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J. Biol. Chem., Vol. 282, Issue 29, 20854-20867, July 20, 2007

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Transcriptional Regulation of the Bovine Leukemia Virus Promoter by the Cyclic AMP-response Element Modulator Isoform^*

Thi Lien-Anh Nguyên¹², Stéphane de Walque²³, Emmanuelle Veithen, Ann Dekoninck⁴, Valérie Martinelli⁵, Yvan de Launoit, Arsène Burny, Robert Harrod^¶, and Carine Van Lint⁴⁶

From the Institut de Biologie et de Médecine Moléculaires, Laboratoire de Virologie Moléculaire, Université Libre de Bruxelles, Rue des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium, the Institut de Biologie de Lille, Institut Pasteur de Lille, Université de Lille 1, UMR 8117 CNRS, BP 447, 1 Rue Calmette, 59021 Lille Cedex, France, and the ^¶Department of Biological Sciences, Laboratory of Molecular Virology, Southern Methodist University, Dallas, Texas 75275-0376

Received for publication, April 11, 2007 , and in revised form, May 23, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bovine leukemia virus (BLV) expression is controlled at the transcriptional level through three Tax_BLV-responsive elements (TxREs) responsive to the viral transactivator Tax_BLV. The cAMP-responsive element (CRE)-binding protein (CREB) has been shown to interact with CRE-like sequences present in the middle of each of these TxREs and to play critical transcriptional roles in both basal and Tax_BLV-transactivated BLV promoter activity. In this study, we have investigated the potential involvement of the cAMP-response element modulator (CREM) in BLV transcriptional regulation, and we have demonstrated that CREM proteins were expressed in BLV-infected cells and bound to the three BLV TxREs in vitro. Chromatin immunoprecipitation assays using BLV-infected cell lines demonstrated in the context of chromatin that CREM proteins were recruited to the BLV promoter TxRE region in vivo. Functional studies, in the absence of Tax_BLV, indicated that ectopic CREM protein had a CRE-dependent stimulatory effect on BLV promoter transcriptional activity. Cross-link of the B-cell receptor potentiated CREM transactivation of the viral promoter. Further experiments supported the notion that this potentiation involved CREM Ser-117 phosphorylation and recruitment of CBP/p300 to the BLV promoter. Although CREB and Tax_BLV synergistically transactivated the BLV promoter, CREM repressed this Tax_BLV/CREB synergism, suggesting that a modulation of the level of Tax_BLV transactivation through opposite actions of CREB and CREM could facilitate immune escape and allow tumor development.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Bovine leukemia virus (BLV)⁷ infection is characterized by viral latency in the large majority of infected cells and by the absence of viremia. These features are thought to be due to the transcriptional repression of viral expression in vivo (1, 2). BLV transcription initiates at the unique promoter located in the 5'-long terminal repeat (5'-LTR) of the BLV genome. The 5'-LTR is composed of the U3, R, and U5 regions and transcription initiates at the U3-R junction. BLV exhibits two distinct functional transcriptional states as follows: a low basal level of transcription ensured by host cellular transcription factors, and a high level of transcription directed by the virus-encoded transcriptional activator Tax_BLV (3, 4). In the early stages of BLV transcription, before Tax_BLV expression and transactivation, the basal transcriptional promoter activity is ensured by several cis-acting elements located in the 5'-LTR. In the U3 region are present the promoter CAAT and TATA boxes (5, 6), and three 21-bp enhancers, each containing an imperfectly conserved 8-bp cyclic AMP-responsive element (CRE), which binds at least three proteins: CRE-binding protein (CREB) and activating transcription factors 1 and 2 (ATF-1 and ATF-2) (7–10). Importantly, the 21-bp enhancers are also called Tax_BLV-responsive elements (TxREs) because transactivation of the BLV LTR by Tax_BLV requires these enhancers. It has been proposed that Tax_BLV activation of transcription could be mediated, as reported for human T-cell leukemia virus-1, through increased binding of the cellular proteins CREB, ATF-1, and ATF-2 (and possibly other factors yet to be identified) to the TxREs (7, 9, 11, 12).

Among the CREB/CREM/ATF family of transcription factors, the cAMP-response element modulator (CREM) has emerged as a unique member because CREM expression is finely regulated, both transcriptionally and post-transcriptionally, leading to the production of various activator and repressor isoforms (13–15). In contrast to CREB and ATF, the CREM isoforms are expressed in a cell- and tissue-specific manner (13, 14, 16–19). Production of the different CREM isoforms depends upon RNA processing and upon selection of transcription initiation sites or translation initiation sites. CREM isoforms are highly homologous to CREB, especially in their DNA-binding domain and kinase-inducible domain (KID). However, with the exception of CREM, CREM1, and CREM2, all other CREM isoforms lack a glutamine-rich transcriptional activating domain and are therefore considered to function as repressors of cAMP-regulated genes, either by competing with CREB proteins for binding to CRE motifs or by heterodimerizing with CREB proteins and abolishing their transcription activating potential.

Expression of the CREM isoforms in BLV-infected cells and recruitment of these CREM isoforms to the BLV promoter through the viral CREs have so far not been investigated. In this study, we have examined the potential expression of CREM proteins in BLV-infected cells and the potential specific binding of CREM proteins to the CRE-like motifs of the U3 BLV LTR region, and we have investigated by transient cotransfection experiments the functional role of the CREM isoform in basal and Tax_BLV-transactivated BLV promoter activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Lines and Cell Culture—The mouse B-lymphoid cell line A20 was obtained from the American Type Culture Collection (Manassas, VA). A20 cells are BALB/c B-lymphoma cells derived from a spontaneous reticulum cell neoplasm found in an old BALB/cAnN mouse. A20 cells were cultured in RPMI 1640 media (Invitrogen) supplemented with 5% fetal bovine serum, 50 units of penicillin/ml, and 50 µg of streptomycin/ml. Human HeLa cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal bovine serum, 50 units of penicillin/ml, and 50 µg of streptomycin/ml. The YR2 B-cell line is derived from leukemic cells of a BLV-infected sheep and contains a single, monoclonally integrated silent provirus with two mutations in Tax_BLV that impair the infectious potential of the integrated provirus (20). The YR2 cell line was maintained in OptiMEM medium (Invitrogen) supplemented with 10% fetal bovine serum, 50 units of penicillin/ml, and 50 µg of streptomycin/ml. All cells were grown at 37 °C in an atmosphere of 5% CO₂.

