cells were lysed and assayed for luciferase activity (Pro-mega). Luciferase activities were normalized with respect toprotein concentrations using the detergent-compatible proteinassay (Bio-Rad). HeLa cells were transfected with jetPEITMTransfection Reagent (Polyplus Transfection) according to themanufacturer’s protocol.In Vitro Coupled Transcription Translation Reaction—Cou-

pled transcription and translation reactions in wheat germextracts were performed with the TNT� coupled wheat germextract system (Promega) under the conditions described bythe manufacturer. Reactions were performed at 30 °C for 90min in the presence of the T7 RNA polymerase with 1 �g ofcoding DNA (pSG-CREB2, pSV-CREM�-wt, or pSG5).Electrophoretic Mobility Shift Assays and Supershift Assays—

Nuclear extracts fromYR2 cells or fromPBMCs isolated from ahealthy sheep were prepared and EMSAs, competition EMSAs,and supershift assays were performed as described previously(10, 21). The DNA sequences of the coding strand of the dou-ble-stranded TxRE probes are as follows (the viral CRE sites areunderlined): TxRE1 (centered at position �148) 5�-CAGACA-GAGACGTCAGCTGCC-3�; TxRE2 (centered at position�123) 5�-AAGCTGGTGACGGCAGCTGGT-3�; TxRE3 (cen-

Regulation of the BLV Promoter by CREM�

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tered at position �48) 5�-GAGCTGCTGACCTCACCTGCT-3�; and TxRE1-mutCRE (5�-CAGACAGAGTGGTCAGCT-GCC-3�) (mutations are highlighted in boldface). Thesequence of the coding strand of the double-stranded Oct-1consensus oligonucleotide used as competitor was 5�-TGTCG-AATGCAAATCACTAGAA-3�). For supershift assays, mono-clonal antibodies against ATF-1 (Santa Cruz Biotechnology,catalog number sc-243X) andATF-2 (catalog number sc-242X)and polyclonal antibodies against human CREB (catalog num-ber sc-240X) and human full-length CREM (catalog numbersc-440X) were added at a final concentration of 2 �g/reactionmixture at the beginning of the binding reaction for 20 minbefore adding the DNA probe (blocking conditions).Western Blot Analysis—Nuclear extracts prepared from

BLV-infected YR2 cells or in vitro translated CREB or CREMproteins were separated by SDS-PAGE and transferred ontopolyvinylidene difluoride membranes. The membranes wereblocked in Tris-buffered saline (TBS) containing 0.1% Tween20 and 5% nonfat dry milk and then incubated with anti-CREB(catalog number sc-240) or anti-CREM (catalog numbersc-440) antibodies in blocking solution. A second antibody,horseradish peroxidase-conjugated goat anti-rabbit IgG (cata-log number sc-2054), was used for enhanced chemilumines-cence detection (Cell Signaling).Preparation of mRNA and cDNA, RT-PCR Amplification,

and Cloning of CREM Isoform—One million YR2 cells wereused for extracting RNA (RNeasymini kit, Qiagen). First strandcDNA synthesis was performed using 2 �g of YR2 total RNAand (dT)16 primers (Invitrogen) with the Superscript II RNaseH reverse transcriptase reagent, according to the manufactur-er’s protocol (Invitrogen). RNaseOUTTM RNase inhibitor(Invitrogen) was added to the RT reaction. The resulting cDNAtemplates were used directly for PCR. After a denaturationperiod of 5 min at 95 °C, PCR was performed as follows: 40cycles for 1min at 95 °C, 1min at 50 °C, 2min at 72 °C, followedby a final elongation at 72 °C for 10 min. Reactions consisted of10�l of PfuDNApolymerase 10� buffer, 3�l of 25mMMgCl2,2 �l of dNTP mixture (10 mM each), 5 units of Pfu DNA poly-merase (all fromPromega), 10�l of sense and antisense primers(20 �M each), and 20 �l of cDNA in a final volume of 100 �l.Primers used for detecting CREM mRNA were designedaccording to themouse cDNA sequence (supplemental Fig. S2)and are as follows: 5�-CCG GAA TTC ATG ACC ATG GAAACAGTTGAATCCCAG-3� (this forward primer localized inCREM exon B at the 5� end of the translational start codon(underlined) contains an added EcoRI restriction site (in bold-face)) and 5�-GC TCT AGA CTA ATC TGT TTT GGG AGAGCA AAT GTC-3� (this reverse primer localized in CREMexon Ib at the 3� end of the DBD II contains an added XbaIrestriction site (in boldface)). The forward primer is located atthe most 5� end of exon B, and the reverse primer is located atthe most 3� end of exon Ib. To control reproducibility of theresults, RNA samples used for RT-PCR were collected fromthree independent cultures of YR2 cells. PCR products wereseparated on a 1.5% agarose gel and purified using theQIAEX IIgel extraction kit (Qiagen). PCR products were restricted withXbaI/EcoRI and inserted into the XbaI/EcoRI sites of theeukaryotic expression vector pcDNA 3.1 (Invitrogen). The

resulting constructs were transformed into TOP10 cells, andseveral independent clones were sequenced. Homologysearches in the nucleotide and genomic DNA data bases wereperformed with BLAST software accessed through the NCBIweb page (ncbi.nlm.nih.gov). The ovine CREM�1� DNAsequence we cloned is deposited in GenBankTM under acces-sion number EF507802.Chromatin Immunoprecipitation Assays—The chromatin

immunoprecipitation (ChIP) assays were performed by usingthe ChIP assay kit (Upstate Biotechnology, Inc.) according tothe manufacturer’s recommendations. Formaldehyde cross-linking reactions from 107 BLV-infected YR2 cells were per-formed for 10 min (or 30 min for the detection of CBP recruit-ment) at room temperature and quenchedwith 125mMglycine.Cells were lysed, and chromatin was sonicated to obtain anaverage DNA length of 600 bp. Following centrifugation, thechromatin was diluted 10-fold and precleared with a proteinA-agarose slurry containing salmon sperm DNA and bovineserum albumin (Upstate Biotechnology, Inc.). Precleared chro-matin (2ml) was incubated overnight at 4 °Cwith 5�g of eitheranti-CREB antibody (catalog number sc-186X), anti-ATF-1antibody (catalog number sc-243X), anti-ATF-2 antibody (cat-alog number sc-242X), anti-CREM antibody (catalog numbersc-440X), anti-CBP (catalog number sc-369X), or normal rab-bit IgG antibody (Upstate Biotechnology, Inc., catalog number12-370) as control. Incubation with the antibodies was followedby immunoprecipitationwithproteinA-agarose. Immunoprecipi-tated complexes were washed and eluted twice with 200�l of elu-tionbuffer.Theprotein-DNAcross-linkswerereversedbyheatingat 65 °C overnight, and 10% of the recovered DNA was used forradioactive PCRamplification (35 cycles of 94 °C for 45 s, 55 °C for30 s, and 72 °C for 1 min) with a primer set amplifying the BLVTxRE promoter region (nt �172 to �29), 5�-nt-172CCGTAAAC-CAGACAGAGACGTCAG-3�/5�-nt�29CACGAGGGTCTCAG-GAGGAGAAC-3�, or with an unrelated primer set amplifying agaggene region located�1.8kbdownstreamof theLTRregion (nt�1736 to �1955): 5�-nt�1736CAGCCCTCTCAGAATCAAGC-3�/5�-nt�1955AATTTGCCATTTATCGAAAG-3�. PCRproductsfrom all reactions were resolved by PAGE and visualized byautoradiography.

