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J Virol. Dec 2004; 78(24): 13848–13864.
PMCID: PMC533944

Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency

Abstract

Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.

In the course of retrovirus infection, the integration of the viral DNA into the host genome is a crucial step in the virus life cycle. This step is important for the efficient expression of progeny virus and is responsible for the ability of retroviruses to persist and cause disease (72). Once integrated, the proviral DNA is organized into chromatin, as are all cellular genes. At least two different, yet highly conserved, mechanisms are at work to alter chromatin structure: chromatin remodeling activities and/or factors and posttranslational modifications of histone proteins, in particular, histone acetylation (reviewed in references 46 and 63). Acetylation of histones allows DNA to become more accessible to the transcriptional machinery and can lead to nucleosome disruption. In contrast, histone deacetylation allows the histones to bind more tightly to DNA and therefore contributes to a transcriptionally repressed chromatin architecture. The level of histone acetylation is determined by the competition between enzymatic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (18). The activity of a de novo integrated retroviral promoter can be strongly affected by the site of integration and by the histone acetylation status at this site.

Previous reports have demonstrated that deacetylases play an important role in transcriptional silencing of several viruses, such as Epstein-Barr virus (EBV) (37, 77), human cytomegalovirus (57), and Kaposi's sarcoma-associated herpesvirus (29). Regarding retroviruses, feline foamy virus was shown to be reactivated following deacetylase inhibitor treatment (32) and histone hyperacetylation induces human immunodeficiency virus type 1 expression by specifically disrupting a single nucleosome positioned immediately downstream of the transcription start site (20, 33, 83). The presence of HDACs in the human T-cell leukemia virus type 1 (HTLV-1) promoter was recently observed, and treatment of HTLV-1-infected cells with deacetylase inhibitors increases the level of histone H4 acetylation in the HTLV-1 promoter and concomitantly increases viral transcription (48).

Bovine leukemia virus (BLV) gene expression is induced at the transcriptional level by the virus-encoded transactivator TaxBLV, which acts through three 21-bp Tax-responsive elements (TxREs) located in the U3 region of the 5′ long terminal repeat (5′ LTR) (22, 40). TaxBLV binds indirectly to DNA through cellular proteins of the CREB/ATF family of transcription factors (2, 45, 88). In the early stages of BLV infection, i.e., in the absence of TaxBLV, a basal transcriptional activity is ensured by several cis-acting elements located in the 5′ LTR. In the U3 region, each TxRE contains an imperfectly conserved cyclic AMP responsive element (CRE), which binds CRE binding protein (CREB), activating transcription factor 1 (ATF1), and ATF2 (1). Besides the imperfect CRE consensus, each TxRE also contains an E box sequence, which overlaps each of the three CRE-like motifs (80, 87). A PU.1/Spi-B binding site (21) and a glucocorticoid responsive element (GRE) (7, 8, 66, 90) are also present in U3. Finally, viral expression is regulated by LTR sequences downstream of the transcription initiation site: an upstream stimulatory factor (USF)-binding site in the R region (14) and an interferon regulatory factor (IRF)-binding site in the U5 region (42).

It was previously reported that treatment with deacetylase inhibitors increased viral expression in peripheral blood mononuclear cells (PBMCs) from BLV-infected sheep or cows (59). The objective of the present study was to further investigate the role of deacetylase inhibitors in the activation of BLV gene expression. More specifically, we performed a systematic analysis of the 5′ LTR cis-acting DNA elements to delineate the sequences responsible for the transcriptional induction of BLV promoter activity in response to different deacetylase inhibitors. Indeed, on one hand, several of the nuclear factors binding to the LTR, including CREB/ATF (4, 19), PU.1/Spi-B (91, 92), USF2 (12), glucocorticoid receptor (38, 44), IRF1 (71), and IRF2 (56), have been shown to interact with acetyltransferases and/or to be acetylated. On the other hand, some of these factors, including PU.1 (43) and glucocorticoid receptor (35), have been shown to interact with deacetylases. These factors therefore represent good candidates for the specific targeting of HATs and HDACs to the BLV promoter, thereby regulating the acetylation level of histones and/or of transcription factors binding to the LTR.

In the present study, we demonstrated that BLV promoter activity was induced by several deacetylase inhibitors, both in the context of episomal LTR constructs and in the context of integrated BLV proviruses. We reported a close correlation between histone H4 hyperacetylation in vivo and BLV transcription following trichostatin A (TSA) treatment. We next investigated whether the stimulatory effect of TSA could be mediated through one of the known cis-regulatory DNA elements located in the 5′ LTR. We showed that, among these elements, the E box motifs overlapping the CRE-like elements in U3 were involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutation of the three E boxes markedly reduced the inducibility of the BLV promoter by TSA, suggesting that deacetylases could be recruited to the BLV 5′ LTR through these E box elements. E boxes are susceptible to recognition by basic helix-loop-helix leucine-zipper (bHLH-ZIP) transcriptional repressors such as Max-Mad-mSin3 complexes (54), which repress transcription by recruiting HDACs (6). However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the U3 E box-specific complexes. Importantly, TSA increased the occupancy of the TxRE CRE-like motifs by CREB/ATF, suggesting that the proteins binding to the E boxes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB and ATF and/or their transcriptional coactivators possessing acetyltransferase activities. These results identify a novel strategy for BLV to minimize viral gene expression and escape from the host immune response, thereby favoring tumor development.

MATERIALS AND METHODS

Animals.

The animals used in this study were BLV-seropositive adult sheep (M2658 and M2311) affected with lymphocytosis presenting a persistently elevated lymphocyte count and an inverted B/T lymphocyte ratio. Sheep were housed at the Veterinary and Agrochemical Research Center (Machelen, Belgium).

Isolation of PBMCs and cell culture conditions.

Blood samples were collected by jugular venipuncture, mixed with EDTA as an anticoagulant, and centrifuged at 1,750 × 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 (PBS) containing 0.075% EDTA, with centrifugation steps at 450 × g for 10 min at room temperature. The cells were washed with PBS (centrifugations at 200 × g for 10 min at room temperature) 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 U of penicillin/ml, and 50 μg of streptomycin/ml and cultured in the absence or presence of TSA (500 nM; Sigma Aldrich) for 22 h at 37°C with 5% CO2.

Cell lines and cell culture.

The EBV-positive B-cell line Raji is derived from a Burkitt's lymphoma. The human epithelial HeLa cell line is derived from a cervical carcinoma and is transformed by human papillomavirus type 18. All media, sera (Myoclone Superplus), and supplements were from Invitrogen. Raji cells were grown in RPMI 1640-Glutamax I medium supplemented with 10% fetal bovine serum, 50 U of penicillin/ml, and 50 μg of streptomycin/ml. HeLa cells were cultured in Dulbecco's modified Eagle's-Glutamax I medium containing 5% fetal bovine serum, 50 U of penicillin/ml, and 50 μg of streptomycin/ml. YR2, a cloned B-lymphoid cell line established from peripheral blood lymphocytes isolated from a BLV-infected sheep (81) and the YR2 derivative cell line, YR2LTaxSN, were maintained in OptiMEM medium supplemented with 10% fetal bovine serum, 50 U of penicillin/ml, and 50 μg of streptomycin/ml. All cells were grown at 37°C in an atmosphere of 5% CO2.