Isolation of PBMCs and Cell Culture Conditions—Blood samples were collected from a healthy sheep by jugular venipuncture, mixed with EDTA as an anticoagulant, and centrifuged at 1,750 x g for 25 min at room temperature. The PBMCs were then isolated by Percoll gradient centrifugation (density, 1.129 g/ml; Amersham Biosciences) and washed twice in phosphate-buffered saline containing 0.075% EDTA. The cells were then washed with phosphate-buffered saline until the supernatant became clear. Cells were resuspended at a concentration of 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 50 units of penicillin/ml, and 50 µg of streptomycin/ml and cultured at 37 °C with 5% CO₂.

Plasmid Constructs—The pLTRwt-luc, containing the luciferase gene under the control of the complete 5'-LTR of the 344 BLV provirus and its derivative pLTR-mutCRE-luc, mutated in the three viral CRE-like motifs, were previously described (9, 10, 21). The eukaryotic expression vectors pSG-Tax_BLV and pSG-CREB2 were gifts from Drs. Luc Willems and Richard Kettmann (12, 22). The pSV-CREM-wt expression vector (kindly provided by Dr. Paolo Sassone-Corsi) was described previously (23). Mutation of CREM serine 117 to alanine was performed by the QuikChange site-directed mutagenesis method (Stratagene) using the pSV-CREM-wt as substrate and the following pair of mutagenic oligonucleotides (mutation is highlighted in boldface and the serine 117 codon is underlined on the coding strand primer): CV 867/868 5'-CTTTCACGAAGACCCGCATATAGAAAAATACTG-3'. The mutated construct was fully resequenced after identification by cycle sequencing using the ThermoSequenase DNA sequencing kit (Amersham Biosciences). The resulting plasmid was designated pSV-CREM-S117A. The expression vectors for the cytomegalovirus wild-type and deleted E1A 12S proteins (p12S-E1A-wt and p12S-E1A/2-36del, respectively) have been described previously (24). The pRc/RSV-CBP expression vector (kindly provided by Dr. D. Trouche) was described previously (25).

Transient Transfection and Luciferase Assays—A20 cells were transfected by using the DEAE-dextran procedure as described previously (26). At 20 h post-transfection, transfected cells were mock-treated or treated with a goat anti-mouse IgG antibody at a concentration of 6.5 µg/ml (reference 115-006-006, Jackson ImmunoResearch). At 42 h post-transfection, cells were lysed and assayed for luciferase activity (Promega). Luciferase activities were normalized with respect to protein concentrations using the detergent-compatible protein assay (Bio-Rad). HeLa cells were transfected with jetPEI^TM Transfection Reagent (Polyplus Transfection) according to the manufacturer's protocol.

In Vitro Coupled Transcription Translation Reaction—Coupled transcription and translation reactions in wheat germ extracts were performed with the TNT® coupled wheat germ extract system (Promega) under the conditions described by the manufacturer. Reactions were performed at 30 °C for 90 min in the presence of the T7 RNA polymerase with 1 µgof coding DNA (pSG-CREB2, pSV-CREM-wt, or pSG5).

Electrophoretic Mobility Shift Assays and Supershift Assays—Nuclear extracts from YR2 cells or from PBMCs isolated from a healthy 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 double-stranded TxRE probes are as follows (the viral CRE sites are underlined): TxRE1 (centered at position –148) 5'-CAGACAGAGACGTCAGCTGCC-3'; TxRE2 (centered at position –123) 5'-AAGCTGGTGACGGCAGCTGGT-3'; TxRE3 (centered at position –48) 5'-GAGCTGCTGACCTCACCTGCT-3'; and TxRE1-mutCRE (5'-CAGACAGAGTGGTCAGCTGCC-3') (mutations are highlighted in boldface). The sequence of the coding strand of the double-stranded Oct-1 consensus oligonucleotide used as competitor was 5'-TGTCGAATGCAAATCACTAGAA-3'). For supershift assays, monoclonal antibodies against ATF-1 (Santa Cruz Biotechnology, catalog number sc-243X) and ATF-2 (catalog number sc-242X) and polyclonal antibodies against human CREB (catalog number sc-240X) and human full-length CREM (catalog number sc-440X) were added at a final concentration of 2 µg/reaction mixture at the beginning of the binding reaction for 20 min before adding the DNA probe (blocking conditions).

Western Blot Analysis—Nuclear extracts prepared from BLV-infected YR2 cells or in vitro translated CREB or CREM proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% nonfat dry milk and then incubated with anti-CREB (catalog number sc-240) or anti-CREM (catalog number sc-440) antibodies in blocking solution. A second antibody, horseradish peroxidase-conjugated goat anti-rabbit IgG (catalog number sc-2054), was used for enhanced chemiluminescence detection (Cell Signaling).

Preparation of mRNA and cDNA, RT-PCR Amplification, and Cloning of CREM Isoform—One million YR2 cells were used for extracting RNA (RNeasy mini kit, Qiagen). First strand cDNA synthesis was performed using 2 µg of YR2 total RNA and (dT)₁₆ primers (Invitrogen) with the Superscript II RNase H reverse transcriptase reagent, according to the manufacturer's protocol (Invitrogen). RNaseOUT^TM RNase inhibitor (Invitrogen) was added to the RT reaction. The resulting cDNA templates were used directly for PCR. After a denaturation period of 5 min at 95 °C, PCR was performed as follows: 40 cycles for 1 min at 95 °C, 1 min at 50 °C, 2 min at 72 °C, followed by a final elongation at 72 °C for 10 min. Reactions consisted of 10 µlof Pfu DNA polymerase 10x buffer, 3 µl of 25 mM MgCl₂, 2 µl of dNTP mixture (10 mM each), 5 units of Pfu DNA polymerase (all from Promega), 10 µl of sense and antisense primers (20 µM each), and 20 µl of cDNA in a final volume of 100 µl. Primers used for detecting CREM mRNA were designed according to the mouse cDNA sequence (supplemental Fig. S2) and are as follows: 5'-CCG GAA TTC ATG ACC ATG GAA ACA GTT GAA TCC CAG-3' (this forward primer localized in CREM exon B at the 5' end of the translational start codon (underlined) contains an added EcoRI restriction site (in boldface)) and 5'-GC TCT AGA CTA ATC TGT TTT GGG AGA GCA AAT GTC-3' (this reverse primer localized in CREM exon Ib at the 3' end of the DBD II contains an added XbaI restriction site (in boldface)). The forward primer is located at the most 5' end of exon B, and the reverse primer is located at the most 3' end of exon Ib. To control reproducibility of the results, RNA samples used for RT-PCR were collected from three independent cultures of YR2 cells. PCR products were separated on a 1.5% agarose gel and purified using the QIAEX II gel extraction kit (Qiagen). PCR products were restricted with XbaI/EcoRI and inserted into the XbaI/EcoRI sites of the eukaryotic expression vector pcDNA 3.1 (Invitrogen). The resulting constructs were transformed into TOP10 cells, and several independent clones were sequenced. Homology searches in the nucleotide and genomic DNA data bases were performed with BLAST software accessed through the NCBI web page (ncbi.nlm.nih.gov). The ovine CREM1 DNA sequence we cloned is deposited in GenBank^TM under accession number EF507802.