RESULTS

Endogenous CREM Proteins from Nuclear Extracts of BLV-infected YR2 Cells and of Ovine PBMCs Specifically Bind to theBLV LTR CRE Motifs in Vitro—The potential role of CREMmembers in regulating BLV gene expression has so far not beenexamined. CREM is a bZIP protein belonging to the CREB/CREM/ATF family of transcription factors that bind to DNA-regulatory regions via CRE target sequences. As CREM expres-sion is known to be finely regulated in a cell- and tissue-specificmanner (16, 19, 27, 28), we decided to investigate whetherCREM proteins were expressed in BLV-infected cells, andwhether these CREM proteins were able to bind to the BLVLTR CRE motifs. To this end, we performed supershift EMSAexperiments using the three 21-bp TxREs as probes. Theseprobes were incubated with nuclear extracts prepared fromYR2 cells derived from leukemic cells of a BLV-infected sheep(20), either mock-treated or treated for 1 h with phorbol ester




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phorbol 12-myristate 13-acetate plus Ca2� ionophore ionomy-cin (P � I), a combination known to activate BLV expression(29, 30). Incubations were carried out in the presence of either apolyclonal anti-CREM antibody (Fig. 1A, lanes 3, 4, 7, 8, 11, and12) or purified rabbit IgG as control (Fig. 1A, lanes 1, 2, 5, 6, 9,and 10). The anti-CREM antibody is raised against the full-length protein sequence, including the CREM DNA-bindingdomain present in all CREM isoforms. Therefore, this antibodyis predicted to be active against all CREM isoforms. Resultspresented in Fig. 1 showed that the mock-treated YR2 nuclearextracts formed three major retarded complexes (C1, C2, andC3) when incubated with the TxRE1, TxRE2, or TxRE3 probe(Fig. 1A, lanes 1, 5, or 9, respectively). Treatment of BLV-in-fected YR2 cells with P� I caused an increased binding activityto the TxREs (Fig. 1A, compare lanes 1 and 2, lanes 5 and 6, andlanes 9 and 10). Because P � I treatment is known to activateBLV expression, these data suggest that initiation of BLV tran-scription could occur, in response to P � I, through increasedrecruitment of CREB/CREM/ATF cellular transcription fac-tors at the viral LTR TxREs. To evaluate the specificity of theseinteractions, unlabeled double-stranded oligonucleotides wereprepared and used as competitors in EMSAs (Fig. 1B). Bindingof proteins in complexes C1, C2, and C3 was shown to besequence-specific to the CREmotif. Indeed, formation of thesecomplexes was inhibited by a 80-fold molar excess of the unla-beled homologous TxRE1 oligonucleotide (Fig. 1B, comparelanes 1 and 4) but not by the samemolar excess of an unlabeledTxRE1 oligonucleotidemutated at the CRE site or an unlabeled

oligonucleotide with an unrelatedsequence containing an Oct-1 con-sensus binding site (Fig. 1B, com-pare lanes 2 and 3 with lane 4).Addition of the anti-CREM anti-body in the binding reactionsresulted in the complete (mock-treated cells) or partial (P �I-treated cells) disappearance of theC2 complex concomitant with theappearance of a supershifted com-plex of lower mobility (S-CREM)but did not modify the migrationprofile of complexes C1 and C3(Fig. 1A, lanes 3, 4, 7, 8, 11, and 12).In contrast, the presence of theIgG control did not affect forma-tion of complexes C1, C2, and C3(data not shown), indicating thespecificity of the protein-antibodyinteraction. These results indi-cated that the C2 complex con-tained CREM isoform(s).To further evaluate the binding of

CREM to the BLV TxREs and itspotential heterodimerization withother members of the CREB/CREM/ATF family of transcriptionfactors, we performed additionalsupershift experiments with anti-

bodies directed against CREB, ATF-1, or ATF-2 alone or incombination with the anti-CREM antibody (Fig. 2A). Additionof the anti-CREB antibody to the binding reaction interferedwith formation of complexC2 and resulted in the appearance ofa supershifted complex (Fig. 2A, lane 2, 1S-CREB), indicatingthat CREB is part of complex C2. As shown in Fig. 1, addition ofthe anti-CREM antibody impaired complex C2 formation con-comitant with a supershifted complex with lower mobility (Fig.2A, lane 3, 2S-CREM), indicating that CREM is part of complexC2. Anti-ATF-1 antibody interferedwith formation of complexC2 and caused the appearance of three supershifted complexes(Fig. 2A, lane 4, 3, 3�, 3�S-ATF-1), suggesting the binding ofATF-1 to the BLV TxRE1 in vitro. Finally, addition of the anti-ATF-2 antibody resulted in the appearance of a supershiftedcomplex (Fig. 2A, lane 5, 4S-ATF-2) and the corresponding dis-appearance of the slower migrating complex C1 (Fig. 2A, lane5), indicating that formation of this complex results fromATF-2 binding to the TxRE1 in vitro. As negative control, weused the same antibodies in supershift experiments performedwith a probe corresponding to the CRE-mutated TxRE1 site(Fig. 2A, lanes 12–16), and we observed that mutation of theCRE motif abolished formation of complexes C1 and C2, indi-cating that binding of CREB, CREM, ATF-1, and ATF-2 to theBLV TxRE1 was specific to the CRE motif.To evaluate bZIP complex formation on the BLV TxREs, we

next performed additional supershift experiments by includingcombinations of the antibodies in the binding reactions. Weobserved a dramatic decrease of complex C2 formation con-

FIGURE 1. Binding of CREM proteins from nuclear extracts of BLV-infected YR2 cells to the three BLVTxREs. A, oligonucleotides corresponding to the BLV TxRE1, TxRE2, and TxRE3 were 5�-end-labeled and used asprobes in EMSAs. Prior to the addition of the TxRE1, -2, or -3 probe, 20 �g of nuclear extracts from BLV-infectedYR2 cells either mock-treated (lanes 1, 3, 5, 7, 9, and 11) or treated with phorbol 12-myristate 13-acetate (P) plusionomycin (I) (P � I: lanes 2, 4, 6, 8, 10, and 12) were incubated with either an anti-CREM antibody (lanes 3, 4, 7,8, 11, and 12) or purified rabbit IgG as negative control (lanes 1, 2, 5, 6, 9, and 10). Binding reactions wereanalyzed by PAGE, and the retarded complexes were visualized by autoradiography. The three major DNA-protein complexes C1, C2, and C3 are indicated by arrows. The free probe was run out of the gel for betterseparation of the complexes. The supershifted (S-CREM) complexes are indicated by an arrow. B, specificity ofthe C1, C2, and C3 complexes was tested by competition experiments. The TxRE1 oligonucleotide was 5�-end-labeled and incubated with 10 �g of nuclear extracts from BLV-infected YR2 cells in the absence of competitor(lane 4) or in the presence of an 80-fold molar excess of either the homologous unlabeled TxRE1 oligonucleo-tide (lane 1), the unlabeled CRE-mutated TxRE1 oligonucleotide (lane 3), or a heterologous Oct-1 consensusoligonucleotide (lane 2). Binding reactions were analyzed by PAGE, and the retarded complexes were visual-ized by autoradiography. The three major DNA-protein complexes C1, C2, and C3 are indicated by arrows. Thefigure shows only the specific retarded bands of interest.