Plasmid constructs.

The plasmid pLTRwt-luc was described previously (14) and contains the luciferase gene under the control of the complete 5′ LTR of the 344 BLV provirus (89). This vector was used as a substrate for mutagenesis with the QuikChange site-directed mutagenesis method (Stratagene). Some of these mutated constructs were previously described: pLTR(E-box1,2,3-mutB)-luc (14), pLTR(E-box4-mutB)-luc (14), pLTR(IRF-mut)-luc (14), and pLTR(PU.1-mut)-luc (previously called pLTRmut1-luc in reference 21). An additional mutation was generated with the following pair of mutagenic oligonucleotide primers (the mutation is highlighted in boldface type, and the GRE motif is underlined on the coding strand primer): cv751-752 (GRE-mut), 5′-CGAAAAATCCTATCCCACAGTAGCTGACCT-3′. Two constructs containing combinations of mutations were also generated by site-directed mutagenesis by simultaneously using two of the following mutagenic oligonucleotide primers (the mutation is highlighted in boldface type, and the CRE motif is underlined on the coding strand primer): cv409-410 (5′-CGTAAACCAGACAGAGTGGTCAGCTGCCAGAAAAGCTGGTGTGGGCAGCTGGTGGCTAGAATCC-3′) and cv415-416 (5′-CCACACCCCGAGCTGCTGTGCTCACCTGCTGATAAAAC-3′) (CRE1,2,3-mut); cv533-534 (5′-GTAAACCAGACAGTGACGTCAGCTGCCAGAAAAGCTGGTGACGTCAGCTGGTGGCTAG-3′) and cv535-536 (5′-CCCGAGCTGCTGACGTCACCTGCTG-3′) (CRE1,2,3-perfect). Mutated constructs were fully resequenced after identification by cycle sequencing with the Thermosequenase DNA sequencing kit (Amersham Biosciences). These mutated plasmids were designated pLTR(GRE-mut)-luc, pLTR(CRE1,2,3-mut)-luc, and pLTR(CRE1,2,3-perfect)-luc, respectively. In addition, pLTR(CRE1,2,3-perfect)-luc was used as a substrate for site-directed mutagenesis by using the following two mutagenic oligonucleotide primers (the mutation is highlighted in boldface type, and the E box motif is underlined on the coding strand primer): cv617-618 (5′-GACAGTGACGTCAGCGACCAGAAAAGCTGGTGACGTCAGCGAGTGGCTAGAATCC-3′) and cv619-620 (5′-GAGCTGCTGACGTCACCGACTGATAAAACAATAA-3′). This mutated plasmid was designated pLTR(CRE1,2,3-perfect/E-box1,2,3-mutB)-luc.

To construct pREP10s, a 29-bp protruding double-stranded oligonucleotide (5′-TCGAA-CAGCTG-AAGCTT-CTCGAG-GGATCC-3′) containing the PvuII-HindIII-XhoI-BamHI multiple cloning site (in boldface type) was cloned in the sense orientation into the SalI-restricted mammalian episomal expression vector pREP10 (Invitrogen), thereby deleting nucleotides 8 to 1097. A 2,530-bp fragment containing the BLV LTR upstream of the luciferase gene, and the simian virus 40 (SV40) late poly(A) signal was prepared from pLTRwt-luc (14) by digestion with KpnI, blunt ending of the 3′ overhang with T4 DNA polymerase, and digestion with BamHI, successively. This fragment was cloned into pREP10s digested with PvuII and BamHI. This cassette, LTR BLV-luciferase gene-SV40 poly(A) signal, is thus cloned in the sense orientation relative to the succession of the different genetic elements present in the pREP10s vector [EBNA-1/OriP/LTR BLV-luciferase gene-SV40 late poly(A) signal]. The resulting plasmid was designed pLTR*wt(direct)-luc.

The eukaryotic HDAC expression vectors (kindly provided by Stéphane Emiliani) contained the human HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, and HDAC6 cDNAs cloned in the pcDNA3.1(+/−) vector (Invitrogen).

Transient transfection and luciferase assays.

Raji cells were transfected by using the DEAE-dextran procedure as described previously (84). Briefly, cells were harvested at density of 106 cells/ml, washed with STBS (25 mM Tris-HCl [pH 7.5], 137 mM NaCl, 5 mM KCl, 700 μM CaCl2, 500 μM MgCl2, 600 μM Na2HPO4), and resuspended at a concentration of 6 × 106 cells in 600 μl of a mixture containing 500 ng of the pGL3-BASIC-derived constructs (with or without cotransfected DNAs) and 450 μg of DEAE-dextran (Amersham Biosciences)/ml in STBS. Cells were incubated for 1 h at 37°C, washed twice with STBS and once with culture medium, and grown in 3 ml of supplemented medium. At 20 h posttransfection, the cells were treated or mock treated with increasing concentrations of TSA (from 62 to 1,000 nM), sodium butyrate (NaB, from 0.61 to 10 mM; Sigma Aldrich), or valproic acid (VPA, from 0.25 to 5 μM; Sigma Aldrich). At 42 h posttransfection, cells were then lysed and assayed for luciferase activity (Promega). Luciferase activities derived from BLV LTRs were normalized with respect to protein concentrations by using the detergent-compatible protein assay (Bio-Rad).

HeLa cells were transfected by using FuGENE-6 (Roche) according to the manufacturer's protocol. Briefly, cells were seeded at a density of 2 × 105 cells/well in six-well plates. FuGENE-6 (3 μl) was added directly to serum-free medium (97 μl) 5 min prior to the addition to the DNA. One hundred microliters of this FuGENE-6-serum-free medium mix was added to the DNA mixture (1 to 2 μg) in microcentrifuge tubes. The mixture was incubated for 15 min at room temperature and, finally, was added to each well of a six-well plate. Transfected cells were grown in 2 ml of supplemented medium. At 20 h posttransfection, the cells were treated or mock treated with increasing concentrations of TSA (from 62 to 1,000 nM), NaB (from 0.61 to 10 mM), or VPA (from 0.25 to 5 μM). At 42 h posttransfection, cells were then lysed and assayed for luciferase activity (Promega). Luciferase activities derived from BLV LTRs were normalized with respect to protein concentrations by using the detergent-compatible protein assay (Bio-Rad).

ChIP assay in vivo.

The chromatin immunoprecipitation (ChIP) assay was performed by using the ChIP assay kit (Upstate Biotechnology) according to the manufacturer's recommendations. Formaldehyde cross-linking reactions (final formaldehyde concentration of 1%) from 107 BLV-positive YR2 cells, mock treated or treated with TSA (500 nM) for 22 h, were quenched with 125 mM glycine after a 10-min incubation at 37°C. Cells were lysed, and chromatin was sonicated to obtain an average DNA length of 500 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 Biotechnology). Precleared chromatin (2 ml) was incubated or not with 5 μg of anti-acetyl-histone H4 ChIP grade antibody (06-866; Upstate Biotechnology) or normal rabbit immunoglobulin G (IgG) control antibody (12-370; Upstate Biotechnology) overnight at 4°C, 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 (30 cycles).

RNase protection assays.