Chromatin Immunoprecipitation Assays—The chromatin immunoprecipitation (ChIP) assays were performed by using the ChIP assay kit (Upstate%20Biotechnology">Upstate Biotechnology, Inc.) according to the manufacturer's recommendations. Formaldehyde cross-linking reactions from 10⁷ BLV-infected YR2 cells were performed for 10 min (or 30 min for the detection of CBP recruitment) at room temperature and quenched with 125 mM glycine. Cells were lysed, and chromatin was sonicated to obtain an average DNA length of 600 bp. Following centrifugation, the chromatin was diluted 10-fold and precleared with a protein A-agarose slurry containing salmon sperm DNA and bovine serum albumin (Upstate%20Biotechnology">Upstate Biotechnology, Inc.). Precleared chromatin (2 ml) was incubated overnight at 4 °C with 5 µg of either anti-CREB antibody (catalog number sc-186X), anti-ATF-1 antibody (catalog number sc-243X), anti-ATF-2 antibody (catalog number sc-242X), anti-CREM antibody (catalog number sc-440X), anti-CBP (catalog number sc-369X), or normal rabbit IgG antibody (Upstate%20Biotechnology">Upstate Biotechnology, Inc., catalog number 12-370) as control. Incubation with the antibodies was followed by immunoprecipitation with protein A-agarose. Immunoprecipitated complexes were washed and eluted twice with 200 µl of elution buffer. The protein-DNA cross-links were reversed by heating at 65 °C overnight, and 10% of the recovered DNA was used for radioactive PCR amplification (35 cycles of 94 °C for 45 s, 55 °C for 30 s, and 72 °C for 1 min) with a primer set amplifying the BLV TxRE promoter region (nt –172 to +29), 5'-^nt-172CCGTAAACCAGACAGAGACGTCAG-3'/5'-^nt+29CACGAGGGTCTCAGGAGGAGAAC-3', or with an unrelated primer set amplifying a gag gene region located 1.8 kb downstream of the LTR region (nt +1736 to +1955): 5'-^nt+1736CAGCCCTCTCAGAATCAAGC-3'/5'-^nt+1955AATTTGCCATTTATCGAAAG-3'. PCR products from all reactions were resolved by PAGE and visualized by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Endogenous CREM Proteins from Nuclear Extracts of BLV-infected YR2 Cells and of Ovine PBMCs Specifically Bind to the BLV LTR CRE Motifs in Vitro—The potential role of CREM members in regulating BLV gene expression has so far not been examined. 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 expression is known to be finely regulated in a cell- and tissue-specific manner (16, 19, 27, 28), we decided to investigate whether CREM proteins were expressed in BLV-infected cells, and whether these CREM proteins were able to bind to the BLV LTR CRE motifs. To this end, we performed supershift EMSA experiments using the three 21-bp TxREs as probes. These probes were incubated with nuclear extracts prepared from YR2 cells derived from leukemic cells of a BLV-infected sheep (20), either mock-treated or treated for 1 h with phorbol ester phorbol 12-myristate 13-acetate plus Ca²⁺ ionophore ionomycin (P + I), a combination known to activate BLV expression (29, 30). Incubations were carried out in the presence of either a polyclonal anti-CREM antibody (Fig. 1A, lanes 3, 4, 7, 8, 11, and 12) 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-binding domain present in all CREM isoforms. Therefore, this antibody is predicted to be active against all CREM isoforms. Results presented in Fig. 1 showed that the mock-treated YR2 nuclear extracts formed three major retarded complexes (C1, C2, and C3) when incubated with the TxRE1, TxRE2, or TxRE3 probe (Fig. 1A, lanes 1, 5, or 9, respectively). Treatment of BLV-infected YR2 cells with P + I caused an increased binding activity to the TxREs (Fig. 1A, compare lanes 1 and 2, lanes 5 and 6, and lanes 9 and 10). Because P + I treatment is known to activate BLV expression, these data suggest that initiation of BLV transcription could occur, in response to P + I, through increased recruitment of CREB/CREM/ATF cellular transcription factors at the viral LTR TxREs. To evaluate the specificity of these interactions, unlabeled double-stranded oligonucleotides were prepared and used as competitors in EMSAs (Fig. 1B). Binding of proteins in complexes C1, C2, and C3 was shown to be sequence-specific to the CRE motif. Indeed, formation of these complexes was inhibited by a 80-fold molar excess of the unlabeled homologous TxRE1 oligonucleotide (Fig. 1B, compare lanes 1 and 4) but not by the same molar excess of an unlabeled TxRE1 oligonucleotide mutated at the CRE site or an unlabeled oligonucleotide with an unrelated sequence containing an Oct-1 consensus binding site (Fig. 1B, compare lanes 2 and 3 with lane 4). Addition of the anti-CREM antibody in the binding reactions resulted in the complete (mocktreated cells) or partial (P + I-treated cells) disappearance of the C2 complex concomitant with the appearance of a supershifted complex of lower mobility (S-CREM) but did not modify the migration profile of complexes C1 and C3 (Fig. 1A, lanes 3, 4, 7, 8, 11, and 12). In contrast, the presence of the IgG control did not affect formation of complexes C1, C2, and C3 (data not shown), indicating the specificity of the protein-antibody interaction. These results indicated that the C2 complex contained CREM isoform(s).