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comitant with the appearance of a super-supershifted com-plex (Fig. 2A, lane 6, 5SS-CREB � CREM) when both theanti-CREB and the anti-CREM antibodies were added to the

same binding reaction mixture,indicating that CREM�CREB het-erocomplexes bind to the BLVTxRE1. As a control for cross-reac-tivity between the anti-CREM anti-body and the CREB protein, we per-formed Western blot analysis onCREB (TNT-CREB) and CREM�(TNT-CREM�) proteins producedin vitro in a transcription-transla-tion-coupled wheat germ extractreaction (Fig. 2B). This controlexperiment shows that the anti-CREM antibody had no cross-reac-tivity with the TNT-CREB protein(Fig. 2B, top panel, lane 3), therebydemonstrating the relevance of theanti-CREM antibody to distinguishbetween CREM- and CREB-relatedproteins. In parallel, the sameamounts of the TNT-control, TNT-CREM�, and TNT-CREB proteinswere used in a similar Western blotexperiment with the anti-CREBantibody, and we observed the spe-cific recognition of the TNT-CREBprotein (Fig. 2B, bottom panel, lane3). Therefore, both the anti-CREMand the anti-CREB antibodiesappear to be highly specific for rec-ognition of their respective targetproteins. Moreover, we performedadditional supershift experimentsfurther confirming the specificity ofthe anti-CREM antibody againstCREM isoforms (supplemental Fig.S1). The addition of both the anti-CREM and anti-ATF-1 antibodiesto the same binding reactionresulted in a decreased complex C2intensity and in the appearance ofthe 2S-CREM supershifted com-plex, but also in the appearance of asuper-supershifted complex (Fig.2A, lane 9, 7SS-CREM � ATF-1),suggesting the presence of CREM�ATF-1 heterocomplexes in complexC2. When both the anti-CREB andthe anti-ATF-1 antibodies wereadded to the same binding reaction,we observed an important decreaseof complex C2 formation concomi-tant with the appearance of the1S-CREB supershifted complex butalso with the appearance of a super-

supershifted complex (Fig. 2A, lane 7, 6SS-CREB � ATF-1),suggesting that CREB�ATF-1 heterocomplexes are present incomplex C2. On the contrary, supershift experiments with the

FIGURE 2. A, characterization of the protein complexes on the BLV TxRE1. Analyses of the protein composition of thethree retarded complexes obtained by incubation of the TxRE1 probe with YR2 nuclear extracts were performed bysupershift assays. Prior to the addition of the TxRE1 probe to the binding reaction, nuclear extracts from P� I-treatedBLV-infected YR2 cells were incubated with purified rabbit IgG as negative control (lane 1), with the anti-CREBantibody (lane 2), the anti-CREM antibody (lane 3), the anti-ATF-1 antibody (lane 4), the anti-ATF-2 antibody (lane 5),or with different combinations of these antibodies (lanes 6 –11). The probe was then added, and the binding reac-tions were analyzed by PAGE. The retarded complexes were visualized by autoradiography. The major DNA-proteincomplexes C1, C2, and C3 are indicated by arrows, and the supershifted (S-CREB, S-CREM, S-ATF-1, S-ATF-2) or super-supershifted (SS-CREB� CREM, SS-CREM�ATF-1, SS-CREB�ATF-1) complexes are indicated by numbers. The CRE-mutated TxRE1 probe was used as control in order to demonstrate that the binding of CREB, CREM, ATF-1, and ATF-2to the TxRE1 probe required intact viral CRE motifs (lanes 12–16). B, as controls for cross-reactivity between theanti-CREM antibody and CREB proteins and between the anti-CREB antibody and CREM proteins, we performedWestern blot (WB) analysis using in vitro translated CREB and CREM proteins and either the anti-CREM antibody (toppanel) or the anti-CREB antibody (bottom panel). The in vitro translated proteins were generated by using the TNTCoupled Wheat Germ Extract System (Promega) with the pSV-CREM�-wt expression vector encoding murine CREM�(TNT-CREM�, lane 1), the pSG-CREB2 expression plasmid encoding bovine CREB2 (TNT-CREB, lane 3), and the pSG5empty vector as control (TNT control, lane 2). C, CREM proteins are expressed in uninfected ovine PBMCs. Supershiftexperiments were performed with nuclear extracts of PBMCs isolated from a healthy sheep. Prior to the addition ofthe TxRE1 probe to the binding reaction, the PBMC nuclear extracts were incubated with either a purified rabbit IgGas negative control (lane 1) or the anti-CREM antibody (lane 2). The probe was then added, and the binding reactionswere analyzed by PAGE. The retarded complexes were visualized by autoradiography. The DNA-protein complexesN1 and N2 and the supershifted complex S-CREM are indicated by arrows.




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anti-ATF-2 antibody combined with either the anti-CREB anti-body or the anti-CREM antibody or the anti-ATF-1 antibody didnot result in the appearance of any super-supershifted complex,suggesting that ATF-2 did not bind to the BLV TxRE1 as hetero-complexes with those bZIP proteins (Fig. 2A, lanes 8, 10, and 11).In addition, we also performed EMSA and supershift exper-

iments using nuclear extracts from PBMCs isolated from ahealthy sheep (Fig. 2C) or from BLV-infected ovine PBMCs(data not shown). Results presented in Fig. 2C showed thatincubation of the TxRE1 probe with the uninfected PBMCnuclear extracts caused the formation of two major retardedcomplexes (called N1 and N2, respectively) (Fig. 2C, lane 1).Addition of the anti-CREM antibody to the binding reactionresulted in a drastic reduction of the N1 complex concomitantwith the appearance of a supershifted complex (S-CREM) butdid notmodify themigration profile of the complexN2 (Fig. 2C,lane 2).Taken together, our in vitro binding studies demonstrate

that CREM proteins are expressed in a BLV-infected B lym-phoma cell line as well as in ovine-infected and uninfectedPBMCs and that these CREMproteins bind to the TxREs of theBLV promoter in vitro in a CRE-dependentmanner.Moreover,our results suggest that complex C1 contains ATF-2homodimers and that formation of complexC2 results from thebinding of the CREB, CREM, and ATF-1 bZIP proteins to theBLV TxREs as homo- or heterodimers or as heterocomplexes.CREB, CREM, and ATF Transcription Factors Are Recruited

to the TxRE Region of the BLV Promoter in Vivo—To demon-strate in vivo, in the context of chromatin, the relevance of ourin vitro binding studies, we performed chromatin immunopre-cipitation (ChIP) assays using BLV-transformed YR2 cells.Formaldehyde cross-linked chromatin from these cells wasused for immunoprecipitation with antibodies directed againstATF-1, ATF-2, CREB, or CREMor with a purified rabbit IgG asnegative control. Following reverse of the cross-links, the puri-fied DNA was subjected to radioactive PCR analysis using a setof primers flanking the TxRE region (nt �172 to �29) of theBLV promoter. Fig. 3 shows PCR amplification of the DNAafter immunoprecipitation (Fig. 3, IP antibody, lanes 3–7), PCRamplification of the input DNA (Fig. 3, lane 2), and PCR ampli-fication in the absence of DNA as negative control (Fig. 3, PCRcontrol, lane 1). Analysis of PCR products from immunopre-cipitated DNA showed significant enrichment of the TxREregion when immunoprecipitation was carried out with theanti-CREB, anti-ATF-1, anti-ATF-2, or anti-CREM antibody(Fig. 3, lanes 4–7, respectively). In contrast, no such enrich-ment was observed following immunoprecipitation of thecross-linked chromatin with purified rabbit IgG (Fig. 3, lane 3).These data confirmed the binding ofCREB to the BLVTxREs invivo, as demonstrated previously by our laboratory (9), anddemonstrated in vivo the binding of other CREB/CREM/ATFmembers, including CREM protein(s), to this LTR region. As acontrol, we used another set of primers amplifying a region ofthe gag gene of the BLV provirus located �1.8 kb downstreamof the TxRE region, which has not been reported so far as bind-ing CREB/ATF family members. Immunoprecipitations withall the antibodies did not enrich eluates with DNA from thiscontrol region in YR2 cell chromatin, demonstrating the spec-

