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Journal of Virology, December 2008, p. 11939-11947, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01356-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Proto-Oncogene Bcl3, Induced by Tax, Represses Tax-Mediated Transcription via p300 Displacement from the Human T-Cell Leukemia Virus Type 1 Promoter{triangledown}

Young-Mi Kim, Neelam Sharma, and Jennifer K. Nyborg*

Department of Biochemistry and Molecular Biology, Campus Box 1870, Colorado State University, Fort Collins, Colorado 80523

Received 27 June 2008/ Accepted 16 September 2008


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ABSTRACT
 
The etiology of human T-cell leukemia virus type 1 (HTLV-1)-induced adult T-cell leukemia is linked to the expression of the viral oncoprotein Tax. Although the mechanism of retroviral transformation is unknown, Tax interferes with fundamental cellular processes, including proliferation and apoptosis, and these events may directly link Tax to early steps in malignant progression. In this study, we examined the interplay between Tax and the potent proto-oncogene B-cell chronic leukemia protein 3 (Bcl3). Bcl3 is a critical regulator of cell survival and proliferation and is overexpressed in HTLV-1-infected cells. We found that Tax induced Bcl3 expression through stimulation of the NF-{kappa}B pathway. An intronic NF-{kappa}B binding site within the Bcl3 gene served as the primary target of Tax-induced NF-{kappa}B activation. We next considered the consequence of Bcl3 overexpression on Tax function. Interestingly, we found that Bcl3 formed a stable complex with Tax and that this complex potently inhibited Tax-dependent HTLV-1 transcription. Importantly, Bcl3 associated with the HTLV-1 promoter in a Tax-dependent manner and inhibited the binding of the critical cellular coactivator p300. The conserved ankyrin repeat domain of Bcl3 mediated both Tax binding and inhibition of p300 recruitment to the HTLV-1 promoter. Together, these data suggest that Tax-induced Bcl3 overexpression benefits the virus in two important ways. First, Bcl3 may promote cell division and thus clonal proliferation of the virus. Second, Bcl3 may attenuate virion production, facilitating immune evasion. One consequence of this regulatory loop may be Bcl3-induced malignant transformation of the host cell.


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INTRODUCTION
 
Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked to an unusually aggressive and ultimately fatal malignancy called adult T-cell leukemia. Adult T-cell leukemia is characterized by the presence of a chromosomally integrated HTLV-1 provirus in highly aneuploid T cells (33). However, the majority of HTLV-1-infected individuals remain lifelong asymptomatic carriers despite the fact that up to 70% of their CD4+ T cells carry integrated provirus (3, 4). The molecular events that lead to HTLV-1-dependent transformation of the infected T cell remain enigmatic.

HTLV-1 encodes a number of nonstructural proteins that participate in the life cycle of the virus. These include p12, p13, p30, HBZ, Rex, and Tax (for a review, see reference 34). Of these regulatory proteins, the oncoprotein Tax has been well characterized as a critical player in both viral and cellular transcription, as well as malignant transformation. Tax is a strong transcriptional activator required for efficient expression of the viral genome. Tax activates HTLV-1 transcription through three conserved cyclic AMP response elements (CREs) located in the transcriptional control region of the virus. These elements, called viral CREs (vCREs) serve as the binding site for a complex composed of Tax and the cellular transcription factor CREB (25, 29, 32). Together, the Tax/CREB complex recruits the cellular coactivators CBP/p300 via direct interaction with their conserved KIX domain (14, 28). Phosphorylation of CREB at serine 133 (pCREB) is essential for strong p300 recruitment by the promoter-bound Tax/CREB complex. As such, pCREB is required for transcriptional activation by Tax (14). Consistent with these observations, Tax has been found to promote CREB phosphorylation in the infected cell, purportedly to ensure sufficient pCREB availability for Tax activation of viral gene expression (24, 55, 56).

Mitotic replication has been established as a major vehicle of viral transmission within an infected individual. The Tax protein is required for the promotion of clonal proliferation and is directly linked to malignant transformation (1). Tax triggers changes in a variety of intracellular signal transduction pathways and deregulates gene expression though interaction with many different cellular proteins (17). For example, Tax-mediated activation of the NF-{kappa}B pathway results in the upregulation of a large number of genes implicated in proliferation and survival (53). Tax achieves constitutive activation of the NF-{kappa}B pathway via several mechanisms, including activation of I{kappa}B kinase and inactivation of NF-{kappa}B inhibitors, including I{kappa}B{alpha} and I{kappa}Bβ (53).