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Binding of CREM proteins from nuclear extracts of BLV-infected YR2 cells to the three BLV TxREs. A, oligonucleotides corresponding to the BLV TxRE1, TxRE2, and TxRE3 were 5'-end-labeled and used as probes in EMSAs. Prior to the addition of the TxRE1, -2, or -3 probe, 20 µg of nuclear extracts from BLV-infected YR2 cells either mock-treated (lanes 1, 3, 5, 7, 9, and 11) or treated with phorbol 12-myristate 13-acetate (P) plus ionomycin (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 were analyzed 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 better separation of the complexes. The supershifted (S-CREM) complexes are indicated by an arrow. B, specificity of the C1, C2, and C3 complexes was tested by competition experiments. The TxRE1 oligonucleotide was 5'-end-labeled and incubated with 10 µg 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 oligonucleotide (lane 1), the unlabeled CRE-mutated TxRE1 oligonucleotide (lane 3), or a heterologous Oct-1 consensus oligonucleotide (lane 2). Binding reactions were analyzed 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 figure shows only the specific retarded bands of interest.

To evaluate bZIP complex formation on the BLV TxREs, we next performed additional supershift experiments by including combinations of the antibodies in the binding reactions. We observed a dramatic decrease of complex C2 formation concomitant with the appearance of a super-supershifted complex (Fig. 2A, lane 6, ⁵SS-CREB + CREM) when both the anti-CREB and the anti-CREM antibodies were added to the same binding reaction mixture, indicating that CREM·CREB heterocomplexes bind to the BLV TxRE1. As a control for cross-reactivity between the anti-CREM antibody and the CREB protein, we performed Western blot analysis on CREB (TNT-CREB) and CREM (TNT-CREM) proteins produced in vitro in a transcription-translation-coupled wheat germ extract reaction (Fig. 2B). This control experiment shows that the anti-CREM antibody had no cross-reactivity with the TNT-CREB protein (Fig. 2B, top panel, lane 3), thereby demonstrating the relevance of the anti-CREM antibody to distinguish between CREM- and CREB-related proteins. In parallel, the same amounts of the TNT-control, TNT-CREM, and TNT-CREB proteins were used in a similar Western blot experiment with the anti-CREB antibody, and we observed the specific recognition of the TNT-CREB protein (Fig. 2B, bottom panel, lane 3). Therefore, both the anti-CREM and the anti-CREB antibodies appear to be highly specific for recognition of their respective target proteins. Moreover, we performed additional supershift experiments further confirming the specificity of the anti-CREM antibody against CREM isoforms (supplemental Fig. S1). The addition of both the anti-CREM and anti-ATF-1 antibodies to the same binding reaction resulted in a decreased complex C2 intensity and in the appearance of the ²S-CREM supershifted complex, but also in the appearance of a super-supershifted complex (Fig. 2A, lane 9, ⁷SS-CREM + ATF-1), suggesting the presence of CREM·ATF-1 heterocomplexes in complex C2. When both the anti-CREB and the anti-ATF-1 antibodies were added to the same binding reaction, we observed an important decrease of complex C2 formation concomitant with the appearance of the ¹S-CREB supershifted complex but also with the appearance of a super-supershifted complex (Fig. 2A, lane 7, ⁶SS-CREB + ATF-1), suggesting that CREB·ATF-1 heterocomplexes are present in complex C2. On the contrary, supershift experiments with the anti-ATF-2 antibody combined with either the anti-CREB antibody or the anti-CREM antibody or the anti-ATF-1 antibody did not result in the appearance of any super-supershifted complex, suggesting that ATF-2 did not bind to the BLV TxRE1 as heterocomplexes with those bZIP proteins (Fig. 2A, lanes 8, 10, and 11).

View larger version (56K): [in this window] [in a new window]   FIGURE 2. A, characterization of the protein complexes on the BLV TxRE1. Analyses of the protein composition of the three retarded complexes obtained by incubation of the TxRE1 probe with YR2 nuclear extracts were performed by supershift assays. Prior to the addition of the TxRE1 probe to the binding reaction, nuclear extracts from P+ I-treated BLV-infected YR2 cells were incubated with purified rabbit IgG as negative control (lane 1), with the anti-CREB antibody (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 reactions were analyzed by PAGE. The retarded complexes were visualized by autoradiography. The major DNA-protein complexes 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-2 to the TxRE1 probe required intact viral CRE motifs (lanes 12–16). B, as controls for cross-reactivity between the anti-CREM antibody and CREB proteins and between the anti-CREB antibody and CREM proteins, we performed Western blot (WB) analysis using in vitro translated CREB and CREM proteins and either the anti-CREM antibody (top panel) or the anti-CREB antibody (bottom panel). The in vitro translated proteins were generated by using the TNT Coupled 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 pSG5 empty vector as control (TNT control, lane 2). C, CREM proteins are expressed in uninfected ovine PBMCs. Supershift experiments were performed with nuclear extracts of PBMCs isolated from a healthy sheep. Prior to the addition of the TxRE1 probe to the binding reaction, the PBMC nuclear extracts were incubated with either a purified rabbit IgG as negative control (lane 1) or the anti-CREM antibody (lane 2). The probe was then added, and the binding reactions were analyzed by PAGE. The retarded complexes were visualized by autoradiography. The DNA-protein complexes N1 and N2 and the supershifted complex S-CREM are indicated by arrows.

