The HTLV-1 tax protein cooperates with phosphorylated CREB, TORC2 and p300 to activate CRE-dependent cyclin D1 transcription.

Adult T-cell leukemia/lymphoma is a fatal malignancy etiologically linked to infection with the human T-cell leukemia virus (HTLV-1). The virally encoded oncoprotein Tax activates the transcription of HTLV-1 and cellular genes by cooperating with cellular transcription factors. Cyclin D1 is a pivotal regulator of cell cycle progression, and increased expression strongly correlates with malignant transformation. Here, we characterize the mechanism of Tax transactivation of cyclin D1. We find that cyclin D1 transcript levels are elevated in HTLV-1 infected cells and that Tax physically associates with the cyclin D1 gene in vivo. Tax binds the cyclin D1 promoter-proximal cyclic AMP response element (CRE) in the presence of phosphorylated CREB (pCREB) in vitro, and together the Tax-pCREB complex recruits the cellular co-activator p300 to the promoter through this unconventional Tax-responsive element. We further show that the transducer of regulated CREB 2 (TORC2) cooperates with Tax to further enhance p300 recruitment to the cyclin D1 promoter in vitro. Tax and TORC2 in combination stimulate cyclin D1 expression in vivo, demonstrating the functional outcome of the binding interactions. Together, our findings support a model in which Tax-induced accumulation of cyclin D1 shortens the G1 phase of the cell cycle, promotes mitotic replication of the virus, and drives selection and expansion of malignant T-cells.

The oncoprotein Tax binds the SRC-1-interacting domain of CBP/p300 to mediate transcriptional activation.

Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax. To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300. While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood. Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter. In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function. Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction. Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP. We show that a double point mutant targeted to one of the putative alpha-helical motifs in this domain significantly compromises the interaction with Tax. We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding. This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function. Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion. Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation.

Human T-cell leukemia virus type I tax repression of p73beta is mediated through competition for the C/H1 domain of CBP.

The Tax protein, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high level viral transcription and HTLV-I-associated malignant transformation. Although the precise mechanism of malignant transformation by Tax is unclear, it is well established that Tax represses the transcription function of the tumor suppressor p53, possibly accelerating the accumulation of genetic mutations that are critical in HTLV-I-mediated malignant transformation. Tax repression of p53 transcription function appears to occur, at least in part, through competition for the cellular coactivator CBP/p300. In this study, we characterize the effect of Tax on the p53 family member, p73. We demonstrate that Tax also represses the transcription function of p73beta and that the repression is reciprocal in vivo, consistent with the idea that both transcription factors may compete for CBP/p300 in vivo. We provide evidence showing that both Tax and p73 interact strongly with the C/H1 domain of CBP and that their binding to this region is mutually exclusive in vitro. This finding provides evidence supporting the idea that reciprocal transcriptional repression between Tax and p73 is mediated through coactivator competition.

Insight into the tumor suppressor function of CBP through the viral oncoprotein tax.

CREB binding protein (CBP) is a cellular coactivator protein that regulates essentially all known pathways of gene expression. The transcriptional coactivator properties of CBP are utilized by at least 25 different transcription factors representing nearly all known classes of DNA binding proteins. Once bound to their target genes, these transcription factors are believed to tether CBP to the promoter, leading to activated transcription. CBP functions to stimulate transcription through direct recruitment of the general transcription machinery as well as acetylation of both histone and transcription factor substrates. Recent observations indicate that a critical dosage of CBP is required for normal development and tumor suppression, and that perturbations in CBP concentrations may disrupt cellular homeostasis. Furthermore, there is accumulating evidence that CBP deregulation plays a direct role in hematopoietic malignancies. However, the molecular events linking CBP deregulation and malignant transformation are unclear. Further insight into the function of CBP, and its role as a tumor suppressor, can be gained through recent studies of the human T-cell leukemia virus, type I (HTLV-I) Tax oncoprotein. Tax is known to utilize CBP to stimulate transcription from the viral promoter. However, recent data suggest that as a consequence of the Tax-CBP interaction, many cellular transcription factor pathways may be deregulated. Tax disruption of CBP function may play a key role in transformation of the HTLV-I-infected cell. Thus, Tax derailment of CBP may lend important information about the tumor suppressor properties of CBP and serve as a model for the role of CBP in hematopoietic malignancies.

Analysis of CREB mutants in Tax complex formation and trans-activation.