Bcl3 (B-cell chronic leukemia protein 3) is a member of the I{kappa}B family of NF-{kappa}B inhibitors and was initially identified as a putative proto-oncogene from chronic B-cell lymphocytic leukemia (35). Increased Bcl3 expression typically results from chromosomal translocations and leads to increased cell survival, proliferation, and malignant potential (9, 36, 42). For example, high levels of Bcl3 were observed in over 90% of nuclei from hepatocarcinoma cells (43). There is compelling evidence that Bcl3 overexpression is sufficient to enhance tumorigenic potential in many cell types, including T cells (37, 57).

Unlike other I{kappa}B family proteins, Bcl3 is a predominantly nuclear protein and contains a transactivation domain. Studies have shown that Bcl3 preferentially binds to p50 or p52 NF-{kappa}B homodimers and functions as a transcriptional activator or repressor via NF-{kappa}B binding sites (6, 8, 12, 23, 27, 54, 59, 60). Additionally, Bcl3 has been reported to physically interact with STAT-1 transcription factor and coactivators such as CBP/p300, Tip60, and TORC3, as well as transcriptional repressors such as the histone deacetylase complexes (10, 20, 21, 39, 58). These data are consistent with a model of malignant transformation whereby overexpressed Bcl3 inappropriately activates specific target genes that promote cell proliferation and survival.

The strong correlation between elevated Bcl3 expression and leukemias, lymphomas, and other types of cancers led us to investigate the mechanism of Tax deregulation of this important proto-oncogene. Previous studies have correlated Tax expression and elevated Bcl3 transcript levels (20, 40). We extended these studies to establish the molecular basis for these observations. We found that Tax transcriptionally upregulates Bcl3 expression mediated primarily through the NF-{kappa}B site located in the second intron of the Bcl3 gene. In this study, we observed that Bcl3 formed a stable complex with Tax and strongly repressed Tax-dependent HTLV-1 transcription. Furthermore, using the immobilized template assay to specifically address the mechanism of Bcl3 repression of Tax transcription function, we found that Bcl3, via its ankyrin repeat domain (ARD), stably associated with the Tax/pCREB complex on the viral promoter. The incorporation of Bcl3 into the DNA-bound Tax/pCREB complex significantly reduced recruitment of full-length p300, providing a molecular mechanism for the observed Bcl3 repression of Tax-dependent HTLV-1 transcription. Notably, these data reveal a tightly regulated interrelationship between Tax and the proto-oncogene Bcl3 that may have implications for both survival of the retrovirus and transformation of the infected host T cell.


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MATERIALS AND METHODS
 
Plasmid construction. The glutathione S-transferase (GST)-tagged Bcl3 ARD (amino acids 125 to 359) was generated by amplification of the corresponding DNA fragment by PCR using the wild-type plasmid as a template and cloned into pGEX-2T (Amersham Biosciences). The FLAG-Bcl3 ARD construct was generated as described above and cloned into pFLAG-CMV-2 (Sigma).

Cell culture. Uninfected Jurkat and CEM human T-cell lines were cultured in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin. CEM nuclear extracts were prepared as previously described (11). Immortalized human 293T kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin.

Antibodies. The following antibodies were used in Western blots: anti-Bcl3 (catalog no. SC-185), anti-phospho Ser133CREB (sc-7978-R), anti-p300 (SC-585), and anti-GST (SC-138). All were purchased from Santa Cruz Biotechnology. A monoclonal Tax antibody (hybridoma 168B17-46-92) was obtained from the National Institutes of Health Aids Research and Reference Reagent Program. Antibody against FLAG (F9291) was purchased from Sigma. Alexa Fluor IR700 and IR800 goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Molecular Probes.

Expression and purification of recombinant protein. Bacterially expressed Tax-His6 (61) was purified to >98% homogeneity as previously described (16). CREB327 was purified to apparent homogeneity, free of contaminating nucleic acids, as recently described (30). Full-length His6-tagged p300 was expressed from recombinant baculovirus in Sf9 cells and purified as previously described (14, 26). GST-Bcl3 (58), GST-Bcl3 ARD, GST-KIX (amino acids 451 to 719) (16), and GST were expressed in Escherichia coli and purified by glutathione-agarose chromatography. All proteins were dialyzed against TM buffer [50 mM Tris, pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 1 mM EDTA, pH 8.0, 20% (vol/vol) glycerol, 0.025% (vol/vol) Tween 20, and 1 mM dithiothreitol], aliquoted, and stored at –70°C. The purified proteins are shown in Fig. 4A. CREB was phosphorylated using the catalytic subunit of protein kinase A by incubating 1.6 µM CREB in a reaction containing 3.3 µM ATP, 5 mM MgCl2, and 55 units of protein kinase A (Sigma) in a 25 mM potassium phosphate buffer, pH 6.6.