Taken together, our in vitro binding studies demonstrate that CREM proteins are expressed in a BLV-infected B lymphoma cell line as well as in ovine-infected and uninfected PBMCs and that these CREM proteins bind to the TxREs of the BLV promoter in vitro in a CRE-dependent manner. Moreover, our results suggest that complex C1 contains ATF-2 homodimers and that formation of complex C2 results from the binding of the CREB, CREM, and ATF-1 bZIP proteins to the BLV 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 demonstrate in vivo, in the context of chromatin, the relevance of our in vitro binding studies, we performed chromatin immunoprecipitation (ChIP) assays using BLV-transformed YR2 cells. Formaldehyde cross-linked chromatin from these cells was used for immunoprecipitation with antibodies directed against ATF-1, ATF-2, CREB, or CREM or with a purified rabbit IgG as negative control. Following reverse of the cross-links, the purified DNA was subjected to radioactive PCR analysis using a set of primers flanking the TxRE region (nt –172 to +29) of the BLV promoter. Fig. 3 shows PCR amplification of the DNA after immunoprecipitation (Fig. 3, IP antibody, lanes 3–7), PCR amplification of the input DNA (Fig. 3, lane 2), and PCR amplification in the absence of DNA as negative control (Fig. 3, PCR control, lane 1). Analysis of PCR products from immunoprecipitated DNA showed significant enrichment of the TxRE region when immunoprecipitation was carried out with the anti-CREB, anti-ATF-1, anti-ATF-2, or anti-CREM antibody (Fig. 3, lanes 4–7, respectively). In contrast, no such enrichment was observed following immunoprecipitation of the cross-linked chromatin with purified rabbit IgG (Fig. 3, lane 3). These data confirmed the binding of CREB to the BLV TxREs in vivo, as demonstrated previously by our laboratory (9), and demonstrated in vivo the binding of other CREB/CREM/ATF members, including CREM protein(s), to this LTR region. As a control, we used another set of primers amplifying a region of the gag gene of the BLV provirus located 1.8 kb downstream of the TxRE region, which has not been reported so far as binding CREB/ATF family members. Immunoprecipitations with all the antibodies did not enrich eluates with DNA from this control region in YR2 cell chromatin, demonstrating the specificity of the TxRE interactions (data not shown). Thus, these data demonstrate the occupancy in vivo of the BLV TxRE region by CREB, ATF-1, ATF-2 and CREM proteins in the context of an integrated, chromatin-assembled BLV provirus.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Recruitment of CREB, CREM, ATF-1, and ATF-2 to the TxRE promoter region in vivo. ChIP assays were used to detect binding of bZIP transcription factors to the TxRE region of the BLV promoter in the chromosomal context of proviruses integrated in YR2 cells. DNA and protein were cross-linked with formaldehyde for 10 min, and DNA was sheared. The cross-linked protein-DNA complexes were immunoprecipitated (IP) with the anti-CREB antibody (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 negative control (lane 3). The protein-DNA cross-links were reversed, and the purified DNAs were amplified by semi-quantitative radioactive PCR using a primer set amplifying the BLV promoter TxRE region (nt –172 to +29). PCR of the input (sample representing PCR amplification from a 1:25 dilution of total input chromatin from the ChIP experiment) is shown in lane 2. The PCR control represents the PCR amplification in the absence of DNA (lane 1).

We conclude from these experiments that ectopic CREM protein has a CRE-dependent stimulatory effect on the BLV promoter. These results thus establish the functional significance of CREM through the CRE sites present in the BLV 5'-LTR. Our data suggest that transcriptional activity of this promoter is positively regulated by CREM and that CREM could initiate BLV transcription in the absence of Tax_BLV in newly infected cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. Response of the BLV promoter to ectopically expressed CREM transcription factor. A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of an LTR luciferase reporter construct wild-type (pLTRwt-luc) or mutated in the three CRE motifs (pLTR-mutCRE-luc) and with increasing amounts (250, 500n and 750 ng of plasmid DNA) of a CREM expression vector (pSV-CREM-wt). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CREM expression vector cotransfected were complemented to 750 ng of DNA by using the empty vector pSG5. Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms indicating the induction by CREM (in fold) with respect to the activity of each LTR reporter construct in the absence of CREM, which was assigned a value of 1. Means ± S.E. are shown. A representative experiment performed in triplicate of three independent transfections is shown.

To assess the role of BCR cross-link in CREM transactivation of the BLV promoter, we transiently cotransfected A20 cells with pLTRwt-luc and with increasing amounts of pSV-CREM-wt. Transfected cells were mock-treated or treated with anti-IgG antibody and then lysed and assayed for luciferase activity. Results presented in Fig. 5, A and B, showed that, in the absence of anti-IgG treatment, CREM transactivated BLV LTR-directed gene expression in a dose-dependent manner (up to 3.7-fold), whereas in the presence of anti-IgG treatment, we observed a dose-dependent increase in luciferase activity by ectopically expressed CREM (up to 5.7-fold) (Fig. 5B). These results indicate that cross-link of BCR with anti-IgG potentiates CREM transactivation of the BLV promoter and suggest that phosphorylation of CREM could modulate its ability to transactivate BLV gene expression.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. BCR cross-link potentiates CREM transactivation of the BLV promoter. A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and increasing amounts (250, 500, and 750 ng of plasmid DNA) of pSV-CREM-wt. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CREM expression vector cotransfected were complemented to 750 ng of DNA by using the empty vector pSG5. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms. A, indicating luciferase activities (arbitrary units) normalized to protein concentrations; B, indicating the inductions by CREM (in fold) with respect to the activity of pLTRwt-luc in the absence of CREM, which was assigned a value of 1. Means ± S.E. from a representative experiment performed in triplicate of three independent transfections are shown.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. A, expression of CREM in BLV-infected YR2 cells. Nuclear extracts from YR2 cells either mock-treated (lane 1) or treated with P + I for 30 min or 1 or 4 h (lanes 2–4) were fractionated by SDS-PAGE, transferred on a polyvinylidene difluoride membrane, and immunoblotted with the anti-CREM antibody. B, functional role of the cloned ovine CREM1 isoform in BLV promoter-driven gene expression. A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of either pLTRwt-luc or pLTR-mutCRE-luc and increasing amounts (250 and 500 ng of plasmid DNA) of pcDNA-CREM1. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CREM1 expression vector cotransfected were complemented to 500 ng of DNA by using the empty vector pcDNA3.1. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms indicating the inductions by CREM1 (in fold) with respect to the activity of pLTRwt-luc in the absence of CREM1, which was assigned a value of 1. Means ± S.E. from a representative experiment performed in triplicate of three independent transfections are shown.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. CREM Ser-117 mutation reduces anti-IgG potentiation of CREM transactivation of the BLV LTR. A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and 500 ng of either pSV-CREM-wt, pSV-CREM-S117A, or the empty vector pSG5. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the 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 of three independent transfections are shown.