HeLa cells were harvested at 42 h posttransfection following the removal of medium and washing in PBS. Cells were then pelleted and kept on ice. Total RNA samples were prepared by using the commercial RNAqueous phenol-free total RNA isolation kit (Ambion) and treated with 2 U of RNase-free DNase I (10 U/μl; Roche)/μg for 30 min at 37°C in a total volume of 240 μl containing 1× NEB2 buffer (New England Biolabs) and 1.6 U of RNasin (40 U/μl; Promega)/μg. RNA was then extracted with phenol-chloroform-isoamyl alcohol, ethanol precipitated, and resuspended in sterile and RNase-free water (United States Biochemical Corp.).

A BLV-specific 32P-labeled antisense riboprobe was synthesized in vitro by transcription of XbaI-restricted pGEM-LTRBLV (14) with SP6 polymerase according to the protocol provided with the riboprobe in vitro transcription system (Promega). As a control, a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antisense probe was synthesized by the same method and used on the same RNA samples.

RNase protection assays were performed by using the RPA II kit (Ambion) according to the manufacturer's recommendations. Briefly, hybridization reaction mixtures (20 μl) containing 40 μg of total cellular RNA and 200,000 cpm of probe in hybridization buffer were heated to 95°C for 4 min to denature the RNA and then incubated at 42°C for 16 h. The reaction mixtures were diluted by the addition of 200 μl of digestion buffer, and the single-stranded sequences were digested with RNase T1 and RNase A for 1 h at 37°C. Following the addition of 300 μl of inactivation buffer and ethanol precipitation, the protected RNA fragments were analyzed by electrophoresis through 6% urea polyacrylamide gels.

Northern blot analysis.

Cultured PBMCs from sheep M2311 were harvested by centrifugation 22 h after TSA induction. Total RNA was prepared by using TriPure reagent (Roche) according to the manufacturer's protocol. Ten micrograms of total RNA was lyophilized, resuspended in denaturing buffer (6 M deionized glyoxal, 50% dimethyl sulfoxide, 0.1 M phosphate buffer [pH 7.0]), and incubated for 10 min on ice and then for 3 min at 55°C. RNAs were separated by electrophoresis through a 1% agarose gel containing 10 mM phosphate buffer, transferred onto a nylon membrane (Amersham Biosciences) for 20 h in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and cross-linked by UV. Membranes were first prehybridized for 3 h at 42°C in hybridization solution (50% deionized formamide, 0.1% sodium dodecyl sulfate [SDS], 10× Denhardt solution, 200 μg of salmon sperm DNA per ml) and then hybridized for 20 h in a fresh hybridization mixture containing a 32P-labeled TaxBLV probe (81) or a GAPDH probe (4 × 108 to 6 × 108 cpm/ml). Membranes were washed three times for 20 min in 2× SSC-0.1% SDS and then three times in 0.2× SSC-0.1% SDS, dried, and exposed.

Electrophoretic mobility shift assays (EMSAs).

Nuclear extracts were prepared from ovine PBMCs as described previously (64). Briefly, cells were washed in PBS and resuspended in buffer A (10 mM Tris-HCl [pH 7.9], 1.5 mM MgCl2, 50 mM NaCl, 1 mM EDTA, 0.5 M sucrose, 10 mM Na2MoO4, 0.5 mM phenylmethylsulfonyl fluoride). Cells were pelleted and resuspended in 4 volumes of buffer A. Nonidet P-40 was added at a concentration of 0.1%, and the cell suspension was incubated for 10 min at 4°C. Cells were washed once with the same buffer. Nuclear proteins were extracted in a high-salt buffer (20 mM Tris-HCl [pH 7.9], 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.6 M KCl, 10 mM Na2MoO4, 0.5 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride) overnight at 4°C with gentle shaking. The nuclear extracts were then centrifuged at 126,300 × g for 30 min, and the supernatants were dialyzed against a low-salt buffer (50 mM Tris-HCl [pH 7.9], 0.5 mM MgCl2, 1 mM EDTA, 20% glycerol, 0.1 M KCl, 10 mM Na2MoO4, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride) for 5 h. Precipitates were removed by centrifugation at 126,300 × g for 30 min. The supernatants were dispensed in aliquots and stored at −80°C until use. Protein concentrations were determined by the method of Bradford (11). The DNA sequences of the coding strand of the double-stranded oligonucleotides used for this study are listed in the figure legends.

EMSAs were performed as described previously (2). Briefly, nuclear extract (5 μg of protein) was first incubated at room temperature for 20 min in the absence of probe and specific competitor DNA in a 20-μl reaction mixture containing 1 μg of poly(dI-dC) (Amersham Biosciences) as a nonspecific competitor DNA, 1 mM DTT, 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA, and 5% (vol/vol) glycerol. Probe (15,000 cpm; 10 to 40 fmol) was then added to the mixture with or without a molar excess of an unlabeled specific DNA competitor, and the mixture was incubated for 30 min at room temperature. Samples were subjected to electrophoresis at room temperature on 6% polyacrylamide gels at 120 V for 2 to 3 h in 1× TGE buffer (25 mM Tris-acetate [pH 8.3], 190 mM glycine, 1 mM EDTA). Gels were dried and autoradiographed for 24 to 48 h at −70°C. For supershift assays, polyclonal antibodies against USF1 (sc-229), USF2 (sc-862), Max (sc-197), Mad-1 (sc-222), CREB-1 (sc-271), ATF1 (sc-243), and ATF2 (sc-242) (Santa Cruz Biochemicals, Santa Cruz, Calif.) were added at a final concentration of 2 μg/reaction to the binding reaction mixture at the end of the binding reaction for an additional 30-min incubation at room temperature before electrophoresis.

In vitro-translated E box-binding proteins Max and Mad1 were used in control EMSAs. These proteins were generated by using the TNT coupled reticulocyte lysate system (Promega) with the pGEM expression plasmid encoding mouse Max (p22 long form) and the pRC/CMV expression vector for human Mad1 (both kindly provided by S. Segal). Eight microliters of in vitro-translated Mad1 was preincubated with 1 μl of in vitro-translated Max for 10 min at 42°C and then for an additional 20-min period at room temperature to promote the formation of Mad1-Max. These complexes were then incubated for 30 min at room temperature in the presence of 30,000 cpm of either 32P-labeled TxRE 1, 2, or 3 probes or CMD (specific E box) probe (an oligonucleotide with a c-Myc E box consensus binding site [10], 5′-AGCTTCAGACCACGTGGTCGGG-3′) in a buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM DTT, 0.05% NP-40, 1% glycerol, and 1 μg of poly(dI-dC) in a total volume of 20 μl. The protein-DNA complexes were then separated from the free probe by electrophoresis on 4% polyacrylamide gels in 0.5× Tris-borate-EDTA at 150 V. The free probe was run out of the gel for better separation of the complexes (see Fig. Fig.7B7B).

FIG. 7.
Characterization of the factors binding to the E box element located in the TxRE2 motif of the BLV LTR. (A) The TxRE2-CREmut oligonucleotide probe was incubated with 5 μg of nuclear extracts from BLV-infected ovine PBMCs (M2658). Next, antibodies ...

RESULTS

Activation of BLV promoter activity by different deacetylase inhibitors.