Figure 4
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  		FIG. 4. Tax and Bcl3 form a complex on the vCREs and inhibit p300 recruitment. (A) Coomassie-stained SDS-polyacrylamide gels of the purified proteins used in the in vitro assays. (B) A 643-bp promoter fragment amplified from the p4TxRE/G-less template (see reference 14) was incubated with increasing amounts of GST-Bcl3 (10, 20, and 30 pmol) in the presence of pCREB, Tax, and p300 (1 pmol each), as indicated. GST alone (30 pmol) was used as a negative control. Bound complexes were washed, and DNA-bound proteins were separated on a 10% SDS-polyacrylamide gel and analyzed by Western blotting. The band that closely comigrates with GST-Bcl3 is a truncation product of p300. (C) The immobilized template assay was performed as described for panel B, except that CEM nuclear extract was used in place of p300.

Transient transfection assays. Human Jurkat T cells were transfected with a constant amount of DNA using Lipofectamine reagent (Invitrogen). After 24 h, the cells were harvested and lysed, and luciferase activity was measured using a dual-luciferase reporter assay system (Promega) with a Turner Designs model TD 20-e luminometer. Firefly luciferase activity was normalized to Renilla luciferase activity from the herpes simplex virus thymidine kinase promoter (pRL-TK; Promega). The Bcl3-luciferase constructs carrying various Bcl3 promoter and enhancer regions (13) were gifts from Timothy McKeithan (University of Nebraska Medical School). The expression plasmid for Tax (pSG-Tax) (47), Bcl3 (CMV-FLAG-Bcl3) (59), CMV-FLAG-Bcl3 ARD, and the vCRE-Luc reporter plasmid (16) have been previously described. The transient transfection assay was performed in triplicate and repeated in three independent experiments.

GST pull-down assays. GST pull-down experiments were performed as previously described (48). Briefly, 20 µl of glutathione-agarose beads were equilibrated in 0.5x Superdex buffer [25 mM HEPES (pH 7.9), 12.5 mM MgCl2, 10 µM ZnSO4, 150 mM KCl, 20% (vol/vol) glycerol, 0.1% Nonidet P-40, and 1 mM EDTA]. Purified GST, GST-Bcl3, or GST-Bcl3 ARD (10 pmol each) was incubated with the beads for 1 to 2 h at 4°C and then washed with 0.5x Superdex buffer. His-tagged purified Tax (40 pmol) protein was then added to the washed beads and incubated overnight at 4°C. The beads were washed as before, and bound proteins were eluted with sodium dodecyl sulfate (SDS) sample dyes. Bound proteins were separated by electrophoresis on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose for subsequent Western blot analysis.

Coimmunoprecipitation assays. 293T cells were transfected with a constant amount of DNA using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Cells were transfected with expression plasmids for Tax (pSG-Tax) and Bcl3 (CMV-FLAG-Bcl3) (59) as indicated in Fig. 4. Total cell lysates were prepared with 1% Triton X-100/phosphate-buffered saline buffer (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) and protease inhibitors (2 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Proteins were immunoprecipitated using anti-FLAG antibodies with protein A/G agarose beads (Santa Cruz Biotechnology) overnight at 4°C and washed several times in 1x phosphate-buffered saline buffer. The bound proteins were analyzed on a 10% SDS-polyacrylamide gel and detected by Western blotting with anti-Tax (hybridoma 168B17-46-92) antibody.

In vitro transcription assays. The promoter fragment was amplified from the plasmid pHTLV-1/G-less, which carries the natural HTLV-1 promoter driving expression of a G-less cassette (2). Primers for PCR amplification of HTLV-1/G-less were 5'-Bio/GTC TGA AAA GGT CAG GGC C-3' and 5'-GGA TAT ATG AGA TGA GTA GG-3'. This PCR-amplified fragment carried the HTLV-1 promoter from –272 through the transcription start site and 314 bp into the G-less cassette. The promoter fragment was immobilized on streptavidin-agarose beads (Dynabeads Dynal Biotech USA) via a biotin group incorporated at the upstream end of the promoter. The immobilized template was assembled into chromatin using native Drosophila histones as previously described (14). The template (75 fmol) was preincubated for 20 min at 30°C in the absence or presence of Tax and/or pCREB (600 fmol) and GST-Bcl3 or GST alone (approximately 1.25 and 2.5 pmol each) in a 30-µl reaction. Nuclear extract (30 µg), prepared from the uninfected human T-cell line CEM, and acetyl-coenzyme A (100 µM) were added to each reaction and incubated an additional 30 min at 30°C. The transcription reactions were carried out as previously described (14). Molecular weight markers (radiolabeled HpaII-digested pBR322) were used to estimate the sizes of the RNA products. A labeled 622-bp DNA fragment was added to each reaction as a recovery standard.