ificity of the TxRE interactions (data not shown). Thus, thesedata demonstrate the occupancy in vivo of the BLV TxREregion by CREB, ATF-1, ATF-2 and CREMproteins in the con-text of an integrated, chromatin-assembled BLV provirus.Ectopic Expression of the CREM� Activator Isoform Up-regu-

lates BLV Promoter Activity through the Three BLV LTR CRE-like Motifs—To assess the potential functional role of CREMfactors in transcriptional regulation of the BLV promoter, westudied the effect of overexpression of a CREM isoform on BLVLTR-driven luciferase reporter gene expression. To this end,mouse B-lymphoid A20 cells were transiently transfected witha pLTRwt-luc reporter construct and with increasing amountsof an expression vector for the murine CREM� activator iso-form (pSV-CREM�-wt) (23) and then assayed for luciferaseactivity (Fig. 4). Consistent with its role as a transcriptionalactivator, cotransfection of the CREM� expression vector withthe pLTRwt-luc construct resulted in a dose-dependent stimu-lation of the luciferase activity (up to 3.3-fold) (Fig. 4). Thiseffect required intact CRE-like motifs located in the middle ofeachTxRE, becausemutations in thesemotifs (pLTR-mutCRE-luc) almost completely abrogated CREM�-mediated transacti-vation of the BLV LTR (Fig. 4). Because transiently transfectedDNA does not always form proper chromatin structure, wenext confirmed the above transient transfection results in thenucleosomal context of an episomally replicating pREP-basedBLV LTR-luc plasmid (10), and we obtained CREM� transacti-vations of the BLV-promoter similar to those observed in Fig. 4with the nonepisomal pLTRwt-luc plasmid (data not shown).We conclude from these experiments that ectopic CREM�

protein has a CRE-dependent stimulatory effect on the BLVpromoter. These results thus establish the functional signifi-cance of CREM� through the CRE sites present in the BLV5�-LTR. Our data suggest that transcriptional activity of thispromoter is positively regulated by CREM� and that CREM�

FIGURE 3. Recruitment of CREB, CREM, ATF-1, and ATF-2 to the TxRE pro-moter region in vivo. ChIP assays were used to detect binding of bZIP tran-scription factors to the TxRE region of the BLV promoter in the chromosomalcontext of proviruses integrated in YR2 cells. DNA and protein were cross-linked with formaldehyde for 10 min, and DNA was sheared. The cross-linkedprotein-DNA complexes were immunoprecipitated (IP) with the anti-CREBantibody (lane 4), the anti-ATF-1 antibody (lane 5), the anti-ATF-2 antibody(lane 6), the anti-CREM antibody (lane 7), or with a purified rabbit IgG as neg-ative control (lane 3). The protein-DNA cross-links were reversed, and thepurified DNAs were amplified by semi-quantitative radioactive PCR using aprimer set amplifying the BLV promoter TxRE region (nt �172 to �29). PCR ofthe input (sample representing PCR amplification from a 1:25 dilution of totalinput chromatin from the ChIP experiment) is shown in lane 2. The PCR con-trol represents the PCR amplification in the absence of DNA (lane 1).




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could initiate BLV transcription in the absence of TaxBLV innewly infected cells.BCR Cross-link Potentiates CREM� Transactivation of the

BLV Promoter—CREM� is known to be phosphorylated on aparticular key serine residue, Ser-117, located in its KIDdomain. Phosphorylation of the KID allows recruitment of twotranscriptional coactivators CBP and p300 and leads to animportant increase in CREM� activating potential (14, 15, 31).It has been demonstrated that, ex vivo, BLVgene expression canbe induced by cross-link of the B-cell receptor (BCR) with anti-IgG antibody (32). InB-cells, signaling events induced followingBCR cross-link can lead to activation of several different path-ways, i.e. the protein kinase C pathway, the Ca2�/calmodulinpathway, the Ras/extracellular signal-regulated kinase (ERK)pathway, and the phosphatidylinositol 3-kinase/Akt pathway(31, 33–44). Although initially described as specific cAMP-re-sponsive factors, CREB and CREM seem to be phosphorylatedby a growing number of kinases, in response to different signal-ing cascades (45). Importantly, the phosphoacceptor sites (Ser-133 in CREB and Ser-117 in CREM) are the same as those tar-geted by protein kinase A (31, 45). All the kinases activated inresponse to BCR cross-link would therefore be susceptible tophosphorylate Ser-117 of CREM�.

To assess the role of BCR cross-link in CREM� transactiva-tion of the BLV promoter, we transiently cotransfected A20cells with pLTRwt-luc and with increasing amounts of pSV-CREM�-wt. Transfected cells were mock-treated or treatedwith anti-IgG antibody and then lysed and assayed for luciferaseactivity. Results presented in Fig. 5,A andB, showed that, in the

absence of anti-IgG treatment, CREM� transactivated BLVLTR-directed gene expression in a dose-dependentmanner (upto 3.7-fold), whereas in the presence of anti-IgG treatment, weobserved a dose-dependent increase in luciferase activity byectopically expressed CREM� (up to 5.7-fold) (Fig. 5B). Theseresults indicate that cross-link of BCRwith anti-IgGpotentiatesCREM� transactivation of the BLV promoter and suggest thatphosphorylation of CREM� could modulate its ability to trans-activate BLV gene expression.CREM� has been extensively studied in spermatogenesis (14,

15, 17, 46–48). Whether this isoform of CREM is also involvedin regulation of B-cell gene expression has been poorly docu-mented. We therefore performed Western blot experimentsusing an anti-CREM antibody and nuclear extracts from YR2cells treated or not with the combination P � I for differenttimes (Fig. 6A). These Western blot analyses showed that YR2cells expressed a CREM isoform with an apparent molecularmass of 43 kDa, which corresponds to the expected molecular

FIGURE 4. Response of the BLV promoter to ectopically expressedCREM� transcription factor. A20 cells were transiently cotransfectedusing the DEAE-dextran procedure with 500 ng of an LTR luciferasereporter construct wild-type (pLTRwt-luc) or mutated in the three CREmotifs (pLTR-mutCRE-luc) and with increasing amounts (250, 500n and750 ng of plasmid DNA) of a CREM� expression vector (pSV-CREM�-wt). Tomaintain the same amount of transfected DNA and to avoid squelchingartifacts, the different amounts of CREM� expression vector cotransfectedwere complemented to 750 ng of DNA by using the empty vector pSG5.Luciferase activities were measured in cell lysates 42 h after transfectionand were normalized with respect to protein concentrations of thelysates. Results are presented as histograms indicating the induction byCREM� (in fold) with respect to the activity of each LTR reporter constructin the absence of CREM�, which was assigned a value of 1. Means � S.E. areshown. A representative experiment performed in triplicate of three inde-pendent transfections is shown.

