Holli A. Giebler

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.

Human T-Cell Leukemia Virus Type 1 Tax Requires Direct Access to DNA for Recruitment of CREB Binding Protein to the Viral Promoter

ABSTRACT 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.

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.

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.

p53 Recruitment of CREB Binding Protein Mediated through Phosphorylated CREB: a Novel Pathway of Tumor Suppressor Regulation

ABSTRACT 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.

Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent

ABSTRACT Robust transcription of human T-cell leukemia virus type 1 (HTLV-1) genome requires the viral transactivator Tax. Although Tax has been previously shown to interact with the KIX domain of CBP/p300 in vitro, the precise functional relevance of this interaction remains unclear. Using two distinct approaches to interrupt the physical interaction between Tax and KIX, we find that Tax transactivation from chromatin templates is strongly dependent on CBP/p300 recruitment via the KIX domain. Additionally, we find that the primary functional contribution of CBP/p300 to Tax transactivation resides in the intrinsic acetyltransferase activity of the coactivators. These studies unexpectedly uncover a specific requirement for CBP/p300 acetyltransferase activity on chromatin templates assembled with nucleosomes lacking their amino-terminal tails. Together, these data indicate that the KIX domain of CBP/p300 is essential for targeting the acetyltransferase activity of the coactivator to the Tax-CREB (Tax/CREB) complex. Significantly, these observations reveal the presence of one or more CBP/p300 acetyltransferase targets that function specifically on chromatin templates, are independent of the histone tails, and are critical to Tax transactivation.

Molecular Characterization of the Tax-containing HTLV-1 Enhancer Complex Reveals a Prominent Role for CREB Phosphorylation in Tax Transactivation *

Transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1) is mediated by the viral oncoprotein Tax, which utilizes cellular transcriptional machinery to perform this function. The viral promoter carries three cyclic AMP-response elements (CREs), which are recognized by the cellular transcription factor cAMP-response element-binding protein (CREB). Tax binds to GC-rich sequences that immediately flank the CREs. The coactivator CREB-binding protein (CBP)/p300 binds to this promoter-bound ternary complex, which promotes the initiation of HTLV-1 transcription. Protein kinase A phosphorylation of CREB at serine 133 facilitates transcription from cellular CREs by recruiting CBP/p300 via its KIX domain. However, it remains controversial whether CREB phosphorylation plays a role in Tax transactivation. In this study, we biochemically characterized the quaternary complex formed by Tax, CREB, KIX, and the viral CRE by examining the individual molecular interactions that contribute to Tax stabilization in the complex. Our data show KIX, Ser133-phosphorylated CREB, and vCRE DNA are all required for stable Tax incorporation into the complex in vitro. Consonant with a fundamental role for CREB phosphorylation in Tax recruitment to the complex, we found that CREB is highly phosphorylated in a panel of HTLV-1-infected human T-cell lines. Significantly, we show that Tax is directly responsible for promoting elevated levels of CREB phosphorylation. Together, these data support a model in which Tax promotes CREB phosphorylation in vivo to ensure availability for Tax transactivation. Because pCREB has been implicated in leukemogenesis, enhancement of CREB phosphorylation by the virus may play a role in the etiology of adult T-cell leukemia.

Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

BackgroundIn the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation.ResultsUsing a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1β stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1β directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1β, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1β is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1β may serve as a critical regulator of H1 dynamics and gene activation in vivo.ConclusionsThese studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation.

Histone Chaperone Nap1 Is a Major Regulator of Histone H2A-H2B Dynamics at the Inducible GAL Locus

ABSTRACT Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use the GAL locus in Saccharomyces cerevisiae to investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When the GAL locus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measured in vitro. When the GAL locus is transcribed, excess H2A-H2B is reversed, and levels of all chromatin-bound histones are depleted in cells lacking Nap1. We developed an in vivo system to measure histone exchange at the GAL locus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability with in vitro nucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2B in vivo.