Immobilized template assay. A 643-bp promoter fragment was amplified by PCR from the 4TxRE/G-less plasmid template that carries four reiterated copies of the HTLV-1 promoter-proximal vCRE cloned upstream of the HTLV-1 core promoter (2). The promoter fragment was immobilized on Dynabeads. Binding reactions were preincubated for 20 min at 30°C in the presence of Tax and pCREB. Recombinant GST-Bcl3 or GST-Bcl3 ARD, GST-KIX, or GST alone and p300 were added for an additional 30 min. The protein amounts are indicated in each figure legend, and binding reactions were carried out as previously described (14). CEM nuclear extract (100 µg) was added in the indicated experiments following preincubation and incubated an additional 1 h at 4°C.

Image processing. Images were processed in Adobe Photoshop, with minor adjustments to brightness/contrast as needed (gamma was kept at 1). No bands were obscured or altered. Images were annotated in PowerPoint.

Statistical analyses. For the transient transfection assays, the statistical significance was determined using the Student's t test with a P value of <0.05. Results are expressed as means with the variances of the means (means ± standard errors).


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RESULTS
 
Tax induces Bcl3 expression through an intronic enhancer element. Two previous studies correlated elevated Bcl3 RNA levels in cell lines expressing Tax (20, 40). We also demonstrated elevated Bcl3 RNA and protein levels in HTLV-1-infected cells, relative to levels in the uninfected cells (data not shown). To determine the regions of the Bcl3 gene that confer Tax responsiveness, we performed transient transfection assays using luciferase reporter plasmid constructs carrying the Bcl3 promoter region (–1700 to +212, relative to the transcription start site) or the promoter region together with two previously defined enhancer sequences (13) (Fig. 1A). The HS3 and HS4 enhancer elements were originally defined based on their conservation between species and hypersensitivity to DNase I (13). Both the HS3 and HS4 hypersensitive sites are located in the second intron of the Bcl3 gene. Jurkat T cells were cotransfected with each of the Bcl3 reporter constructs in the absence or presence of an expression plasmid for Tax (pSG-Tax). Figure 1B shows that Tax only modestly activated Bcl3 expression through the upstream promoter; however, inclusion of the HS3 region from the second intron conferred Tax responsiveness to the Bcl3 reporter construct. For reasons that are not clear, we observed levels of Tax transactivation with HS3 alone that were slightly higher than those of HS3 and HS4 together (Fig. 2B).


Figure 1
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  		FIG. 1. Tax mediates Bcl3 induction by intronic enhancers. (A) Schematic diagram of the luciferase reporter containing the Bcl3 promoter either alone or in combination with enhancers HS3, HS4, and HS3/4. The presence of the NF-{kappa}B and CREB binding site in HS3 is indicated. (B) Tax transactivates Bcl3 through the enhancer HS3. Jurkat cells were cotransfected with the indicated reporter constructs in the absence or presence of a Tax expression plasmid (pSG-Tax). (C) M22 is defective for Tax transactivation of Bcl3. Jurkat cells were transiently transfected with the reporter constructs in the absence or presence of expression plasmids for wild-type (wt) Tax (pSG-Tax) or the M22 or M47 mutants (pSG-M22 and pSG-M47), as indicated. Luciferase activity was measured 24 h after transfection and n-fold induction was quantified. The graph represents the averages of three independent experiments. (D) Tax requires the NF-{kappa}B binding site in enhancer HS3 for transactivation of Bcl3. Jurkat cells were cotransfected, in the absence or presence of a Tax expression plasmid, with the Bcl3 promoter/HS3-Luc constructs carrying mutations in the {kappa}B or CRE sites, as indicated. Luciferase activity was measured 24 h after transfection and n-fold induction was quantified. Averages from three independent experiments are presented. The P values with asterisks were determined by Student's t test (P of <0.01).


Figure 2
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  		FIG. 2. Tax and Bcl3 interact in a stable complex. (A) GST pull-down assay reveals that Tax and Bcl3 interact in vitro. Recombinant purified Tax (40 pmol) was incubated with GST (10 pmol) or GST-Bcl3 (10 pmol) immobilized on glutathione-agarose beads. Bound complexes were washed and analyzed by Western blotting. Tax binding was detected using an anti-Tax antibody (upper panel), and GST-Bcl3 was detected using an anti-Bcl3 antibody (lower panel). (B) Coimmunoprecipitation assay reveals that Tax and Bcl3 are present in a complex in vivo. 293T cells were transfected with pSG-Tax (3 µg) in the absence or presence of pCMV2-FLAG-Bcl3 (3 µg). Twenty-four hours after transfection, proteins were immunoprecipitated (IP) from whole-cell lysates with an anti-FLAG antibody and analyzed by Western blotting (WB) with an anti-Tax antibody. IgG, immunoglobulin G.