The human T cell leukemia virus type 1 (HTLV-1) oncoprotein Tax interacts with cellular transcription factors to facilitate viral replication in infected cells. Tax binds to the cellular transcription factor CREB and the cellular coactivator protein CBP to form a stable nucleoprotein complex on the viral enhancer elements. The formation of this complex is believed to promote strong Tax-dependent transcriptional activation of viral gene expression. In this study, we characterize a series of internal CREB deletion mutants with respect to Tax and CBP recruitment and transcriptional activation. We find that, although several of these mutants are unable to support ternary complex formation with Tax and the viral CRE DNA, they are fully competent for cooperation with Tax in CBP recruitment. Unexpectedly, CREB proteins that carry deletions in a carboxyterminal region of the KID domain, while competent for ternary and quaternary complex formation, were defective for Tax trans-activation in vivo. These studies suggest that CREB may serve more than just a "scaffolding" role in Tax trans-activation, cooperating directly with Tax (and CBP) to mediate strong transcriptional activation of the provirus.

p53 recruitment of CREB binding protein mediated through phosphorylated CREB: a novel pathway of tumor suppressor regulation.

CREB binding protein (CBP) is a 270-kDa nuclear protein required for activated transcription of a large number of cellular genes. Although CBP was originally discovered through its interaction with phosphorylated CREB (pCREB), it is utilized by a multitude of cellular transcription factors and viral oncoproteins. Both CREB and the tumor suppressor p53 have been shown to directly interact with the KIX domain of CBP. Although coactivator competition is an emerging theme in transcriptional regulation, we have made the fortuitous observation that protein kinase A-phosphorylated CREB strongly enhances p53 association with KIX. Phosphorylated CREB also facilitates interaction of a p53 mutant, defective for KIX binding, indicating that CREB functions in a novel way to bridge p53 and the coactivator. This is accomplished through direct interaction between the bZIP domain of CREB and the amino terminus of p53; a protein-protein interaction that is also detected in vivo. Consistent with our biochemical observations, we show that stimulation of the intracellular cyclic AMP (cAMP) pathway, which leads to CREB phosphorylation, strongly enhances both the transcriptional activation and apoptotic properties of p53. We propose that phosphorylated CREB mediates recruitment of CBP to p53-responsive promoters through direct interaction with p53. These observations provide evidence for a novel pathway that integrates cAMP signaling and p53 transcriptional activity.

Binding of p53 to the KIX domain of CREB binding protein. A potential link to human T-cell leukemia virus, type I-associated leukemogenesis.

The pleiotropic cellular coactivator CREB binding protein (CBP) plays a critical role in supporting p53-dependent tumor suppressor functions. p53 has been shown to directly interact with a carboxyl-terminal region of CBP for recruitment of the coactivator to p53-responsive genes. In this report, we identify the KIX domain as a new p53 contact point on CBP. We show that both recombinant and endogenous forms of p53 specifically interact with KIX. We demonstrate that the activation domain of p53 participates in KIX binding and provide evidence showing that this interaction is critical for p53 transactivation function. The human T-cell leukemia virus, type-I-encoded oncoprotein Tax is a well established repressor of p53 transcription function. Like p53, Tax also binds to KIX. The finding that both transcription factors bind to a common region of CBP suggests that coactivator competition may account for the observed repression. We demonstrate reciprocal repression between Tax and p53 in transient transfection assays, supporting the idea of intracellular coactivator competition. We biochemically confirm coactivator competition by directly showing that both transcription factors bind to KIX in a mutually exclusive fashion. These data provide molecular evidence for the observed intracellular competition and suggest that Tax inhibits p53 function by abrogating a novel p53-KIX interaction. Thus, Tax competition for the p53-KIX complex may be a pivotal event in the human T-cell leukemia virus, type I transformation pathway.

The tax protein-DNA interaction is essential for HTLV-I transactivation in vitro.

The human T-cell leukemia virus type-1 (HTLV-I)-encoded Tax protein enhances viral gene transcription through interaction with three repeated DNA elements located in the viral promoter. These elements, called viral CREs, are composed of an off-consensus eight base-pair cyclic AMP response element (CRE), immediately flanked by sequences that are rich in guanine and cytosine residues. Recent biochemical experiments have demonstrated that in the presence of the cellular protein CREB, Tax directly binds the viral CRE G+C-rich sequences via interaction with the minor groove. To determine the functional significance of the Tax-DNA interaction, we synthesized minor groove-binding pyrrole-imidazole polyamides which bind specifically to the G+C-rich sequences in the viral CREs. At concentrations where the polyamides specifically protect the G+C-rich sequences from MPE:Fe cleavage, the polyamides block the Tax-DNA interaction. At precisely these same concentrations, the polyamides specifically inhibit Tax transactivation in vitro, without altering CREB-activated transcription or basal transcription from the same promoter. Together, these data provide strong evidence that Tax-viral CRE interaction is essential for Tax function in vitro, and suggest that targeted disruption of the Tax-DNA minor groove interaction with polyamides may provide a novel approach for inhibiting viral replication in vivo.