FIGURE 5. BCR cross-link potentiates CREM� transactivation of the BLV pro-moter. A20 cells were transiently cotransfected using the DEAE-dextran proce-dure with 500 ng of pLTRwt-luc and increasing amounts (250, 500, and 750 ng ofplasmid DNA) of pSV-CREM�-wt. To maintain the same amount of transfectedDNA and to avoid squelching artifacts, the different amounts of CREM�expression vector cotransfected were complemented to 750 ng of DNA byusing the empty vector pSG5. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 �g/ml). Luciferase activitieswere measured in cell lysates 42 h after transfection and were normalizedwith respect to protein concentrations of the lysates. Results are presented ashistograms. A, indicating luciferase activities (arbitrary units) normalized toprotein concentrations; B, indicating the inductions by CREM� (in fold) withrespect to the activity of pLTRwt-luc in the absence of CREM�, which wasassigned a value of 1. Means � S.E. from a representative experiment per-formed in triplicate of three independent transfections are shown.




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mass of the CREM� isoform (49). However, the expression ofthis CREM isoform did not seem to be influenced by P � Itreatment (Fig. 6A, compare lane 1 with lanes 2–4). To furtheraddress the relevance of CREM� involvement in BLV regula-tion in the YR2 BLV-infected cells, we have cloned a CREM�1�cDNA by RT-PCR from YR2 cell mRNA. Cloned sequenceswere translated in amino acid sequences, and homologysearches in protein and cDNA data bases led to the identifica-tion of a clone (found in three independent experiments) with atranslated cDNA sequence harboring 92% of identity with themurine CREM�1� isoform (supplemental Fig. S2). The ovineCREM�1� cDNA sequencewas then cloned into the eukaryoticexpression vector pcDNA 3.1 (pCREM�1�) and tested in tran-sient transfection experiments to confirm its transactivationpotential on BLV promoter activity. A20 cells were transientlytransfected with the pLTRwt-luc reporter construct and withincreasing amounts of the ovine pCREM�1� expression vector(Fig. 6B). Transfected cells were mock-treated or treated withanti-IgG antibody and then lysed and assayed for luciferaseactivity. Our results showed that, in the presence of the anti-IgG treatment, the ovine CREM�1� isoform isolated from YR2cells was able to activate BLV LTR-driven expression up to4.8-fold (Fig. 6B). Moreover, similarly to what we observed in

Figs. 4 and 5 with the murine pSV-CREM�-wt, this effectrequired intact CRE-like motifs located in the middle of eachTxRE, because mutations in these motifs (pLTR-mutCRE-luc)abrogatedCREM�1�-mediated transactivation of the BLVLTR(Fig. 6B).Together, our data demonstrated that YR2 BLV-infected

B-cells express aCREM�1� isoformand that this isoform is ableto transactivate BLV promoter-driven transcription followinganti-IgG activation through the three CRE elements present atthe middle of each TxRE.CREM� Ser-117Mutation Reduces the BCRCross-link Poten-

tiation of CREM� Transactivation of the BLV Promoter—Toaddress the role of CREM� phosphorylation at Ser-117 inCREM�-mediated anti-IgG stimulation of BLV LTR gene reg-ulation, we substituted the Ser-117 residue for an alanine resi-due in the context of the pSV-CREM�-wt vector, thereby gen-erating pSV-CREM�-S117A.We next transiently cotransfectedA20 B-cells with pLTRwt-luc and with either the pSV-CREM�-wt or the pSV-CREM�-S117A expression vector. Cellswere mock-treated or treated with anti-IgG antibody, andluciferase activities were measured in cell lysates (Fig. 7). Weobserved that mutation of CREM� Ser-117 resulted in adecrease in CREM� transactivation of the BLV promoter inthe presence of anti-IgG treatment (Fig. 7, anti-IgG, com-pare pSV-CREM� and pSV-CREM�-S117A). These dataindicate that CREM� Ser-117 mutation reduced anti-IgGpotentiation of CREM� transactivation of the BLV 5�-LTR,suggesting that this potentiation was mediated, at least inpart, through CREM� Ser-117 phosphorylation.CBP/p300 Coactivators Are Involved in CREM�Transactiva-

tion of the BLV 5�-LTR—Ser-117 phosphorylation is known toactivate CREM�-directed transcription through the recruit-ment of two coactivators, CBP and p300. Therefore, we decided

FIGURE 6. A, expression of CREM in BLV-infected YR2 cells. Nuclear extractsfrom YR2 cells either mock-treated (lane 1) or treated with P � I for 30 min or1 or 4 h (lanes 2– 4) were fractionated by SDS-PAGE, transferred on a polyvi-nylidene difluoride membrane, and immunoblotted with the anti-CREM anti-body. B, functional role of the cloned ovine CREM�1� isoform in BLV promot-er-driven gene expression. A20 cells were transiently cotransfected using theDEAE-dextran procedure with 500 ng of either pLTRwt-luc or pLTR-mutCRE-luc and increasing amounts (250 and 500 ng of plasmid DNA) ofpcDNA-CREM�1�. To maintain the same amount of transfected DNA and toavoid squelching artifacts, the different amounts of CREM�1� expression vec-tor cotransfected were complemented to 500 ng of DNA by using the emptyvector pcDNA3.1. At 20 h post-transfection, cells were mock-treated ortreated with anti-IgG antibody (6.5 �g/ml). Luciferase activities were meas-ured in cell lysates 42 h after transfection and were normalized with respect toprotein concentrations of the lysates. Results are presented as histogramsindicating the inductions by CREM�1� (in fold) with respect to the activity ofpLTRwt-luc in the absence of CREM�1�, which was assigned a value of 1.Means � S.E. from a representative experiment performed in triplicate ofthree independent transfections are shown.

FIGURE 7. CREM� Ser-117 mutation reduces anti-IgG potentiation ofCREM� transactivation of the BLV LTR. A20 cells were transiently cotrans-fected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and 500ng of either pSV-CREM�-wt, pSV-CREM�-S117A, or the empty vector pSG5. At20 h post-transfection, cells were mock-treated or treated with anti-IgG anti-body (6.5 �g/ml). Luciferase activities were measured in cell lysates 42 h aftertransfection and were normalized with respect to protein concentrations ofthe lysates. Results are presented as histograms indicating relative light units(RLU) with respect to the basal activity of pLTRwt-luc in the absence of CREM�and in the absence of anti-IgG treatment, which was assigned a value of 1.Means � S.E. from a representative experiment performed in triplicate ofthree independent transfections are shown.




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to examine the potential functional effect of CBP/p300 onCREM�-mediated transactivation of the BLVpromoter. To thisend, A20 cells were cotransfected with pLTRwt-luc, with pSV-CREM�-wt, and with an expression vector coding for the ade-novirus wild-type E1A 12S protein (pE1A-12S-wt) or anexpression vector coding for a mutant form of the E1A 12Sprotein (pE1A-12S/2-36del) as negative control. The adenovi-rus E1A oncoprotein is known to bind to CBP/p300 and toinhibit their capacity to coactivate transcription (50–52). ThepE1A-12S/2-36del expression vector codes for an E1A mutantprotein that lacks the ability to interact with CBP/p300 and failsto inhibit CBP/p300 activity. Results presented in Fig. 8Ashowed that coexpression of the wild-type E1A 12S proteindecreased (by �2-fold) CREM�-mediated transactivation ofthe BLV promoter, both in the presence and in the absence ofanti-IgG treatment, when compared with coexpression of themutant E1A/2-36del protein (Fig. 8A). These results suggestthat CBP/p300 are functionally involved in CREM� transacti-vation of the BLV LTR.To determine whether CBP is recruited at the BLV promoter

region in BLV-infected YR2 cells, we performed ChIP assays.For these assays, YR2 cells were treated with P � I for 30 minand then formaldehyde cross-linked for another 30 min. Chro-matin from these cells was used for immunoprecipitation withanti-CBP antibody or with a purified rabbit IgG as negativecontrol. Following reverse of the cross-links, the purified DNAwas subjected to radioactive PCR analysis with primers flankingthe TxRE region of the BLV promoter. Fig. 8B shows PCRamplification of the DNA after immunoprecipitation. Enrich-ment of the TxRE region when immunoprecipitation was car-ried out with the anti-CBP antibody demonstrated that CBP isrecruited at the BLV promoter in vivo (Fig. 8B, lane 4). As acontrol, we used another set of primers amplifying a region ofthe gag gene of the BLV provirus located �1.8 kb downstreamof the TxRE region, which has not been reported so far as bind-ing CBP. Immunoprecipitations with the CBP antibody did notenrich eluates with DNA from this control region in YR2 cellchromatin, demonstrating the specificity of the TxRE interac-tions (data not shown). Moreover, we performed functionalstudies to further illustrate the involvement of CBP/p300 in