Tax induces Bcl3 expression primarily via activation of the NF-{kappa}B pathway. The Tax-responsive Bcl3 HS3 region carries both conserved NF-{kappa}B and ATF/CREB recognition elements (Fig. 1A). Tax has previously been shown to activate transcription through both of these transcription factor pathways, although Tax activation of cellular genes through the NF-{kappa}B pathway is the most prevalent (52). To determine which pathway was utilized by Tax in the activation of Bcl3 transcription, we performed transient transfection assays using the mutant Tax expression plasmids M22 and M47. M22 Tax is defective for activation through the NF-{kappa}B pathway, whereas M47 Tax is defective for activation of HTLV-1 transcription via the vCREs (51). Figure 1C shows that Tax mutant M22 was defective for the activation of Bcl3 transcription through the HS3 region (and to a lesser extent through the upstream Bcl3 promoter). Tax mutant M47 had no effect on expression through the upstream promoter or the promoter/intronic enhancer construct. These data indicate that Tax stimulation of the NF-{kappa}B pathway is the primary route for upregulation of Bcl3 transcription. To further confirm that the NF-{kappa}B pathway was responsible for Tax-induced expression of Bcl3, we performed a parallel experiment testing Bcl3 reporter constructs carrying mutations in the {kappa}B and CRE transcription factor recognition sites (13). Figure 1D shows that the NF-{kappa}B site in the second intron is the primary element responsible for Tax induction of the Bcl3 gene. Although we did not detect an effect using the M47 Tax mutant (Fig. 2C), deletion of CRE did reduce Tax transactivation approximately twofold.

Tax physically associates with Bcl3. As a member of the I{kappa}B family, the Bcl3 protein contains seven ankyrin repeats, which form the ARD. Since Tax has previously been shown to bind the ARDs of I{kappa}B{alpha}, I{kappa}B{gamma}, and NF-{kappa}B p105 (18, 19, 44), we were interested in testing whether Tax interacts directly with Bcl3. To investigate this possibility, we first performed GST pull-down assays using bacterially expressed GST-Bcl3 (see Fig. 4A) (58). Purified GST-Bcl3 was bound to glutathione-agarose beads and incubated with purified recombinant Tax (see Fig. 4A). Figure 2A shows that Tax bound to GST-Bcl3 but not to GST alone. This result indicates that Tax and Bcl3 directly interact in vitro. To determine whether Bcl3 interacts with Tax in vivo, we performed coimmunoprecipitation assays. Because the commercially available antibody against Bcl3 was inadequate for analysis of the native protein, we were unable to show that Tax interacts with Bcl3 in HTLV-1-infected cells. Therefore, 293T cells were cotransfected with expression plasmids for Tax and FLAG-tagged Bcl3. Whole-cell extracts prepared from transfected 293T cells were immunoprecipitated with an anti-FLAG antibody, and the presence of Tax in the complex was analyzed by Western blotting. Figure 2B shows that Tax was coimmunoprecipitated with Bcl3, indicating that Tax and Bcl3 interact in vivo.

Bcl3 expression reduces Tax transactivation through the vCRE. The observation that Tax physically interacts with Bcl3 raised the question as to whether Bcl3 binding to Tax might affect Tax transcription function. We performed luciferase assays that measured Tax-dependent transcription from a chimeric promoter carrying the three tandem copies of the vCREs upstream of a core promoter (16). This construct enabled analysis of transcriptional effects mediated through the vCREs (and thus Tax), without contribution from other regions of the HTLV-1 promoter. Jurkat cells were cotransfected with expression plasmids encoding Tax and Bcl3, together with the vCRE-luciferase reporter plasmid. After 24 h, cells were harvested and luciferase activity was measured. Figure 3A shows that Bcl3 potently suppressed Tax-dependent transcription from the vCRE promoter in a dose-dependent manner. We obtained similar results with the natural HTLV-1 promoter/luciferase construct (data not shown). We next investigated the effect of Bcl3 on Tax transactivation in an in vitro transcription assay. We utilized an HTLV-1 promoter fragment carrying a biotin group incorporated at the upstream end of the DNA (Fig. 3B). The immobilized template was assembled into chromatin, followed by preincubation with pCREB, Tax, and increasing concentrations of GST-Bcl3 (or GST alone as a control). The reactions were then supplemented with T-cell nuclear extract (HTLV-1-negative CEM cells) and assayed by in vitro transcription. Figure 3C shows that the addition of Bcl3, compared to GST alone, strongly repressed Tax/pCREB activation in a dose-dependent manner. These data corroborate the findings from the luciferase assays.