Binding of the human T-cell leukemia virus Tax protein to the coactivator CBP interferes with CBP-mediated transcriptional control.

The HTLV-I oncoprotein Tax is required for high level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Tax stimulates HTLV-I transcription through high affinity binding to the KIX domain of CBP, a pleiotropic coactivator. Several cellular proteins, including c-jun, also bind to KIX and utilize CBP as a coactivator. To test whether Tax binding to KIX may disable cellular CBP function, we examined the potential interplay between Tax and c-jun for binding to KIX. We show that Tax represses the transcription function of c-jun in vivo and demonstrate that both transcription factors bind to an overlapping minimal region of KIX in vitro. c-jun binding to KIX is displaced by Tax, indicating that their binding is mutually exclusive and providing a molecular basis for the observed repression. The competition between Tax and cellular transcription factors for CBP represents a novel pathway for HTLV-I dependent deregulation of gene expression, and may have significant implications for cellular homeostasis and transformation in the HTLV-I infected T-cell.

The human T-cell leukemia virus type 1 oncoprotein Tax inhibits the transcriptional activity of c-Myb through competition for the CREB binding protein.

Tax, the transforming protein of human T-cell leukemia virus type 1 (HTLV-1), is required for strong activation of HTLV-1 transcription. This activation is mediated through interaction with the KIX domain of the cellular coactivator CREB binding protein (CBP). In this study we examined the possibility that the Tax-KIX interaction may mediate effects on cellular gene transcription in vivo, as a growing number of cellular transcription factors have been shown to utilize CBP as a coactivator. We tested the ability of Tax to deregulate the activity of the cellular transcription factor, c-Myb, since both Tax and c-Myb interact with the KIX domain of CBP. Our results show that in vivo, Tax antagonizes the transcriptional activity of c-Myb and, reciprocally, c-Myb antagonizes the transcriptional activity of Tax. Furthermore, c-Myb competes for KIX binding to Tax in vitro, indicating that these two transcription factors bind CBP in a mutually exclusive manner. This novel mechanism of transcriptional interference by Tax may promote globally deregulated cellular gene expression in the HTLV-1-infected cell, possibly leading to leukemogenesis.

Molecular interactions between the coactivator CBP and the human T-cell leukemia virus Tax protein.

The oncoprotein Tax, encoded by the human T-cell leukemia virus type I (HTLV-I), is required for high-level viral transcription and is strongly linked to HTLV-I-associated malignant transformation. Recent evidence suggests that Tax stimulates HTLV-I transcription through recruitment of the cellular coactivator protein CBP to the HTLV-I promoter, promoting high-level viral replication via the transcriptional activation properties associated with CBP. Tax directly contacts the KIX domain of CBP to stably anchor the coactivator to nucleoprotein complexes at the promoter. Here, we identify KIX amino acid residues 588 to 683 as the minimal region sufficient for interaction with Tax. This region is similar to the minimal KIX amino acid residues necessary for strong interaction with phosphorylated CREB, and is composed of a structural domain that forms an extensive hydrophobic core. We further show that a double point mutation in KIX differentially affects the binding of Tax and phosphorylated CREB, suggesting that these transcription factors may recognize unique amino acid residues within the KIX domain. These observations suggest that Tax directly contacts the hydrophobic core of KIX, and provides a structural framework to further define the molecular interactions between Tax and CBP.

CLoning of equine interleukin 1 alpha and equine interleukin 1 beta and determination of their full-length cDNA sequences.