CREM Ser-117 Mutation Reduces the BCR Cross-link Potentiation of CREM Transactivation of the BLV Promoter—To address the role of CREM phosphorylation at Ser-117 in CREM-mediated anti-IgG stimulation of BLV LTR gene regulation, we substituted the Ser-117 residue for an alanine residue in the context of the pSV-CREM-wt vector, thereby generating pSV-CREM-S117A. We next transiently cotransfected A20 B-cells with pLTRwt-luc and with either the pSV-CREM-wt or the pSV-CREM-S117A expression vector. Cells were mock-treated or treated with anti-IgG antibody, and luciferase activities were measured in cell lysates (Fig. 7). We observed that mutation of CREM Ser-117 resulted in a decrease in CREM transactivation of the BLV promoter in the presence of anti-IgG treatment (Fig. 7, anti-IgG, compare pSV-CREM and pSV-CREM-S117A). These data indicate that CREM Ser-117 mutation reduced anti-IgG potentiation of CREM transactivation of the BLV 5'-LTR, suggesting that this potentiation was mediated, at least in part, through CREM Ser-117 phosphorylation.

CBP/p300 Coactivators Are Involved in CREM Transactivation of the BLV 5'-LTR—Ser-117 phosphorylation is known to activate CREM-directed transcription through the recruitment of two coactivators, CBP and p300. Therefore, we decided to examine the potential functional effect of CBP/p300 on CREM-mediated transactivation of the BLV promoter. To this end, A20 cells were cotransfected with pLTRwt-luc, with pSV-CREM-wt, and with an expression vector coding for the adenovirus wild-type E1A 12S protein (pE1A-12S-wt) or an expression vector coding for a mutant form of the E1A 12S protein (pE1A-12S/2-36del) as negative control. The adenovirus E1A oncoprotein is known to bind to CBP/p300 and to inhibit their capacity to coactivate transcription (50–52). The pE1A-12S/2-36del expression vector codes for an E1A mutant protein that lacks the ability to interact with CBP/p300 and fails to inhibit CBP/p300 activity. Results presented in Fig. 8A showed that coexpression of the wild-type E1A 12S protein decreased (by 2-fold) CREM-mediated transactivation of the BLV promoter, both in the presence and in the absence of anti-IgG treatment, when compared with coexpression of the mutant E1A/2-36del protein (Fig. 8A). These results suggest that CBP/p300 are functionally involved in CREM transactivation of the BLV LTR.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Functional involvement of CBP in transactivation of the BLV LTR 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 or pE1A-12S/2-36del. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the 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. B, ChIP assays were performed in order to demonstrate CBP recruitment to the BLV promoter. Cells were treated with P + I for 1 h; DNA and protein were cross-linked with formaldehyde for 30 min, and DNA was sheared. The cross-linked DNA-protein complexes were immunoprecipitated (IP) with the anti-CBP antibody (lane 4) or with purified rabbit IgG as negative control (lane 3). Cross-links were reversed, and purified DNA was amplified by semi-quantitative radioactive PCR using a primer set amplifying the BLV promoter TxRE region (nt –172 to +29). PCR of the input (sample representing PCR amplification from a 1:25 dilution of total input chromatin from the ChIP experiment) is shown in lane 2. The PCR control represents the PCR amplification in the absence of DNA (lane 1). C, HeLa cells were transiently cotransfected using the jetPEITM procedure with 500 ng of pLTRwt-luc and increasing amounts (from 0 to 200 ng of plasmid DNA) of pRc-RSV-CBP. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CBP expression vector cotransfected were complemented to 200 ng of DNA by using the parental empty vector. Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms indicating relative light units (RLU). D, HeLa cells were transiently cotransfected using the jetPEITM procedure with 500 ng of pLTRwt-luc and increasing amounts (from 0 to 60 ng of plasmid DNA) of pSV-CREM-wt, in the presence or the absence of 50 ng of pRc-RSV-CBP. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CREM expression vector cotransfected were complemented to 60 ng of DNA by using the empty vector pSG5. Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms indicating relative light units (RLU). Means ± S.E. from a representative experiment performed in triplicate of three independent transfections are shown.

CREM Has No Effect on Transactivation of the BLV Promoter by the Viral Transactivator Tax_BLV, whereas CREB and Tax_BLV Synergistically Activate BLV Promoter Transcriptional Activity—Several studies have suggested that Tax_BLV transactivation of the BLV promoter is mediated through interactions of Tax_BLV with cellular factors that bind to the CRE motifs of the viral TxREs. Our laboratory has reported previously that a dominant negative inhibitor of CREB (A-CREB) efficiently inhibits Tax_BLV transactivation of the BLV promoter, suggesting that recruitment of CREB to the BLV TxREs plays a critical role in the mechanism of Tax_BLV transactivation (9). However, the effect of CREM expression on Tax_BLV-mediated transactivation of the BLV 5'-LTR has not been examined so far. Therefore, we transiently cotransfected the B-lymphoid A20 cell line with pLTRwt-luc, the CREM expression vector, and a Tax_BLV expression vector (pSG-Tax_BLV). Because Ser-117 phosphorylation of CREM could also intervene in Tax_BLV-mediated transactivation, transfected cells were mock-treated or treated with anti-IgG antibody. As control, we compared in the same transfection experiment the effect of CREB expression on Tax_BLV-mediated transactivation of the BLV LTR, by including cotransfections with a CREB expression vector (pSG-CREB) instead of the CREM expression vector. Results presented in Fig. 9 showed that, in the absence of anti-IgG treatment, cotransfection of 20 ng of the pSG-Tax_BLV alone resulted in a 40.7-fold stimulation of the luciferase activity (Fig. 9) and that cotransfection of pSV-CREM-wt or pSG-CREB-wt alone resulted in a 1.6- or 10.9-fold activation of transcription, respectively (Fig. 9). Remarkably, when cells were cotransfected with both the pSG-Tax_BLV and pSG-CREB-wt, a strong synergism was observed between Tax_BLV and CREB, resulting in a 197-fold activation of the BLV promoter activity (Fig. 9). Transcriptional activators synergize when their combination produces a transcriptional rate that is greater than the sum of the effects produced by the individual activators (53). In the latter case, the amount of transcription in the presence of both Tax_BLV and CREB (197-fold) is 4.9-fold greater (fold synergism) than the sum of the effect produced by each activator separately (10.9 + 40.7). These results demonstrate that CREB synergistically enhances transcriptional activation of the BLV promoter by Tax_BLV, and we confirm that CREB plays an important role in the mechanism of Tax_BLV transactivation. On the contrary, when cells were cotransfected with both pSG-Tax_BLV and pSG-CREM-wt, no synergism between Tax_BLV and CREM was observed. Indeed cotransfection of both expression vectors led to a 38-fold stimulation of the luciferase activity, which is similar to the sum of the effect produced by Tax_BLV and CREM individually (40.7 + 1.6) (Fig. 9), suggesting that CREM is not involved in Tax_BLV transactivation of the BLV LTR.