Figure 3
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  		FIG. 3. Bcl3 inhibits Tax transactivation in vivo and in vitro. (A) HTLV-1-negative Jurkat T cells were cotransfected with the vCRE-Luc reporter plasmid (100 ng) and a Tax expression plasmid (pSG-Tax; 10 ng) or Bcl3 expression plasmid, as indicated. Transient transfections were performed in triplicate, and each experiment was repeated three times. (B) Schematic representation of the HTLV-1 promoter templates used in the in vitro transcription reaction. (C) In vitro transcription assay showing Bcl3 inhibition of Tax/pCREB-dependent activation. The immobilized HTLV-1 template fragment was assembled into chromatin and then preincubated with purified, recombinant pCREB, Tax, GST-Bcl3, or GST, followed by incubation with nuclear extract (30 µg) from the HTLV-1-negative human T-lymphocyte cell line CEM, as indicated. The positions of the recovery standard (RS), the transcript, and radiolabeled DNA markers are indicated.

Bcl3 inhibits Tax transactivation through blocked recruitment of p300. To investigate the molecular basis for Bcl3 inhibition of Tax transactivation function, we analyzed the interactions of purified proteins on an immobilized promoter template carrying four tandem copies of the vCREs (p4TxRE/G-less) upstream of the core HTLV-1 promoter (2). Purified, recombinant Tax, pCREB, and Bcl3 proteins were preincubated with the DNA template, followed by incubation with purified recombinant full-length p300 (14). The purified proteins are shown in Fig. 4A. The templates were washed, and the bound proteins were analyzed by Western blotting. Figure 4B shows that p300 associated with the Tax/pCREB complex bound to the template (lane 5). This observation is in agreement with the requirement for both Tax and pCREB for efficient p300 recruitment and strong Tax transactivation (14). Consistent with the Tax/Bcl3 protein-protein interaction data presented in Fig. 2, Bcl3 associated with the vCRE template only in the presence of Tax (Fig. 4B, lane 4). Unexpectedly, titration of Bcl3 into the reactions containing Tax and pCREB significantly reduced the binding of p300 to the immobilized template, with a concomitant increase in Bcl3 binding (Fig. 4B, lanes 6 to 8). Essentially identical results were obtained using an immobilized template carrying the natural HTLV-1 promoter (data not shown). We next examined whether Bcl3 similarly displaced native p300 from the HTLV-1 promoter template. We performed the DNA pull-down experiment as described above, except that the immobilized fragment was incubated with a T-cell nuclear extract derived from HTLV-1-negative CEM cells. Figure 4C shows that Bcl3 also reduced the binding of endogenous p300 to the HTLV-1 promoter, similar to that observed with the full-length recombinant protein.

The ankyrin repeat of Bcl3 interacts with Tax and blocks p300 recruitment. The results of these binding assays prompted us to further investigate the molecular basis for the Tax/Bcl3 interaction. As mentioned above, Bcl3 contains seven repeats of the ankyrin domain. The ~33-amino-acid ankyrin repeat is a tandem array of {alpha}-helices responsible for mediating protein-protein interactions. Since there is precedent for Tax interaction with the ARD of I{kappa}B{alpha} (44), we expressed and purified a truncated form of Bcl3 carrying only the seven ankyrin repeats (see Fig. 4A). A schematic of the Bcl3 ARD is shown in Fig. 5A. To investigate whether the ARD was sufficient for interaction with Tax, we compared the binding of ARD (GST-Bcl3 ARD) with the wild-type full-length protein (GST-Bcl3 wt) in GST pull-down assays. Figure 5B shows that the ARD interacts with purified Tax, albeit at a modestly reduced level. There are two possible explanations for the observed reduction in the interaction between Tax and the ARD. First, additional regions of Bcl3 may participate in Tax binding. Second, the reduced interaction may be due to fusion of the GST moiety immediately adjacent to the amino terminus of the ARD. We next compared the effect of the FLAG-tagged ARD versus wild-type full-length Bcl3 on Tax transactivation using transient transfection assays. In the functional assays, both proteins reduced Tax transcriptional activation mediated through the vCREs in a dose-dependent manner (Fig. 5C). We also examined whether the Bcl3 ARD bound to the HTLV-1 promoter in a Tax-dependent fashion and similarly reduced p300 recruitment. Figure 5D reveals that the ARD of Bcl3 was competent for association with the promoter and that the ARD similarly reduced p300 recruitment.