To test the involvement of deacetylases in the transcriptional regulation of the BLV promoter, we tested the effect of three deacetylase inhibitors on viral gene expression in transient transfection assays. The pLTRwt-luc reporter plasmid, containing the firefly luciferase (luc) gene under the control of the complete BLV 5′ LTR, was transiently transfected into the human B-lymphoid Raji cell line. Transfected cells were mock treated or treated with increasing concentrations of TSA (0, 62, 125, 250, 500, and 1,000 nM), NaB (0, 0.61, 1.25, 2.5, 5, and 10 mM), or VPA (0, 0.25, 0.5, 1, 2.5, and 5 μM) and assayed for luciferase activity (Fig. (Fig.1A).1A). The BLV LTR was activated by each of the three deacetylase inhibitors in a dose-dependent manner: pLTRwt-luc presented TSA inductions varying from 1.2- to 47-fold, NaB inductions varying from 5.1- to 46-fold, and VPA inductions varying from 3.0- to 12-fold. Of note, the maximal acetylation of Raji histone H4, visualized on a Triton-acetic acid-urea gel (23), occurred at 500 nM, the same TSA concentration at which maximal BLV LTR-driven gene expression was observed (data not shown). Similar results were observed with the human B-lymphoid Daudi cell line (data not shown).

FIG. 1.
Activation of BLV LTR activity by deacetylase inhibitors. (A) Raji cells were transiently transfected by the DEAE-dextran procedure with 500 ng of pLTRwt-luc. At 20 h posttransfection, cells were mock treated or treated with increasing concentrations ...

Since transiently transfected DNA does not always form proper chromatin structure, we next wanted to confirm the above transient transfection results in a nucleosomal context. To this end, we cloned the luciferase gene under the control of the BLV LTR into a modified pREP10 episomal vector that contained the EBV replication origin and encoded nuclear antigen EBNA-1. Indeed, several studies have reported that pREP-based episomal constructs display hallmarks of proper chromatin structure when transiently transfected into cells (51, 82). The episomally replicating BLV LTR-luc plasmid we generated [named pLTR*wt(direct)-luc] was transiently transfected into Raji cells. Transfected cells were mock treated or treated with increasing concentrations of deacetylase inhibitors (as described above) and assayed for luciferase activity (Fig. (Fig.1B).1B). The BLV promoter was activated in a dose-dependent manner by TSA (up to 6.7-fold), NaB (up to 7.4-fold), and VPA (up to 5.2-fold).

We used a ChIP assay to investigate changes in histone H4 acetylation at the BLV promoter following TSA treatment of the ovine BLV-positive leukemia cell line YR2. Cross-linked YR2 chromatin was immunoprecipitated with the relevant acetyl-histone H4 antibody, and the purified genomic DNA was amplified with PCR primers that bracketed the viral CRE region. Figure Figure1C1C shows the amplification of the input DNA from the BLV LTR region used in the ChIP assay in the absence and presence of TSA (Fig. (Fig.1C,1C, lanes 6 and 7, respectively) and of the DNA after immunoprecipitation (Fig. (Fig.1C,1C, lanes 1 to 5). Interestingly, we observed an enrichment of the CRE promoter sequences with an immunoprecipitate prepared with the acetyl-histone H4 antibody in the presence of TSA (500 nM) (Fig. (Fig.1C,1C, lane 5) compared with the control samples in the absence of TSA (Fig. (Fig.1C,1C, lanes 1 to 4). These results thus indicate a close correlation between the degree of histone acetylation at the BLV 5′ LTR in the ovine BLV-positive YR2 cell line and transcriptional activation of the viral promoter by TSA in Raji cells.

In conclusion, our results demonstrate the activation of BLV LTR-directed gene reporter expression in response to several deacetylase inhibitors. Of note, our transient transfection system reflects what we observe when we use episomal constructs that are physiologically relevant in terms of proper chromatin structure. Remarkably, ChIP assays with BLV-infected B cells link in vivo-enhanced BLV transcription to the hyperacetylation of histone H4.

Ectopically expressed HDAC1 to HDAC5 downregulate BLV promoter activity.

To confirm results in a non-B-cell line, the reporter construct pLTRwt-luc was transiently transfected into the human epithelial HeLa cell line. Treatment of transfected HeLa cells with increasing concentrations of TSA, NaB, or VPA also resulted in a marked transcriptional activation of the BLV promoter (Fig. (Fig.2A).2A). To analyze whether the activity of the BLV promoter was actually modulated in an acetylation- or deacetylation-dependent manner, cotransfections of HeLa cells were performed with pLTRwt-luc and increasing amounts of expression vectors coding for different human deacetylases, HDAC1 to HDAC6. As shown in Fig. Fig.2B,2B, HDAC1 to HDAC5 strongly repressed basal LTR-directed gene expression, suggesting that these deacetylases could be recruited to the BLV promoter and could negatively regulate its activity. In contrast, HDAC6 failed to repress BLV basal gene expression, suggesting a certain specificity in the recruitment of HDACs to the BLV promoter.

FIG. 2.
Response of the BLV promoter to ectopically expressed deacetylases. (A) HeLa cells were transiently transfected by the FuGENE procedure with 500 ng of pLTRwt-luc. At 20 h posttransfection, cells were mock treated or treated with increasing concentrations ...

Taken together, our results suggest that the activity of the BLV promoter is negatively regulated by deacetylases. BLV activation by deacetylase inhibitors could be explained by histone hyperacetylation and/or acetylation events involved in the regulation of transcription factors binding to the BLV LTR.

Activation of the BLV promoter by deacetylase inhibitors takes place at the transcriptional level in vitro and in vivo.

To verify that the effect of deacetylase inhibitors occurred at the level of transcription, BLV transcript levels were measured in transiently transfected cells by RNase protection with a probe proximal to the viral promoter. As previously reported (14), hybridization to the promoter-proximal probe allows the detection of all transcripts that initiate at the LTR and provides an approximate measure of total transcription levels. We failed to observe any reporter transcripts in transiently transfected Raji cells with the BLV promoter-specific probe (data not shown), probably as a consequence of the weak BLV promoter activity in the absence of the viral transactivator TaxBLV and of the weak transfection efficiency by use of the DEAE-dextran procedure. We therefore decided to analyze BLV RNA synthesis in HeLa cells, which we transiently transfected by the FuGENE procedure, allowing a higher transfection efficiency. To this end, we performed RNase protection assays with RNAs extracted from HeLa cells transiently transfected with pLTRwt-luc and mock treated or treated with TSA (500 nM) or NaB (5 mM). As expected, TSA and NaB treatment increased the luciferase activity by 15- and 11-fold, respectively (Fig. (Fig.3A,3A, upper panel). In the same experiment, an increase in the level of transcripts detected by RNase protection with the 5′ LTRBLV proximal probe was observed after treatment with TSA (5.5-fold) and NaB (6.1-fold) (Fig. (Fig.3A,3A, lower right panel), indicating that the total transcription levels from the BLV LTR were increased following treatment with deacetylase inhibitors. As an internal control, RNase protection analysis of the same RNA samples with an antisense riboprobe corresponding to the GAPDH gene showed no change in the level of mRNA following treatment with TSA or NaB (Fig. (Fig.3A,3A, lower right panel).