OBJECTIVES: To clone equine interleukin 1 alpha (IL-1 alpha) and equine interleukin 1 beta (IL-1 beta) and determine their full-length cDNA sequences. PROCEDURES: The mRNA isolated from lipopolysaccharide-stimulated cultured equine monocytes was reverse transcribed, and a cDNA library was constructed in a lambda phage. The cDNA library was screened by means of plaque hybridization with radiolabeled human IL-1 alpha and IL-1 beta cDNA probes. The cDNA nucleotide sequences for equine IL-1 alpha and equine IL-1 beta were determined by use of the dideoxy chain termination technique. The cDNA sequences were analyzed, using computer software, for sequence characteristics and compared with sequences reported for other species. RESULTS: The cDNA for equine IL-1 alpha was 1,728 base pairs in length with an ORF encoding a peptide of 270 amino acids with a predicted molecular mass of 30.823 kd. The cDNA for equine IL-1 beta was 1,473 base pairs in length with an ORF encoding a peptide of 268 amino acids with a predicted molecular mass of 30.342 kd. Similarity between amino acid sequence of equine IL-1 alpha and sequences for IL-1 alpha of other species ranged from 62.5 to 82.2%; similarity between amino acid sequence of equine IL-1 beta and sequences for IL-1 beta of other species ranged from 62.5 to 82.2%; similarity between amino acid sequences of equine IL-1 alpha and equine IL-1 beta was 26%. CONCLUSIONS AND CLINICAL RELEVANCE: Results establish a basis for studying the roles of interleukin 1 in healthy and diseased joints in horses.

Cloning of equine interleukin 1 receptor antagonist and determination of its full-length cDNA sequence.

OBJECTIVES: To clone equine interleukin 1 receptor antagonist (IL-1ra) and determine its full-length cDNA sequence. PROCEDURE: A cDNA library derived from lipopolysaccharide-stimulated equine monocytes was screened by means of plaque hybridization to radiolabeled equine IL-1ra DNA probes generated by means of the polymerase chain reaction. The cDNA nucleotide sequence for equine IL-1ra was determined by use of the dideoxy chain termination technique, analyzed by use of computer software for sequence characteristics, and compared with sequences reported for IL-1ra of other species. RESULTS: The cDNA of equine IL-1ra was 1,614 base pairs in length with an ORF encoding a peptide of 177 amino acids with a predicted molecular mass of 20.427 kd. Similarity between the amino acid sequence of equine IL-1ra and sequences for human, murine, rat, and lapine IL-1ra was 76%. Similarity between sequence for equine IL-1ra and sequences for equine interleukin-1 alpha and equine interleukin-1 beta were 22.6 and 24.6%, respectively. CONCLUSION: Comparison of the sequence for equine IL-1ra with sequences for IL-1ra of other species indicated a high degree of conservation. CLINICAL RELEVANCE: Results establish a basis for studying the roles of interleukin-1 in healthy and diseased joints in horses.

Activation of BLV transcription by NF-kappa B and Tax.

Bovine leukemia virus (BLV) is the causative agent of bovine leukosis, a naturally occurring fatal disease in cattle. BLV transcription is regulated by cellular transcription factors and the virally encoded oncoprotein Tax. In this report, we investigated the functional role of the putative NF-kappa B binding site recently identified in the BLV promoter. Our studies indicate that the kappa B binding motif acts as a functional enhancer in the presence of the cellular NF-kappa B proteins. Furthermore, the kappa B site together with a single 21-bp repeat confers strong activation of BLV transcription in the presence the NF-kappa B proteins and Tax. These results suggest that cellular NF-kappa B may be involved in the regulation of BLV transcription and activation of the virus from latency.

Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

Anchoring of CREB binding protein to the human T-cell leukemia virus type 1 promoter: a molecular mechanism of Tax transactivation.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates viral transcription through interaction with the cellular transcription factor CREB (cyclic AMP response element [CRE] binding protein). Although Tax stabilizes the binding of CREB to the Tax-responsive viral CREs in the HTLV-1 promoter, the precise molecular mechanism by which Tax mediates strong transcriptional activation through CREB remains unclear. In this report, we show that Tax promotes high-affinity binding of the KIX domain of CREB binding protein (CBP) to CREB-viral CRE complexes, increasing the stability of KIX in these nucleoprotein complexes by up to 4.4 kcal/mol. Comparable KIX binding affinities were measured for both phosphorylated and unphosphorylated forms of CREB, and in all cases high-affinity binding was dependent upon both Tax and the viral CRE. Tax also promoted association of KIX to a truncated form of CREB containing only the 73-amino-acid basic leucine zipper (bZIP) domain, indicating that the entire amino-terminal CBP-interacting domain of CREB is nonessential in the presence of Tax. Functional studies upheld the binding studies, as expression of the bZIP domain of CREB was sufficient to support Tax transactivation of HTLV-1 transcription in vivo. Finally, we show that transfection of a KIX expression plasmid, which lacks activation properties, inhibited Tax transactivation in vivo. This suggests that KIX occupies the CBP binding site on Tax, and therefore CBP is likely a cofactor in mediating Tax stimulation of HTLV-1 transcription. Together, these data support a model in which Tax anchors CBP to the HTLV-1 promoter, with strong transcriptional activation resulting from the CBP-associated activities of nucleosome remodeling and recruitment of the general transcription machinery.