Figure 5
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  		FIG. 5. The ankyrin repeat of Bcl3 mediates Tax binding and represses recruitment of p300 to the HTLV-1 promoter. (A) Schematic diagram of full-length GST-Bcl3 (GST-Bcl3 wt) and the truncated protein carrying the ARD (GST-Bcl3 ARD). (B) GST pull-down assay was performed as described for Fig. 2A, except that GST-Bcl3 wt or GST-Bcl3 ARD was incubated with recombinant Tax. (C) The vCRE-Luc reporter plasmid (100 ng) was cotransfected into Jurkat T cells with pSG-Tax (10 ng) and increasing amounts of pCMV-FLAG-Bcl3 wt or pCMV-FLAG-Bcl3 ARD (10, 50, and 100 ng), as indicated. Luciferase activity was measured 24 h after transfection. (D) The immobilized template assay was performed as described for Fig. 4B, except that Bcl3 wt or Bcl3 ARD was titrated in the presence of pCREB, Tax, and p300. GST was used as a negative control. (E) Immobilized template assay performed as described for panel D, except that GST-KIX (CBP amino acids 451 to 719) (10 pmol) was assayed in the presence of Bcl3 wt, Bcl3 ARD, or GST alone (10 pmol each). The upper panel shows a Western blot using a combination of the anti-GST and anti-Tax antibodies. The lower panel shows a Western blot of the same gel using the anti-CREB antibody.

The highly conserved KIX domain of CBP/p300 is the primary region of the coactivators involved in their recruitment to the HTLV-1 promoter (16, 24, 28, 46). In addition, the KIX domain significantly stabilizes Tax in complex with pCREB at the vCRE (16, 24, 46). Based on these observations, we were interested in testing whether Bcl3 blocks the recruitment of the purified KIX domain to the promoter-bound Tax/pCREB complex. Figure 5E shows that purified GST-KIX (see Fig. 4A) bound to the immobilized HTLV-1 promoter and, as expected, enhanced Tax binding to the template (see lane 5; also see reference 24). Importantly, the addition of full-length Bcl3 as well as the Bcl3 ARD, resulted in strong inhibition of KIX binding to the template (Fig. 5E, lanes 5 to 7). The remarkable inhibition of KIX binding by Bcl3 indicates that the oncoprotein forms a very stable complex with Tax, possibly with significantly greater affinity than the association of the KIX domain with the DNA-bound Tax/pCREB complex. Finally, Bcl3 inhibition of CBP-derived KIX domain recruitment suggests that the proto-oncogene Bcl3 inhibits recruitment of both full-length p300 and CBP.

Together, these data strongly support a model of Bcl3 inhibition of Tax transcription function that is accomplished as a consequence of a direct interaction between Tax and the Bcl3 ARD. This complex forms in solution and on the transcriptionally poised HTLV-1 promoter. The promoter-bound Tax/Bcl3 complex physically blocks CBP/p300 recruitment, via the KIX domain, and this is likely responsible for the observed strong inhibition of Tax transactivation. A model depicting Bcl3 inhibition of Tax transcription function at the HTLV-1 promoter is shown in Fig. 6.


Figure 6
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  		FIG. 6. Schematic showing Bcl3 inhibition of p300 recruitment to the HTLV-1 promoter. For simplicity, only one of the three vCREs is shown.


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DISCUSSION
 
Expression of the Tax protein is directly responsible for transformation of HTLV-1-infected cells. Compelling evidence suggests that the oncogenicity of Tax is linked to its ability to promote cell survival and proliferation. This is accomplished through the deregulation of cellular gene expression and through protein-protein interactions with critical host-cell regulatory molecules. The proto-oncogene Bcl3 is expressed in many different tissue types, most notably the spleen and other lymphoid organs (41). In both B cells and T cells, mitogenic stimuli induce Bcl3 expression (42). Evidence supporting a role for Bcl3 in human T-cell malignancies includes the observation that Bcl3 overexpression enhances the survival of activated T cells and that elevated levels of Bcl3 have been detected in many lymphoid cancers (7, 37, 57). The link between Bcl3 overexpression and malignant transformation may stem from the transcriptional activation properties of Bcl3 on the cell cycle regulator cyclin D1 (59). Cyclin D1 expression is strongly correlated with the development and progression of many cancers. Furthermore, Bcl3 also suppresses p53 activation in response to DNA damage (22). It is noteworthy that Tax has also been shown to increase the expression of cyclin D1 as well as inhibit the function of p53 (38, 45).

Based on these observations, it is perhaps not surprising that elevated Bcl3 levels have been identified in HTLV-1-infected/transformed cells and correlated with Tax expression (20, 40; also data not shown). Due to the potential significance of these observations on HTLV-1-associated malignancies, we investigated the transcriptional basis for Tax-induced Bcl3 overexpression. In the studies described herein, we demonstrate that Tax activates Bcl3 transcription primarily through the NF-{kappa}B site located in the second intron of the gene. These data provide a molecular basis for the observed Tax-induced overexpression of Bcl3.