FIG. 3.
Activation of BLV promoter by deacetylase inhibitors occurs at the mRNA level. (A) Upper panel, HeLa cells were transiently transfected with 500 ng of pLTRwt-luc. At 20 h posttransfection, cells were mock treated or treated with TSA (500 nM) or NaB (5 ...

To assess whether inhibition of deacetylases also increased BLV mRNA levels in the context of an integrated provirus, PBMCs were isolated from a BLV-infected sheep (sheep M2311) and cultured for 5 or 22 h in the absence or presence of TSA (500 nM). The effect of TSA treatment on the quiescent state of BLV was studied by Northern blotting (Fig. (Fig.3B).3B). The major BLV transcripts include an 8.4-kb full-length mRNA which acts both as genomic RNA and as the Gag-Pol protein-encoding mRNA, a 4.4-kb singly spliced RNA encoding the Env protein, and a 2.1-kb doubly spliced RNA encoding the Tax and Rex regulatory proteins. Northern blot hybridization of total RNA derived from cultured BLV-infected ovine PBMCs revealed all three RNA species with a BLV TAX probe corresponding to the 1-kb XbaI fragment of pGEM7zfLOR1 (81) (Fig. (Fig.3B,3B, lanes 2 to 5), in agreement with the observation that the in vitro culture of freshly isolated BLV-infected ovine PBMCs provokes a massive production of viral antigens (62). BLV transcripts were undetectable in uncultured PBMCs (Fig. (Fig.3B,3B, lane 1) (negative control) and were highly produced in total RNA derived from the YR2LTaxSN cell line (a BLV-producing B-cell line resulting from retrovirus vector-mediated tax gene transfer into native YR2 cells) (81) used as a positive control (Fig. (Fig.3B,3B, lane 6). While the levels of BLV transcripts in the absence or presence of TSA treatment were very similar after a 5-h induction (Fig. (Fig.3B,3B, compare lane 2 to 3), a marked increase in all of the major BLV transcripts was observed after a 22-h TSA treatment compared to those under unstimulated conditions (Fig. (Fig.3B,3B, compare lane 4 to 5), demonstrating that the transcription of the BLV genes can be specifically activated in BLV-infected ovine PBMCs following treatment with deacetylase inhibitors. As an internal control, Northern blot hybridization of the same RNA samples with a GAPDH probe showed no change in the level of BLV transcripts (Fig. (Fig.3B,3B, lower panel).

Thus, our results demonstrate that deacetylase inhibitors increase the amount of transcription directed by the BLV promoter, stimulating the initiation transcriptional level. Moreover, we report the possibility of reactivating BLV proviral gene expression in BLV-infected ovine PBMCs by TSA treatment.

Activation of BLV gene expression by TSA is mediated in part through the three E box motifs in the LTR U3 region.

We next investigated whether the stimulatory effect of TSA could be mediated through one of the cis-regulatory DNA elements located in the BLV 5′ LTR. To this end, individual or combined mutations in all of the known LTR cis-acting elements (Fig. (Fig.4A)4A) were introduced by site-directed mutagenesis in the context of the pLTRwt-luc construct. These mutated constructs were transiently transfected into Raji cells. Transfected cells were mock treated or treated with TSA (500 nM) and assayed for luciferase activity. As shown in Fig. Fig.4B4B (upper panel), mutations in the CREs, PU box, GRE, E box 4, or IRF motifs caused a decrease of BLV LTR-driven basal gene expression, in agreement with previous reports (14, 21, 42, 58, 66, 90). In contrast, the combined mutation of E boxes 1, 2, and 3 caused an increase of basal BLV gene expression by approximately 4.5-fold (Fig. (Fig.4B,4B, upper panel), consistent with previous results showing a 2-fold effect in the D17 cell line (58), supporting the notion that the three E boxes overlapping the CREs in U3 were involved in transcriptional repression of BLV basal gene expression. The maximal increase in LTR-directed luciferase expression was observed when E boxes 1, 2, and 3 were mutated in combination, and individual mutations revealed no particular contribution of one of the three E box motifs to this transcriptional repression (data not shown). We confirmed these observations in several B-cell and non-B-cell lines (data not shown).

FIG. 4.
Study of the cis-acting elements involved in the TSA inducibility of the BLV promoter. (A) All the transcription factor binding sites in the 5′ LTR of the BLV genome (exhaustively described in the introduction) and their respective point mutations ...

Importantly, when we compared the TSA responsiveness of the wild-type LTR with that of the LTR mutated in E boxes 1, 2, and 3 (Fig. (Fig.4B,4B, lower panel), we observed a marked reduction (60%) in the TSA inducibility of the BLV promoter. This was specific, since inducibility by TSA did not seem to be affected by any of the mutations introduced in the other transcription factor-binding sites, with the exception of the PU.1/Spi-B-binding site, whose mutation caused a similar 60% decrease in TSA inducibility (Fig. (Fig.4B,4B, lower panel). Interestingly, it has recently been reported that the C-terminal region of PU.1 forms a complex with mSin3A and HDAC1 in vivo (43). In future experiments, we will investigate the potential role of PU.1 in the recruitment of HDAC1 to the BLV promoter.

To further confirm the functional role of the three U3 E box motifs in the TSA inducibility of the BLV LTR, we performed additional transient transfection experiments into the Raji cell line with LTR luciferase reporter plasmids containing or not containing point mutations in the three E boxes. The DNA sequence of the coding strand of each E box motif within the BLV promoter, as well as the point mutations we used, are listed in Fig. Fig.5A.5A. Raji cells were then mock treated or treated with increasing concentrations of TSA (from 62 to 1,000 nM) and assayed for luciferase activity (Fig. (Fig.5B).5B). We observed that the combined mutation in E boxes 1, 2, and 3 (E box-mutB) decreased the TSA responsiveness of the BLV LTR. This was observed at all of the TSA concentrations tested, with a maximal effect at 500 nM (2.7-fold). Similar results were obtained with the epithelial HeLa cell line (data not shown).

FIG. 5.
Differential TSA response of pLTRwt-luc and of pLTR(E-box1,2,3mutB)-luc. (A) Nucleotide sequences of BLV LTR E box motifs and overlapping CRE motifs. For each TxRE, the bases with boldface arrows indicate the E box motif and the bases with dotted arrows ...

Our functional results thus support a model in which deacetylases are recruited to the BLV promoter through the three E box motifs located in the U3 region, thereby resulting in transcriptional repression of viral gene expression.

Specific binding of nuclear factors to CREs and E boxes 1, 2, and 3 is mutually exclusive.