FIGURE 8. Functional involvement of CBP in transactivation of the BLVLTR by CREM�. A, A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and 500 ng of either pSV-CREM�-wt or the empty vector pSG5 in the presence of either pE1A-12S-wt orpE1A-12S/2-36del. At 20 h post-transfection, cells were mock-treated ortreated with anti-IgG antibody (6.5 �g/ml). Luciferase activities were meas-ured in cell lysates 42 h after transfection and were normalized with respect toprotein concentrations of the lysates. Results are presented as histogramsindicating relative light units (RLU) with respect to the basal activity ofpLTRwt-luc in the absence of CREM� and in the absence of anti-IgG treatment,which was assigned a value of 1. B, ChIP assays were performed in order todemonstrate CBP recruitment to the BLV promoter. Cells were treated with P � Ifor 1 h; DNA and protein were cross-linked with formaldehyde for 30 min, andDNA was sheared. The cross-linked DNA-protein complexes were immuno-precipitated (IP) with the anti-CBP antibody (lane 4) or with purified rabbit IgGas negative control (lane 3). Cross-links were reversed, and purified DNA wasamplified by semi-quantitative radioactive PCR using a primer set amplifyingthe BLV promoter TxRE region (nt �172 to �29). PCR of the input (sample

representing PCR amplification from a 1:25 dilution of total input chromatinfrom the ChIP experiment) is shown in lane 2. The PCR control represents thePCR amplification in the absence of DNA (lane 1). C, HeLa cells were transientlycotransfected using the jetPEITM procedure with 500 ng of pLTRwt-luc andincreasing amounts (from 0 to 200 ng of plasmid DNA) of pRc-RSV-CBP. Tomaintain the same amount of transfected DNA and to avoid squelching arti-facts, the different amounts of CBP expression vector cotransfected werecomplemented to 200 ng of DNA by using the parental empty vector. Lucif-erase activities were measured in cell lysates 42 h after transfection and werenormalized with respect to protein concentrations of the lysates. Results arepresented as histograms indicating relative light units (RLU). D, HeLa cellswere transiently cotransfected using the jetPEITM procedure with 500 ng ofpLTRwt-luc and increasing amounts (from 0 to 60 ng of plasmid DNA) ofpSV-CREM�-wt, in the presence or the absence of 50 ng of pRc-RSV-CBP. Tomaintain the same amount of transfected DNA and to avoid squelching arti-facts, the different amounts of CREM expression vector cotransfected werecomplemented to 60 ng of DNA by using the empty vector pSG5. Luciferaseactivities were measured in cell lysates 42 h after transfection and were nor-malized with respect to protein concentrations of the lysates. Results are pre-sented as histograms indicating relative light units (RLU). Means � S.E. from arepresentative experiment performed in triplicate of three independenttransfections are shown.




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CREM� transactivation of the BLV promoter. HeLa cells werecotransfectedwith the pLTRwt-luc reporter construct togetherwith increasing amounts of an expression vector for the CBPcoactivator (pSV-CBP), and assayed for luciferase activity (Fig.8C). Cotransfection of the CBP expression vector resulted in adose-dependent stimulation of the BLV LTR-driven lucifer-ase activity (up to 7-fold) (Fig. 8C). Additional cotransfec-tion experiments with the CBP expression vector along withincreasing amounts of the pSV-CREM�-wt expression vec-tor demonstrated that CBP overexpression potentiatedCREM� transactivation of the BLV promoter (Fig. 8D).Taken together, these data demonstrate for the first time therole of CBP in BLV transcriptional regulation and suggestthat CBP recruitment to the BLV promoter is involved in themechanism of CREM� transactivation.CREM� Has No Effect on Transactivation of the BLV Pro-

moter by the Viral Transactivator TaxBLV, whereas CREB andTaxBLV Synergistically Activate BLV Promoter TranscriptionalActivity—Several studies have suggested that TaxBLV transacti-vation of the BLVpromoter ismediated through interactions ofTaxBLV with cellular factors that bind to the CRE motifs of theviral TxREs. Our laboratory has reported previously that adominant negative inhibitor of CREB (A-CREB) efficientlyinhibits TaxBLV transactivation of the BLV promoter, suggest-ing that recruitment of CREB to the BLV TxREs plays a criticalrole in the mechanism of TaxBLV transactivation (9). However,the effect of CREM� expression on TaxBLV-mediated transac-tivation of the BLV 5�-LTR has not been examined so far.Therefore, we transiently cotransfected the B-lymphoid A20cell line with pLTRwt-luc, the CREM� expression vector, and aTaxBLV expression vector (pSG-TaxBLV). Because Ser-117phosphorylation of CREM� could also intervene inTaxBLV-me-diated transactivation, transfected cells were mock-treated ortreated with anti-IgG antibody. As control, we compared in thesame transfection experiment the effect of CREB expression onTaxBLV-mediated transactivation of the BLV LTR, by includingcotransfections with a CREB expression vector (pSG-CREB)instead of the CREM� expression vector. Results presented inFig. 9 showed that, in the absence of anti-IgG treatment,cotransfection of 20 ng of the pSG-TaxBLV alone resulted in a40.7-fold stimulation of the luciferase activity (Fig. 9) and thatcotransfection of pSV-CREM�-wt or pSG-CREB-wt aloneresulted in a 1.6- or 10.9-fold activation of transcription,respectively (Fig. 9). Remarkably, when cells were cotransfectedwith both the pSG-TaxBLV and pSG-CREB-wt, a strong syner-gism was observed between TaxBLV and CREB, resulting in a197-fold activation of the BLV promoter activity (Fig. 9). Tran-scriptional activators synergize when their combination pro-duces a transcriptional rate that is greater than the sum of theeffects produced by the individual activators (53). In the lattercase, the amount of transcription in the presence of both TaxBLVand CREB (197-fold) is 4.9-fold greater (fold synergism) thanthe sum of the effect produced by each activator separately(10.9 � 40.7). These results demonstrate that CREB synergisti-cally enhances transcriptional activation of the BLV promoterby TaxBLV, and we confirm that CREB plays an important rolein the mechanism of TaxBLV transactivation. On the contrary,when cells were cotransfected with both pSG-TaxBLV and pSG-