We next considered the consequence of Bcl3 overexpression in the HTLV-1-infected T cell. We made the interesting observation that Tax interacts with Bcl3 in vivo and forms a direct protein-protein complex in vitro. We also found that Bcl3 potently inhibited Tax-dependent HTLV-1 transcriptional activation both in vivo and in vitro. To investigate the potential mechanism of Bcl3 inhibition of Tax transcription function, we characterized transcriptional regulatory protein binding to the HTLV-1 promoter using the immobilized template assay. We found that Bcl3 binding was dependent upon Tax (in the context of the Tax/pCREB complex) and that upon binding, Bcl3 blocked recruitment of the critical coactivator p300 to the HTLV-1 promoter. We have previously shown that the complex of Tax and pCREB recruits p300 to the HTLV-1 promoter and potently activates viral transcription (14). Therefore, the dramatic reduction in p300 recruitment by Bcl3 likely produces the observed Bcl3 inhibition of Tax transactivation. This interpretation is consistent with a significant number of studies demonstrating a vital role for CBP/p300 in Tax-mediated HTLV-1 transcription (14, 15, 31, 49, 50).

We found that the ARD of Bcl3 participated in Tax binding and was sufficient for inhibition of p300 recruitment. Previous studies have shown that Tax interacts with the ARDs of I{kappa}B{alpha}, I{kappa}B{gamma}, as well as NF-{kappa}B1 p105, ultimately leading to NF-{kappa}B activation (18, 19, 44). Interestingly, a previous study found that the ARD protein NF-kB2 p100 interacts with Tax in the cytoplasm and inhibits HTLV-1 Tax transactivation (5). The authors suggested that an ARD at the carboxy terminus of p100 was critical to inhibition of viral transcription. These data support the notion that Tax forms a stable complex with ARDs. Therefore, it will be of interest to examine whether Tax interacts with additional ankyrin repeat-containing proteins and whether the interaction results in functional dysregulation.

A recent study found that Bcl3 inhibits HTLV-1 transcription through the CREB coactivator TORC3 (20). In this study, the authors excluded the possibility that Bcl3 directly affected Tax transcription function by examining the effect of Bcl3 on a chimeric Gal4-Tax construct driving expression of the artificial Gal4-luciferase reporter. Perhaps the Bcl3 binding site on the Tax protein was inaccessible in the fusion construct, and thus, Bcl3 was unable to form a complex with Tax. Additionally, there is no evidence that Gal4-Tax is competent for CBP/p300 recruitment; therefore, Gal4-Tax activation does not recapitulate the natural mechanism of Tax transcription function from the HTLV-1 promoter. However, the data presented by Hishiki et al. (20) provide compelling evidence that Bcl3 inhibits Tax-independent transcription via direct binding to TORC3. This observation, taken together with our observation that Bcl3 inhibits Tax transactivation via suppression of p300 recruitment, indicates that Bcl3 functions as a potent repressor of viral gene expression. It will be of interest to determine whether Bcl3 represses proviral gene expression in HTLV-1-infected cells.

In summary, the data presented herein establish that Tax expression leads to Bcl3 overexpression via activation of the NF-{kappa}B pathway. The Bcl3 protein, via its ARD, then associates with the promoter-bound Tax/pCREB complex to block recruitment of the essential coactivator p300, resulting in repression of viral transcription. We propose that the interrelationship between Tax and Bcl3 has the following consequences in an HTLV-1-infected cell. First, Tax-induced overexpression of Bcl3 promotes cell proliferation via the transcriptional upregulation of cyclin D1 and/or other key regulators of cell division. Second, Tax-induced overexpression of Bcl3 results in downregulation of HTLV-1 gene expression via inhibition of CBP/p300 recruitment to the promoter. While these observations are seemingly paradoxical, it is likely that the reciprocal relationship between Tax and Bcl3 results in a fine balance that ultimately endows a net evolutionary benefit to the virus. We propose that Tax activation of Bcl3 expression enhances mitotic replication of the provirus while concomitant Bcl3 inhibition of HTLV-1 transcription produces a negative feedback that represses viral gene expression, thus contributing to immune evasion. Through these two routes, Bcl3 ultimately enhances survival of the retrovirus.


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ACKNOWLEDGMENTS
 
We are most grateful to Patrick Viatour and Albert Baldwin for the Bcl3 plasmids and Timothy McKeithan for the Bcl3 reporter constructs. We thank Dinaida Lopez and Jason Rivest for purified proteins and technical assistance and Julita Ramìrez, Holli Giebler, and other members of the lab for critical reading of the manuscript.

This work was supported by a grant from the National Institutes of Health (CA55035).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Campus Box 1870, Colorado State University, Fort Collins, CO 80523-1870. Phone: (970) 491-0420. Fax: (970) 491-0494. E-mail: Jennifer.Nyborg{at}ColoState.Edu Back

{triangledown} Published ahead of print on 24 September 2008. Back


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Journal of Virology, December 2008, p. 11939-11947, Vol. 82, No. 23
0022-538X/08/$08.00+0     doi:10.1128/JVI.01356-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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