Repression of bax gene expression by the HTLV-1 Tax protein: implications for suppression of apoptosis in virally infected cells.

The human T-cell leukemia virus-encoded oncoprotein Tax is a potent deregulator of cellular gene expression. Here we report that Tax represses transcription of the human bax gene, a gene whose protein product accelerates apoptosis. This repression is mediated through a 27-bp sequence in the bax promoter that contains a putative basic helix-loop-helix binding site. Deletion of this sequence abolishes Tax-mediated repression of bax. Repression of the bax gene may be biologically significant, as we also show that HTLV-I-infected cell lines are resistant to a variety of physical, chemical, and biological stimuli which induce apoptosis in uninfected T-cells. The repression of genes involved in promoting apoptosis, including the bax gene, may contribute to retroviral survival, and initiate a pathway toward malignant transformation.

In vitro transcription of human T-cell leukemia virus type 1 is RNA polymerase II dependent.

The HTLV-1 promoter directs RNA polymerase II transcription of viral genomic RNA in vivo. However, it has been reported that in vitro, a unique RNA polymerase, with characteristics of RNA polymerases II and III, is capable of HTLV-1 transcription (G. Piras, F. Kashanchi, M. F. Radonovich, J. F. Duvall, and J. N. Brady, J. Virol. 68:6170-6179, 1994). To further characterize the polymerase involved in HTLV-1 transcription in vitro, runoff transcription assays were performed with a variety of extracts and RNA polymerase inhibitors. Under all in vitro reaction conditions tested, RNA polymerase II appeared to be the only polymerase capable of correct transcriptional initiation from the HTLV-1 promoter. Synthesis of the specific HTLV-1 RNA transcript showed sensitivities to the RNA polymerase inhibitors tagetitoxin and alpha-amanitin that are consistent with RNA polymerase II transcription. Together, these data indicate that in vitro, as in vivo, the HTLV-1 promoter directs transcription by RNA polymerase II.

Transcriptional activation of RNA polymerase III-dependent genes by the human T-cell leukemia virus type 1 tax protein.

The human T-cell leukemia virus-encoded tax protein is a potent activator of many viral and cellular genes transcribed by RNA polymerase II. We find that both chromatin and cell extracts derived from human T-cell leukemia virus type 1-infected human T lymphocytes support higher levels of 5S rRNA and tRNA gene transcription than chromatin or extracts from uninfected T lymphocytes. The viral protein Tax was likely responsible for this higher level of class II gene transcription, as purified Tax was found to stimulate both genes when added to the uninfected cell extract or in reconstituted systems. Both limiting-component transcription assays and DNA binding assays identified the class III gene transcription factor TFIIIB as the principle target of Tax activity. Surprisingly, we find that Tax increases the effective concentration of active TFIIIB molecules. These data suggest that Tax stimulates RNA polymerase III-dependent gene expression by accelerating the rate and/or extent of transcription initiation complex assembly.

Transcriptional repression of p53 by human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I oncoprotein Tax transcriptionally deregulates a wide variety of viral and cellular genes. Tax deregulation of gene expression is mediated through interaction with a variety of structurally unrelated cellular transcription factors, as Tax does not bind DNA in a sequence-specific manner. Although most of these cellular transcription factors have been shown to mediate activation by Tax, we have recently demonstrated that members of the basic helix-loop-helix (bHLH) family of transcription factors, which play a critical role in progression through the cell cycle, mediate repression by Tax. In this report, we examined whether Tax might repress transcription of the tumor suppressor p53, as the p53 gene has recently been demonstrated to be regulated by the bHLH protein c-Myc. Furthermore, loss or inactivation of the p53 gene has been shown to be causally associated with oncogenic transformation. We show that Tax represses transcription of the p53 gene and that this repression is dependent upon the bHLH recognition element in the p53 promoter. Together, these results suggest that Tax may promote malignant transformation through repression of p53 transcription.