CREM�-wt, no synergism between TaxBLV and CREM� wasobserved. Indeed cotransfection of both expression vectors ledto a 38-fold stimulation of the luciferase activity, which is sim-ilar to the sum of the effect produced by TaxBLV and CREM�individually (40.7 � 1.6) (Fig. 9), suggesting that CREM� is notinvolved in TaxBLV transactivation of the BLV LTR.The TaxBLV/CREB synergism was also observed following

anti-IgG treatment. Indeed, when cells were cotransfected withboth the TaxBLV expression vector and the CREB expressionvector, anti-IgG treatment resulted in a 1242-fold stimulationof the luciferase activity, which was 6.5-fold (fold synergism)greater than the sum of the activations by TaxBLV alone andCREB alone (19.5 � 192) in the presence of anti-IgG treatment(Fig. 9). On the contrary, no synergism was observed betweenTaxBLV and CREM� in the presence of anti-IgG treatment.Indeed, in the presence of anti-IgG treatment, cotransfection ofTaxBLV and CREM� expression vectors resulted in a 210-foldincrease of the luciferase activity, which was approximatelyequal to the sumof the activations by TaxBLV alone andCREM�alone (192 � 3.8) (Fig. 9). This suggests that, in the presence ofIgG-treatment, CREM� is not involved in TaxBLV transactiva-tion of the 5�-LTR .Thus, our results demonstrate a strong synergism between

TaxBLV and CREB in transcriptional transactivation of the BLVpromoter, both in the absence and presence of BCR cross-link,and confirm previous studies from our laboratory indicatingthatCREB recruitment at theBLVpromoter plays an importantrole in the mechanism of transactivation by TaxBLV (9). In con-trast, our results show no synergism between TaxBLV and

FIGURE 9. CREB and TaxBLV synergistically activate BLV promoter tran-scriptional activity, whereas CREM� has no effect on TaxBLV-mediatedtransactivation of the BLV promoter. A20 cells were transiently cotrans-fected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and with20 ng of a TaxBLV expression vector (pSG-TaxBLV) in the presence or theabsence of 500 ng of pSV-CREM�-wt or pSG-CREB-wt. To maintain the sameamount of transfected DNA and to avoid squelching artifacts, the differentamounts of expression vectors cotransfected were complemented to 520 ngof DNA by using the empty vector pSG5. At 20 h post-transfection, cells weremock-treated or treated with anti-IgG antibody (6.5 �g/ml). Luciferase activ-ities were measured in cell lysates 42 h after transfection and were normalizedwith respect to protein concentrations of the lysates. Results are presented ashistograms indicating relative light units (RLU) with respect to the basal activ-ity of pLTRwt-luc in the absence of CREM�, CREB, TaxBLV, and anti-IgG treat-ment, which was assigned a value of 1. Means � S.E. from a representativeexperiment performed in triplicate of three independent transfections areshown.




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CREM�, suggesting that CREM� is not involved in the mecha-nism of TaxBLV-mediated transactivation.CREM� Counteracts the Functional Action of CREB and

Affects the TaxBLV/CREB Synergistic Activation of the BLVPromoter—Because both CREB and CREM� are recruited tothe BLV promoter through the CRE-like motifs of the TxREs,we next wanted to determine their mutual influence on TaxBLV-mediated transactivation. We therefore tested whetherCREM� could counteract the action of CREB and affect theTaxBLV/CREB synergistic activation of BLV promoter tran-scriptional activity and/or whether CREB could counteract theaction of CREM� and synergize with TaxBLV in the presence ofCREM�. To this end, we performed cotransfection experimentsin A20 B-cells with pLTRwt-luc, with pSG-TaxBLV, and with afixed amount of pSG-CREB in the presence of increasingamounts of pSV-CREM� (Fig. 10A), or with pLTRwt-luc, withpSG-TaxBLV, and with a fixed amount of pSV-CREM� in thepresence of increasing amounts of pSG-CREB (Fig. 10B).Results showed that, in both the absence and the presence ofanti-IgG treatment, theTaxBLV/CREB synergismwas repressedby increasing amounts of ectopically expressed CREM� (Fig.10A).Moreover, cotransfection of increasing amounts of CREBin the presence of a fixed amount of CREM� activated synergis-tically withTaxBLV LTR-directed reporter gene expression (Fig.10B). Taken together, our results suggest that CREB andCREM� exert opposite roles in TaxBLV-mediated transactiva-tion of the BLV promoter and that a modulation of the level ofTaxBLV transactivation takes place through a competitionbetween the actions of CREB and CREM�. This competitionwould depend on the relative spatio-temporal expression levelsof those proteins in BLV-infected cells.

DISCUSSION

In this study, we have shown that CREM proteins wereexpressed in the YR2 cell line derived from leukemic cells of aBLV-infected sheep as well as in infected and uninfected ovinePBMCs and that these CREM proteins bound to the BLV pro-moter in vitro, through the three CRE-likemotifs present in themiddle of each viral TxRE. Supershift analysis suggested that invitroCREM formed heterocomplexes with CREB andATF-1 tobind to the BLV TxREs. Moreover, chromatin immunoprecipi-tation analysis using the BLV-infected YR2 B-cell line demon-strated the recruitment of CREMproteins to the BLVpromoterin vivo, in the chromatin context of a BLV-integrated provirus.Functionally, we showed that in the absence of the viral TaxBLVprotein, ectopic expression of the CREM� activator isoformup-regulated BLV promoter activity through the three BLVLTR CRE-like motifs. Cross-link of the B-cell receptor potenti-ated CREM� transactivation of the viral promoter. Furtherexperiments supported the notion that this potentiation couldinvolve CREM� Ser-117 phosphorylation and recruitment ofCBP/p300 by CREM�. Finally, CREB and CREM� seem to playopposite roles in TaxBLV-mediated transactivation of the BLVpromoter. AlthoughCREB andTaxBLV synergistically activatedthe LTR, CREM� repressed this TaxBLV/CREB synergism.

In conclusion, these results suggested that activationofTaxBLV-independent BLV transcription could occur in vivo, inresponse to B-cell activation. CREM� could thus initiate a low

level of BLV transcription and lead to the synthesis of smallamounts of the TaxBLV transactivator, which would thenamplify transcription from the 5�-LTR. In this regard, we havedemonstrated that CREM proteins are expressed in PBMCsisolated from a healthy sheep and are able to bind to the BLVpromoter in vitro, supporting our hypothesis that CREM couldinitiate BLV transcription in the absence of TaxBLV in newlyinfected cells. Moreover, we have shown that CREM� did notpotentiate TaxBLV-mediated transactivation of BLV LTR-di-rected gene expression. Indeed, our cotransfection experi-

FIGURE 10. Opposite roles of CREB and CREM� during TaxBLV-mediatedtransactivation of the BLV promoter. A, A20 cells were transiently cotrans-fected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc, 250 ngof pSG-CREB, 20 ng of pSG-TaxBLV, and increasing amounts of pSV-CREM�(from 0 to 750 ng of plasmid DNA). To maintain the same amount of trans-fected DNA and to avoid squelching artifacts, the different amounts of CREM�expression vector cotransfected were complemented to 750 ng of DNA byusing the empty vector pSG5. B, A20 cells were transiently cotransfectedusing the DEAE-dextran procedure with 500 ng of pLTRwt-luc, 250 ng of pSV-CREM�, 20 ng of pSG-TaxBLV, and increasing amounts of pSG-CREB (from 0 to750 ng of plasmid DNA). To maintain the same amount of transfected DNAand to avoid squelching artifacts, the different amounts of CREB expressionvector cotransfected were complemented to 750 ng of DNA by using theparental empty vector pSG5. A and B, at 20 h post-transfection, cells weremock-treated or treated with anti-IgG antibody (6.5 �g/ml). Luciferase activ-ities were measured in cell lysates 42 h after transfection and were normalizedwith respect to protein concentrations of the lysates. Results are presented ashistograms indicating relative light units (RLU) with respect to the basal activ-ity of pLTRwt-luc in the absence of CREB, CREM�, TaxBLV, and anti-IgG treat-ment, which was assigned a value of 1. Means � S.E. from a representativeexperiment performed in triplicate of three independent transfections areshown.