To identify the cellular factors binding to E boxes 1, 2, and 3 and responsible for the transcriptional repression of BLV promoter activity, EMSAs were performed by using, as probes, the wild-type and several mutated versions of a 21-bp TxRE oligonucleotide corresponding to the TxRE 2 (nucleotides −139 to −119) of the BLV 5′ LTR (Fig. (Fig.6A).6A). In addition to the wild-type version of the TxRE 2 oligonucleotide (referred to as TxRE2-wt), we used oligonucleotides containing mutations within either the CRE (referred to as TxRE2-CREmut) or the E box motif (referred to as TxRE2-EboxmutB or TxRE2-EboxmutA). These wild-type and mutated versions of the TxRE 2 probe were incubated with nuclear extracts from PBMCs derived from a BLV-infected sheep (M2658). Two retarded protein-DNA complexes were detected with the TxRE2-wt probe: a major slower-migrating band (Fig. (Fig.6A,6A, lane 1), corresponding to the CREB/ATF complex, as demonstrated by supershift assays (data not shown), and a fainter faster-migrating band (Fig. (Fig.6A,6A, lane 1). Interestingly, when the CRE was mutated, the intensity of the faster-migrating band increased significantly, concomitant with a decrease in the intensity of the CREB/ATF complex (Fig. (Fig.6A,6A, lane 2). Conversely, when the E box motif was mutated, CREB/ATF binding increased strongly, concomitant with the disappearance of the E box complex (Fig. (Fig.6A,6A, lanes 3 and 4). These DNA-protein interaction profiles thus suggested that binding of nuclear factors to the overlapping CRE and E box elements was mutually exclusive. Similar in vitro binding results were obtained when studying TxRE 1 and 3 (data not shown).

FIG. 6.
Mutagenesis of the CRE and E box elements located in the TxRE2 motif of the BLV LTR U3 region. (A) The TxRE2-wt (5′-AAGCTGGTGACGGCAGCTGGT-3′), TxRE2-CREmut (5′-AAGCTGGTGTGGGCAGCTGGT-3′), TxRE2-EboxmutB (5′-AAGCTGGTGACGG ...

To better evaluate the specificity of the faster-migrating E box complex, unlabeled double-stranded oligonucleotides were prepared and used as competitors in EMSAs with TxRE2-CREmut as a probe and nuclear extracts from sheep PBMCs (Fig. (Fig.6B).6B). The binding of proteins to E box 2 was shown to be sequence specific by competition EMSAs. Indeed, the formation of the E box complex was competed for by an excess of the TxRE2-CREmut unlabeled homologous oligonucleotide (Fig. (Fig.6B,6B, lanes 2 to 5) but not by the same molar excess of a heterologous oligonucleotide of unrelated sequence (Fig. (Fig.6B,6B, lanes 10 to 13). Moreover, the E box complex was not competed by a mutated version of the TxRE2-CREmut oligonucleotide containing an additional mutation within the E box motif (named TxRE2-CREmut/Ebox-mutB) (Fig. (Fig.6B,6B, lanes 6 to 9), confirming that formation of the E box complex was dependent on the presence of an intact E box sequence (Fig. (Fig.6A,6A, lane 3).

To identify directly the factors present in the E box-specific retarded complex, we performed supershift assays with antibodies directed against individual members of the bHLH family of transcription factors (Fig. (Fig.7A).7A). Some of these transcription factors can act as either transactivators or repressors of gene expression, depending on the gene promoter or on their dimerization partner (reviewed in reference 54). Because our functional results indicated a role for E boxes 1, 2, and 3 in transcriptional silencing of the BLV LTR (Fig. (Fig.4B4B and and5),5), we first focused on the Mad/Mxi proteins that, as heterodimers with Max, function as repressors when bound to E box elements (74). The Mad repression domain (SID) interacts directly with the mSin3A and mSin3B proteins, thereby regulating repression through a SID-dependent association with a complex containing HDAC activity (6, 73). We incubated the TxRE2-CREmut probe with nuclear extracts from BLV-infected ovine PBMCs. Polyclonal antibodies directed against Max and Mad-1 were added to the binding reaction mixture, but no supershifted complex was observed (Fig. (Fig.7A,7A, lanes 4 and 5), although the validities of these antibodies were already controlled in a previous study from our laboratory (14). The binding pattern was not affected by the addition of antibodies directed either against other members of the Mad family (Mad-2, Mad-3, and Mad-4) or against a recently identified B-cell-specific HLH repressor, the ABF-1 protein (55) (data not shown). Previously, functional studies from our laboratory have demonstrated that E boxes 1, 2, and 3 could contribute to the BLV LTR response to USF1 and USF2a (14). However, the addition of polyclonal antibodies directed against USF1 or USF2 in the binding reaction mixture did not generate any supershifted complexes (Fig. (Fig.7A,7A, lanes 2 and 3). To further demonstrate that the E box-specific faster-migrating complex did not involve the Mad-Max proteins, additional EMSAs were performed by incubating in vitro-translated Max or Mad1-Max with each of the three TxREs as probes (Fig. (Fig.7B).7B). None of the TxRE probes were able to bind Max or Mad-1 (Fig. (Fig.7B,7B, lanes 2 to 7), whereas a CMD (specific E box) probe (used as a positive control) (10) bound Max homodimers as well as Mad1-Max heterodimers (Fig. (Fig.7B,7B, lanes 8 and 9, respectively). In vitro-translated USF1, USF2, or AP4 proteins also failed to bind to any of the three TxREs (data not shown).

Altogether, these in vitro binding studies indicate that the binding to the overlapping CRE and E box elements within the BLV TxREs is mutually exclusive. Moreover, specific nuclear factors binding to the E box motifs seem to negatively modulate the binding of CREB/ATF to the overlapping CREs and thereby seem to be responsible for a reduced transcriptional activity of the BLV promoter. However, despite many attempts, we did not succeed in the identification of these E box-specific nuclear factors.

Mutations in BLV E boxes 1, 2, and 3 stimulate viral gene expression via enhanced recruitment of CREB/ATF.

Based on the above in vitro studies, we considered the possibility that the factors interacting with the E box elements could repress BLV LTR transcriptional activity by decreasing the binding of CREB/ATF. To assess the respective transcriptional regulatory function of the BLV CRE and E box motifs, we performed functional experiments with LTR-directed reporter constructs harboring individual or combined mutations within the CRE and E box elements in U3. It should be stressed that none of the three CRE-like motifs within the TxRE enhancers fit perfectly with the well-characterized consensus sequence TGACGTCA; the distal, middle, and proximal TxREs were, respectively, AGACGTCA, TGACGGCA, and TGACCTCA (with differing nucleotides underlined). It has previously been demonstrated that basal BLV transcriptional activity is strongly induced when these wild-type CRE-like motifs are mutated to a perfect consensus CRE. This higher basal transcriptional activity parallels the increased binding of the CREB/ATF proteins (58). We constructed, by site-directed mutagenesis, a recombinant LTR harboring perfect consensus CREs in all three TxREs in the context of both the pLTRwt-luc and the pLTR(E-box1,2,3mutB)-luc. The two mutated plasmids were designated pLTR(CRE1,2,3 perfect)-luc and pLTR(CRE1,2,3-perfect/E-box1,2,3-mutB)-luc, respectively, and were transiently transfected into Raji cells. As shown in Fig. Fig.8A,8A, mutation of the wild-type CRE-like motifs into perfect consensus CREs markedly increased the basal activity of the BLV promoter (by 19-fold). Importantly, mutation of E boxes 1, 2, and 3 in the context of pLTR(CRE1,2,3 perfect)-luc further increased basal LTR-directed gene expression, since it caused a 35-fold increase in luciferase activity. These results were in agreement with our in vitro binding studies (Fig. (Fig.6A)6A) and suggested that additional mutation of these E box motifs further potentiated the activation by CREB/ATF. Similar results were obtained with HeLa cells (data not shown).