View larger version (16K): [in this window] [in a new window]   FIGURE 9. CREB and TaxBLV synergistically activate BLV promoter transcriptional activity, whereas CREM has no effect on TaxBLV-mediated transactivation of the BLV promoter. A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc and with 20 ng of a TaxBLV expression vector (pSG-TaxBLV) in the presence or the absence of 500 ng of pSV-CREM-wt or pSG-CREB-wt. To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of expression vectors cotransfected were complemented to 520 ng of DNA by using the empty vector pSG5. At 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the 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, CREB, TaxBLV, and anti-IgG treatment, which was assigned a value of 1. Means ± S.E. from a representative experiment performed in triplicate of three independent transfections are shown.

Thus, our results demonstrate a strong synergism between Tax_BLV and CREB in transcriptional transactivation of the BLV promoter, both in the absence and presence of BCR cross-link, and confirm previous studies from our laboratory indicating that CREB recruitment at the BLV promoter plays an important role in the mechanism of transactivation by Tax_BLV (9). In contrast, our results show no synergism between Tax_BLV and CREM, suggesting that CREM is not involved in the mechanism of Tax_BLV-mediated transactivation.

CREM Counteracts the Functional Action of CREB and Affects the Tax_BLV/CREB Synergistic Activation of the BLV Promoter—Because both CREB and CREM are recruited to the BLV promoter through the CRE-like motifs of the TxREs, we next wanted to determine their mutual influence on Tax_BLV-mediated transactivation. We therefore tested whether CREM could counteract the action of CREB and affect the Tax_BLV/CREB synergistic activation of BLV promoter transcriptional activity and/or whether CREB could counteract the action of CREM and synergize with Tax_BLV in the presence of CREM. To this end, we performed cotransfection experiments in A20 B-cells with pLTRwt-luc, with pSG-Tax_BLV, and with a fixed amount of pSG-CREB in the presence of increasing amounts of pSV-CREM (Fig. 10A), or with pLTRwt-luc, with pSG-Tax_BLV, and with a fixed amount of pSV-CREM in the presence of increasing amounts of pSG-CREB (Fig. 10B). Results showed that, in both the absence and the presence of anti-IgG treatment, the Tax_BLV/CREB synergism was repressed by increasing amounts of ectopically expressed CREM (Fig. 10A). Moreover, cotransfection of increasing amounts of CREB in the presence of a fixed amount of CREM activated synergistically with Tax_BLV LTR-directed reporter gene expression (Fig. 10B). Taken together, our results suggest that CREB and CREM exert opposite roles in Tax_BLV-mediated transactivation of the BLV promoter and that a modulation of the level of Tax_BLV transactivation takes place through a competition between the actions of CREB and CREM. This competition would depend on the relative spatio-temporal expression levels of those proteins in BLV-infected cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have shown that CREM proteins were expressed in the YR2 cell line derived from leukemic cells of a BLV-infected sheep as well as in infected and uninfected ovine PBMCs and that these CREM proteins bound to the BLV promoter in vitro, through the three CRE-like motifs present in the middle of each viral TxRE. Supershift analysis suggested that in vitro CREM formed heterocomplexes with CREB and ATF-1 to bind to the BLV TxREs. Moreover, chromatin immunoprecipitation analysis using the BLV-infected YR2 B-cell line demonstrated the recruitment of CREM proteins to the BLV promoter in vivo, in the chromatin context of a BLV-integrated provirus. Functionally, we showed that in the absence of the viral Tax_BLV protein, ectopic expression of the CREM activator isoform up-regulated BLV promoter activity through the three BLV LTR CRE-like motifs. Cross-link of the B-cell receptor potentiated CREM transactivation of the viral promoter. Further experiments supported the notion that this potentiation could involve CREM Ser-117 phosphorylation and recruitment of CBP/p300 by CREM. Finally, CREB and CREM seem to play opposite roles in Tax_BLV-mediated transactivation of the BLV promoter. Although CREB and Tax_BLV synergistically activated the LTR, CREM repressed this Tax_BLV/CREB synergism.

In conclusion, these results suggested that activation of Tax_BLV-independent BLV transcription could occur in vivo, in response to B-cell activation. CREM could thus initiate a low level of BLV transcription and lead to the synthesis of small amounts of the Tax_BLV transactivator, which would then amplify transcription from the 5'-LTR. In this regard, we have demonstrated that CREM proteins are expressed in PBMCs isolated from a healthy sheep and are able to bind to the BLV promoter in vitro, supporting our hypothesis that CREM could initiate BLV transcription in the absence of Tax_BLV in newly infected cells. Moreover, we have shown that CREM did not potentiate Tax_BLV-mediated transactivation of BLV LTR-directed gene expression. Indeed, our cotransfection experiments indicated that overexpression of CREM repressed the synergistic activation of the BLV promoter by Tax_BLV and CREB. The absence of viral proteins at the surface of BLV-infected cells is necessary to escape from an efficient host immune attack and to allow BLV-induced tumor development. However, because the tax_BLV gene product is considered to account for viral leukemogenicity (22, 54–56), a low level of basal BLV expression is likely necessary at the early stage of BLV infection to induce transformation of the infected cells. Based on our results we suggest a bimodal role of CREM in BLV LTR-directed transcription: in the absence of Tax_BLV, CREM would initiate BLV transcription in response to activation of the BLV-infected B-cells, and on the contrary, in the presence of Tax_BLV, CREM would decrease Tax_BLV-mediated transactivation of the BLV promoter by competing with CREB for binding to the viral CREs. CREM would thus be able to initiate a low level of BLV expression necessary for Tax_BLV-induced transformation of the infected cells, but in the presence of Tax_BLV, CREM would reduce BLV expression and thus facilitate immune escape and allow tumor development.