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ments indicated that overexpression of CREM� repressed thesynergistic activation of the BLV promoter by TaxBLV andCREB. The absence of viral proteins at the surface of BLV-infected cells is necessary to escape from an efficient hostimmune attack and to allow BLV-induced tumor development.However, because the taxBLV gene product is considered toaccount for viral leukemogenicity (22, 54–56), a low level ofbasal BLV expression is likely necessary at the early stage ofBLV infection to induce transformation of the infected cells.Based on our results we suggest a bimodal role of CREM� inBLV LTR-directed transcription: in the absence of TaxBLV,CREM� would initiate BLV transcription in response to activa-tion of the BLV-infected B-cells, and on the contrary, in thepresence of TaxBLV, CREM� would decrease TaxBLV-mediatedtransactivation of the BLV promoter by competing with CREBfor binding to the viral CREs. CREM� would thus be able toinitiate a low level of BLV expression necessary for TaxBLV-induced transformation of the infected cells, but in the pres-ence of TaxBLV, CREM�would reduce BLV expression and thusfacilitate immune escape and allow tumor development.BLV infection is characterized by viral latency, rendering the

infected animals asymptomatic seropositive carriers, for whomonly a small fraction will develop persistent lymphocytosis orB-cell lymphosarcoma several years later (32, 56). During thislatency period, expression of the virus is thought to be blockedat the transcriptional level (32, 57, 58). However, little is knownabout specific molecular events responsible for this virallatency. Some studies have suggested that a blocking factorcould be implicated in the inhibition of viral synthesis in vivo(59–62), but the nature of this factor and its mechanism ofaction have never been established. To date, all the regulatoryproteins known to be recruited to the BLV promoter and toinfluence BLV transcriptional activity are activating transcrip-tion factors (CREB, ATF-1, PU.1/Spi-B, USF1, IRF, etc.). In thisstudy, we propose a role for CREM proteins in transcriptionalrepression of the BLV LTR in the presence of TaxBLV. More-over, even though the CREM� isoform transactivates the BLVpromoter, we did not investigate the potential functional role ofother CREM isoforms. The CREM gene consists of 14 exons(63, 64). Alternative exon splicing and utilization of alternativepromoters and translation initiation codons result in the pro-duction of functionally different CREM proteins with eitheractivating or repressing potential on target gene expression (23,46). However, as all CREM isoforms contain at least the bZIPdomain, they potentially could be recruited to the BLV pro-moter through the three CRE-like motifs and could then up- ordown-regulate BLV provirus transcription. Preliminary resultsfrom our laboratory suggest that a CREM repressor isoform isexpressed in latently BLV-infected cells.8 Recruitment of suchrepressor to the BLV promoter could contribute to the silentstatus of the BLV promoter observed in the majority of BLV-infected cells in vivo.Regulation of the expression of the different activator and

repressor isoforms of the CREM gene has been extensivelystudied in spermatogenesis (14, 15, 17, 46–48). During sper-

matogenesis, only CREM repressors are expressed in pre-mei-otic germcells, but a switch to the expression of theCREM� andCREM�2 activators occurs in post-meiotic germ cells (15, 17,46, 65) and allows expression of testis-specific genes such ascalspermine, TP1, and RT7 (66–68). However, little is knownabout regulation of CREM expression in B-cells. In this studywe have shown that BLV-infected YR2 B-cells express aCREM�1� activator isoform and demonstrate the functionalrole of this isoform in BLV expression. Two different studieshave studied specific expression of the CREMgene in B-lymph-oid cells. The first study, published by Pongubala and Atchison(69), has suggested through RNA analysis that the CREM geneis expressed differentially in the B-cell lineage. More specifi-cally, their hypothesis is that repressor isoforms of CREMcouldpredominate at the pre-B-cell stage and that an activator formof CREMcould replace these repressive forms of CREMat laterstages of B-cell development. The second study, published byvan der Stoep et al. (70)., has demonstrated that ICER down-regulates the constitutive transcriptional activity of the CIITA-PIII promoter in B-cells, indicating the implication of anotherCREM isoform (ICER) during B-cell specific gene expression.Together, these two studies suggest that, like in germ cells,expression of the different CREM isoforms would be finely reg-ulated during differentiation and activation of B-cells. At theearly stage of BLV infection, predominant expression of CREMrepressor isoforms in the infected B-cells would explain theabsence of BLV expression. However, a switch in CREM geneexpression following B-cell activation or differentiation wouldallow initiation of BLV transcription, thereby generating thefirst TaxBLVmolecules. In this regard, it has been demonstratedthat, in BLV-infected but asymptomatic sheep, BLV integratedboth in CD5� and CD5� B-cells. In lymphoma, however, BLVprovirus was detected only in CD5�- B-cells but not in CD5�B-cells (71). Furthermore, van den Broeke et al. (72) have dem-onstrated that a variety of ovine B-cell populations can supporta productive BLV infection, suggesting that BLV can infect andreplicate in both immature and mature B-cells.In the case of the closely related HTLV-I retrovirus, several

studies have investigated the potential role of different CREMisoforms during basal and TaxHTLV-I-activated transcription oftheHTLV-I promoter. Bodor et al. (73) have demonstrated thatCREM binds to all three HTLV-I CRE-like motifs and attenu-ates the activation of aHTLV-I LTR-CATreporter construct byTaxHTLV-I and protein kinase A. Further studies by the samegroup have shown that elevated cAMP levels in T cells correlatewith expression of the potent transcriptional repressor ICER(74). Moreover, it has been shown that quiescent PBMCsrespond to cell activation by producing sustained ICER RNAlevels through 18 h post-stimulation and that ICER is able toinhibit TaxHTLV-I-mediated transactivation of theHTLV-I pro-moter (75), suggesting a role for ICER in the establishment andmaintenance of a persistentHTLV-I infection. In contrast, Lau-rance et al. (76) have shown that binding of theCREM� isoformto the HTLV-I CREs allows TaxHTLV-I to stimulate viral LTRexpression, but with concomitant inhibition of TaxHTLV-Ieffects on other CRE-containing genes. Together, these studiesindicate that the level of HTLV-I transcription is influenced bythe expression pattern of the different CREM isoforms accord-8 T. L.-A. Nguyen and C. Van Lint, unpublished results.




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ing to the state of T-cell activation and/or differentiation. BothBLV and HTLV-I oncoretroviruses could therefore use similarstrategies, depending on the CREB and CREM gene expressionlevels, to inhibit and/or activate viral transcription according tothe activation state of the infected cell.

Acknowledgments—We thank Drs. Luc Willems and Richard Kett-mann (Faculty of Agronomy, Gembloux, Belgium), Dr. DidierTrouche (CNRS, Toulouse, France), and Dr. Paolo Sassone-Corsi(Institut de Genetique et de Biologie Moleculaire et Cellulaire, B.P.10142, 67404 Illkirch-Strasbourg, France) for reagents used in thisstudy.

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Valérie Martinelli, Yvan de Launoit, Arsène Burny, Robert Harrod and Carine Van LintThi Lien-Anh Nguyên, Stéphane de Walque, Emmanuelle Veithen, Ann Dekoninck,

IsoformτAMP-response Element Modulator Transcriptional Regulation of the Bovine Leukemia Virus Promoter by the Cyclic

doi: 10.1074/jbc.M703060200 originally published online May 25, 20072007, 282:20854-20867.J. Biol. Chem.

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transcriptionalregulationofthebovineleukemiavirus ... · a modulation of the level of tax blv...

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