FIG. 8.
Effects of perfect consensus CREs on basal and TSA-induced BLV promoter activity. (A) Raji cells were transiently transfected with 500 ng of either pLTRwt-luc, pLTR(CRE1,2,3mut)-luc, pLTR(E-box1,2,3mutB)-luc, pLTR(CRE 1,2,3 perfect)-luc, or pLTR(CRE1,2,3-perfect/E-box1,2,3-mutB)-luc. ...

Previously in this report, we hypothesized that deacetylases could be recruited to the BLV promoter through E boxes 1, 2 and 3, thereby resulting in transcriptional repression of viral gene expression (Fig. (Fig.4B4B and and5B).5B). Such a hypothesis cannot be excluded as long as nuclear factors binding to these E boxes remained unidentified. To gain additional insight into the respective functional roles of the CREs in U3, we compared the TSA response of the pLTRwt-luc and pLTR(CRE1,2,3 perfect)-luc constructs in transient transfection experiments into Raji cells. The cells were then mock treated or treated with increasing concentrations of TSA (from 62 to 1,000 nM) and assayed for luciferase activity (Fig. (Fig.8B).8B). We observed that mutations converting the BLV CRE-like elements to perfect consensus CREs [pLTR(CRE1,2,3 perfect-luc)] decreased the TSA responsiveness of the BLV LTR at all of the TSA concentrations tested, with a maximal effect at 500 nM (2.8-fold) (Fig. (Fig.8B).8B). These functional results were in agreement with the results we obtained with the reporter plasmid mutated within E boxes 1, 2, and 3, pLTR(E-box1,2,3mutB)-luc (Fig. (Fig.5B),5B), demonstrating that a stronger binding of CREB/ATF to the CREs resulted in a decreased TSA inducibility of the BLV promoter. Similar results were obtained with the HeLa cell line (data not shown).

We conclude from these experiments that the factors associating with the E box motifs in U3 exert a negative cooperative effect on the activity of the BLV promoter through the CREs, most likely by sterically inhibiting the binding of CREB/ATF to these CREs. Moreover, our data suggest that the increased binding of CREB/ATF, following either a mutation abolishing binding to E boxes 1, 2, and 3 or a substitution of the BLV CRE-like motifs into perfect consensus CREs, is associated functionally with a decreased TSA responsiveness of the BLV promoter. This viral strategy (presence of E boxes overlapping the CRE-like motifs and reducing their accessibility by steric hindrance) could allow a better silencing of BLV transcription, thereby facilitating hiding from recognition by the host immune response.

TSA treatment increases CREB/ATF binding activity.

The CREB/ATFs play a central role in the transcriptional activation of the BLV promoter (1, 2, 58, 88). To further investigate the role of these factors in the activation of BLV by deacetylase inhibitors, EMSAs were performed by using oligonucleotide probes corresponding either to wild-type TxREs or to TxREs harboring perfect consensus CREs. These probes were incubated with nuclear extracts prepared from BLV-infected ovine PBMCs either mock treated or treated with TSA (500 nM) for 22 h (Fig. (Fig.9).9). As expected, we observed the specific binding of members of the CREB/ATF family, as shown by supershift or blocking assays with antibodies directed against CREB1, ATF1, or ATF2 (Fig. (Fig.9,9, right panel). Treatment of cells with TSA caused an increased binding activity of CREB1 and ATF2 homodimers. This increased binding was more strongly observed with the wild-type versions of the three TxREs (Fig. (Fig.9,9, left panel, lanes 1, 2, 5, 6, 9, and 10) but was also detectable with the TxREs harboring perfect consensus CREs (Fig. (Fig.9,9, left panel, lanes 3, 4, 7, 8, 11, and 12). Additionally, supershift analysis of the CREB/ATF complexes in the absence or presence of TSA treatment showed no change in the dimer composition of the CREB/ATF retarded complexes (Fig. (Fig.9,9, right panel). Of note, TSA did not alter the binding of the constitutively expressed Sp1 transcription factor (Fig. (Fig.9,9, lower left panel). These results indicate that TSA enhances CREB/ATF binding activity, even to optimal CRE consensus sequences, and are thus consistent with our previous functional results presented in Fig. Fig.8B8B.

FIG. 9.
TSA treatment increases CREB/ATF binding activity. Nuclear extracts were prepared from BLV-infected ovine PBMCs (M2658) mock treated or treated with TSA (500 nM) for 22 h. The TxRE1-wt, TxRE2-wt, and TxRE3-wt oligonucleotides (referred to as CRE1wt, CRE2wt, ...

These results thus demonstrate that treatment with deacetylase inhibitors increases CREB/ATF binding to the BLV promoter, suggesting that some proteins involved in the CREB/ATF signaling pathway may have their expression and/or action modulated by TSA.

DISCUSSION

In summary, our results demonstrate that BLV transcriptional promoter activity is strongly activated following treatment with deacetylase inhibitors in the context of transiently transfected constructs, of episomally replicating plasmids, and of an integrated BLV provirus. Importantly, treatment of BLV-infected B cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping CREs in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. Although we could not identify, despite many attempts, the nature of the BLV LTR E box-specific complexes, our data imply that these complexes competitively inhibited, by steric hindrance, protein binding to the TxRE CRE-like motifs. Interestingly, we demonstrated that TSA increased the occupancy of these CREs by CREB/ATF. Altogether, our results suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow a better silencing of viral transcription and to escape from the host immune response.

It has been reported that several proteins from animal viruses use deacetylases to repress their own viral promoters and to modify host cell growth. For example, the E7 oncoprotein from human papillomavirus interacts with Mi2 and the HDAC complex to promote cell growth (13) and the EBV nuclear antigen 3C interacts with HDAC to repress transcription of the latency-associated viral Cp promoter (69). Recently, Ego et al. have demonstrated that TaxHTLV-1, a regulatory oncoprotein encoded by HTLV-1 and closely related to TaxBLV, interacts with HDAC1 and negatively regulates viral gene expression (26). Conversely, many viral proteins either utilize CBP and its paralog p300, which possess intrinsic HAT activity, as coactivators or target these coactivators as integrators of transcriptional regulation. These viral proteins include adenovirus E1A protein (24, 52), human immunodeficiency virus type 1 Tat protein (34), EBV nuclear protein 2 and Zta proteins (86, 95), Kaposi's sarcoma-associated herpesvirus vIRF protein (29, 36, 76), and the SV40 large T antigen (25). Interestingly, TaxHTLV-1 was shown to recruit CBP/p300, through binding to multiple independent sites (39, 47, 75), and the acetyltransferase p300/CBP-associated factor (31).

In this study, we decided to examine the potential role of deacetylase recruitment to the BLV promoter in transcriptional repression of viral gene expression. We first examined the effects of treatment with NaB (9, 15, 85), TSA (93, 94), and VPA (27, 68) on BLV LTR-driven basal gene expression, i.e., in the absence of BLV proteins. The effect of these drugs in the presence of the viral transactivator TaxBLV is reported by another group from our laboratory (T. L.-A. Nguyên and C. Van Lint, submitted for publication). All of the deacetylase inhibitors we tested dramatically increased LTR-directed reporter activity of both nonepisomal and episomal constructs. Although we did not investigate in the present study the chromatin organization of the transiently transfected LTR templates, our results also demonstrated that TSA potentiated the expression of the BLV LTR in stably transfected D17 canine osteosarcoma cells (59) and in stably transfected DG-75 human EBV-negative B-lymphoid cells (A. Dekoninck and C. Van Lint, data not shown) and in the context of an integrated BLV provirus, thereby indicating that our transient transfection system reflected what we observed when the LTR was properly organized into chromatin. We also demonstrated that ectopically overexpressed HDACs negatively regulated BLV promoter activity. BLV activation by deacetylase inhibitors could be explained by histone hyperacetylation and/or acetylation events involved in the regulation of transcription factors binding to the BLV LTR.