View larger version (19K): [in this window] [in a new window]   FIGURE 10. Opposite roles of CREB and CREM during TaxBLV-mediated transactivation of the BLV promoter. A, A20 cells were transiently cotransfected using the DEAE-dextran procedure with 500 ng of pLTRwt-luc, 250 ng of 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 transfected DNA and to avoid squelching artifacts, the different amounts of CREM expression vector cotransfected were complemented to 750 ng of DNA by using the empty vector pSG5. B, A20 cells were transiently cotransfected using 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 to 750 ng of plasmid DNA). To maintain the same amount of transfected DNA and to avoid squelching artifacts, the different amounts of CREB expression vector cotransfected were complemented to 750 ng of DNA by using the parental empty vector pSG5. A and B, at 20 h post-transfection, cells were mock-treated or treated with anti-IgG antibody (6.5 µg/ml). Luciferase activities were measured in cell lysates 42 h after transfection and were normalized with respect to protein concentrations of the lysates. Results are presented as histograms indicating relative light units (RLU) with respect to the basal activity of pLTRwt-luc in the absence of CREB, CREM, TaxBLV, and anti-IgG treatment, which was assigned a value of 1. Means ± S.E. from a representative experiment performed in triplicate of three independent transfections are shown.

Regulation of the expression of the different activator and repressor isoforms of the CREM gene has been extensively studied in spermatogenesis (14, 15, 17, 46–48). During spermatogenesis, only CREM repressors are expressed in pre-meiotic germ cells, but a switch to the expression of the CREM and CREM2 activators occurs in post-meiotic germ cells (15, 17, 46, 65) and allows expression of testis-specific genes such as calspermine, TP1, and RT7 (66–68). However, little is known about regulation of CREM expression in B-cells. In this study we have shown that BLV-infected YR2 B-cells express a CREM1 activator isoform and demonstrate the functional role of this isoform in BLV expression. Two different studies have studied specific expression of the CREM gene in B-lymphoid cells. The first study, published by Pongubala and Atchison (69), has suggested through RNA analysis that the CREM gene is expressed differentially in the B-cell lineage. More specifically, their hypothesis is that repressor isoforms of CREM could predominate at the pre-B-cell stage and that an activator form of CREM could replace these repressive forms of CREM at later stages of B-cell development. The second study, published by van 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 another CREM 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 regulated during differentiation and activation of B-cells. At the early stage of BLV infection, predominant expression of CREM repressor isoforms in the infected B-cells would explain the absence of BLV expression. However, a switch in CREM gene expression following B-cell activation or differentiation would allow initiation of BLV transcription, thereby generating the first Tax_BLV molecules. In this regard, it has been demonstrated that, in BLV-infected but asymptomatic sheep, BLV integrated both in CD5– and CD5+ B-cells. In lymphoma, however, BLV provirus was detected only in CD5–-B-cells but not in CD5+ B-cells (71). Furthermore, van den Broeke et al. (72) have demonstrated that a variety of ovine B-cell populations can support a productive BLV infection, suggesting that BLV can infect and replicate 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 CREM isoforms during basal and Tax_HTLV-I-activated transcription of the HTLV-I promoter. Bodor et al. (73) have demonstrated that CREM binds to all three HTLV-I CRE-like motifs and attenuates the activation of a HTLV-I LTR-CAT reporter construct by Tax_HTLV-I and protein kinase A. Further studies by the same group have shown that elevated cAMP levels in T cells correlate with expression of the potent transcriptional repressor ICER (74). Moreover, it has been shown that quiescent PBMCs respond to cell activation by producing sustained ICER RNA levels through 18 h post-stimulation and that ICER is able to inhibit Tax_HTLV-I-mediated transactivation of the HTLV-I promoter (75), suggesting a role for ICER in the establishment and maintenance of a persistent HTLV-I infection. In contrast, Laurance et al. (76) have shown that binding of the CREM isoform to the HTLV-I CREs allows Tax_HTLV-I to stimulate viral LTR expression, but with concomitant inhibition of Tax_HTLV-I effects on other CRE-containing genes. Together, these studies indicate that the level of HTLV-I transcription is influenced by the expression pattern of the different CREM isoforms according to the state of T-cell activation and/or differentiation. Both BLV and HTLV-I oncoretroviruses could therefore use similar strategies, depending on the CREB and CREM gene expression levels, to inhibit and/or activate viral transcription according to the activation state of the infected cell.

	FOOTNOTES

 
^* This work was supported in part by grants (to C. V. L.) from the "Fonds National de la Recherche Scientifique" (Belgium), the Télévie Program of the Fonds National de la Recherche Scientifique, the "Action de Recherche Concertée du Ministère de la Communauté Franaise," ULB, ARC Program 04/09-309, the Internationale Brachet Stiftung, the Interreg III Program ("Intergenes" Project), the Theyskens-Mineur Foundation, the "Fortis Banque Assurance," and the "Fédération Belge Contre le Cancer." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) EF507802.

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

¹ Fellow of the Belgian Fonds pour la Recherche dans l'Industrie et l'Agriculture (FRIA) and of the FNRS Télévie Program. Present address: Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, 3755 Cote Ste. Catherine, Montreal, Quebec H3T 1E2, Canada.

² Both authors contributed equally to this work.

³ Supported by a postdoctoral fellowship from the Région Wallonne Program Waleo2 616295.

⁴ Chargé de Recherches of the Fonds National de la Recherche Scientifique.

⁵ Fellow of the Fonds National de la Recherche Scientifique Télévie Program.

⁶ Directeur de Recherches of the Fonds National de la Recherche Scientifique. To whom correspondence should be addressed: Université Libre de Bruxelles, Institut de Biologie et de Médecine Moléculaires, Laboratoire de Virologie Moléculaire, Rue des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium. Tel.: 32-2-650-9807; Fax: 32-2-650-9800; E-mail: cvlint{at}ulb.ac.be.

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

⁸ T. L.-A. Nguyen and C. Van Lint, unpublished results.

	ACKNOWLEDGMENTS

 
We thank Drs. Luc Willems and Richard Kettmann (Faculty of Agronomy, Gembloux, Belgium), Dr. Didier Trouche (CNRS, Toulouse, France), and Dr. Paolo Sassone-Corsi (Institut de Genetique et de Biologie Moléculaire et Cellulaire, B.P. 10142, 67404 Illkirch-Strasbourg, France) for reagents used in this study.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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