By mutational analysis, we demonstrated a regulatory link between the full responsiveness of the BLV LTR to TSA stimulation and the presence in U3 of intact E box motifs 1, 2, and 3. Originally, these motifs were described as binding sites for the cellular transcription factor AP4 (80, 90). The assumption that AP4 could be implicated in BLV promoter activity was based on transdominant and antisense inhibition of transcription, but direct binding of this particular factor has not been reported. Here, we first postulated that a transcriptional repressor harboring deacetylase activity could bind to the three E boxes. Our in vitro binding studies revealed a faster-migrating complex specifically associated with the E box motifs located in the TxREs. The Mad-Max transcription factors therefore represented good candidates for the specific targeting of HDACs to the BLV promoter. Mad1-mediated repression requires the formation of a ternary complex with Max and the corepressor mSin3 through its mSin3 interaction domain (SID) (5, 28, 74). Nuclear receptor corepressor and mSin3 are present in a protein complex with HDAC1, with the result of this association being the recruitment of HDAC1 to the local environment of a promoter (3, 65). Mad-Max heterodimers have already been shown to repress E box-containing viral promoters, like the latent membrane protein 1 gene promoter in the EBV genome (77). However, our supershift assays with antibodies directed against Max or Mad1 to Mad4 and our EMSAs with in vitro-translated Max or Mad1-Max proteins revealed that none of these transcription factors were able to bind to the E box motifs located in the BLV LTR U3 region. Although we also tested a large panel of other E box binding proteins, either activators (USF1, USF2, and AP4, etc.) or repressors (ABF-1, etc.), the identity of the factors binding to E boxes 1, 2, and 3 still remains to be established. Additional microsequencing studies are actually under way to identify the nature of the proteins binding to the BLV E box motifs.

We next investigated the direct role of E-box-specific complexes in transcriptional repression of the BLV promoter. Our results actually support a model in which the U3 E box-specific complexes negatively regulate BLV transcription by steric hindrance with the CREB/ATF activators. The promoter regions of some other genes were shown to possess juxtaposed or overlapping CRE and E box elements. The cooperativity between their respective binding factors could be either positive, as for the follicle-stimulating hormone-mediated activation of the transferrin promoter (16) or for the mouse renin promoter (67), negative, as for the cholecystokinin gene promoter (70) or for the macrophage cyclooxygenase-2 promoter (60), or context dependent, as for the vgf gene promoter (53). In the BLV promoter, the CRE consensus within the three TxREs is never strictly conserved in any viral strain. It was recently demonstrated that reconstitution of perfect CRE sites within the BLV LTR induces a drastic reduction in the efficiency of viral propagation in vivo (58). These results suggest that suboptimal CRE sequences were selected during evolution to reduce transcriptional activation by cellular CREB/ATF factors. Here we report another viral strategy to lower BLV transcriptional activation: repression of BLV transcription by E boxes overlapping and competitively inhibiting the binding to the LTR CRE-like motifs. Together, both strategies (the imperfect CRE consensus sequences in each TxRE and the presence of E boxes overlapping these CRE-like motifs) could act in concert to allow a better silencing of BLV transcription, thereby facilitating hiding from recognition by the host immune response and allowing tumor development.

We further investigated the consequence of enhanced CREB/ATF recruitment on the TSA inducibility of the BLV promoter. We observed that the LTR harboring perfect consensus CREs responded to TSA as badly as the LTR mutated in E boxes 1, 2, and 3. Thus, two distinct mutations, which both lead to an increased binding of CREB/ATF to the CREs, cause a similar decrease in the TSA response of the BLV promoter. Mechanistically, we also showed by gel retardation assays that TSA increased CREB/ATF binding to the BLV promoter, suggesting that some proteins involved in the CREB/ATF signaling pathway could have their expression and/or action modulated by deacetylase inhibitor treatment. In this regard, Michael et al. have previously demonstrated that HDAC inhibition potentiated gene expression via cyclic AMP by enhancing phosphorylation of CREB in a chromatin-dependent manner (61). In this study, the increased binding of CREB to the three BLV CREs following TSA treatment could be explained either by direct acetylation of CREB, as reported for a variety of other transcription factors (17, 78), or by modulation of the expression and/or action of proteins involved in the CREB signaling pathway, such as CBP/p300. The intrinsic HAT activity of ATF2 (41) could also play a role in BLV transcriptional activity, since ATF2 binding to CRE 1, 2, and 3 is enhanced by TSA. Paradoxically, our functional studies revealed that the increased binding of CREB/ATF transcription factors decreased the TSA inducibility of the BLV LTR. It is of note that although, for many factors, acetylation leads to enhanced transcriptional activity, acetylation can also have a negative regulatory effect (30, 49, 50, 56, 79). Thus, regarding the BLV promoter, a simple interpretation for the decreased TSA inducibility associated with increased CREB binding to the LTR would be that CREB, by acting as a substrate for HATs like CBP/p300, competitively inhibits histone acetylation. ChIP assays, which allow measurement of the histone acetylation state of specific chromosomal sites in living cells, will be necessary to characterize in vivo the influence of CREB/ATF on the transcriptional activity of a chromosomal BLV promoter template.

In conclusion, the results described in this report provide new insights into the activation of BLV transcription by deacetylase inhibitors and, more generally, into the molecular mechanisms of CREB/ATF-mediated transactivation. The competition between HDAC and HAT activities in infected cells could thus play an important role in controlling BLV latent and productive cycles, respectively.

Acknowledgments

We are grateful to members of the C.V.L. laboratory for critical reading of the manuscript. We thank Stéphane Emiliani (Département des maladies infectieuses, Institut Cochin, Paris, France) for reagents used in this study. Special thanks to Pierre Kerkhofs for management of sheep at the Veterinary and Agrochemical Research Center.

This work was supported by grants to C.V.L. from the Fonds National de la Recherche Scientifique (FNRS; Belgium), the Télévie-Program, the Université Libre de Bruxelles (ULB; ARC program no. 98/03-224), the Internationale Brachet Stiftung (I.B.S.), the CGRI-INSERM cooperation, the Région Wallonne-Commission Européenne FEDER and the Theyskens-Mineur Foundation. C.V.L. is Maître de Recherches of the FNRS. C.C. is Chargé de Recherches of the FNRS. A.D. is a designated Aspirant of the FNRS. S.N. was a fellow of the FNRS-Télévie Program. E.A. was supported by a postdoctoral fellowship from the ULB (ARC program no. 98/03-224). T.L.-A.N. is a fellow of the Belgian Fonds pour la Recherche dans l'Industrie et l'Agriculture (FRIA). L.W. and R.K. are Directeurs de Recherches of the FNRS.

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