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

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.

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

BACKGROUND: In 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. RESULTS: Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1ß 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/Taf1ß directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1ß, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1ß is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1ß may serve as a critical regulator of H1 dynamics and gene activation in vivo. CONCLUSIONS: These 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.

Human T-cell leukemia virus type 1 (HTLV-1) tax requires CADM1/TSLC1 for inactivation of the NF-κB inhibitor A20 and constitutive NF-κB signaling.

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.

Class I HDAC inhibition stimulates cardiac protein SUMOylation through a post-translational mechanism.

Lysine residues are subject to a multitude of reversible post-translational modifications, including acetylation and SUMOylation. In the heart, enhancement of lysine acetylation or SUMOylation using histone deacetylase (HDAC) inhibitors or SUMO-1 gene transfer, respectively, has been shown to be cardioprotective. Here, we addressed whether there is crosstalk between lysine acetylation and SUMOylation in the heart. Treatment of cardiomyocytes and cardiac fibroblasts with pharmacological inhibitors of HDAC catalytic activity robustly increased conjugation of SUMO-1, but not SUMO-2/3, to several high molecular weight proteins in both cell types. The use of a battery of selective HDAC inhibitors and short hairpin RNAs demonstrated that HDAC2, which is a class I HDAC, is the primary HDAC isoform that controls cardiac protein SUMOylation. HDAC inhibitors stimulated protein SUMOylation in the absence of de novo gene transcription or protein synthesis, revealing a post-translational mechanism of HDAC inhibitor action. HDAC inhibition did not suppress the activity of de-SUMOylating enzymes, suggesting that increased protein SUMOylation in HDAC inhibitor-treated cells is due to stimulation of SUMO-1 conjugation rather than blockade of SUMO-1 cleavage. Consistent with this, multiple components of the SUMO conjugation machinery were capable of being acetylated in vitro. These findings reveal a novel role for reversible lysine acetylation in the control of SUMOylation in the heart, and suggest that cardioprotective actions of HDAC inhibitors are in part due to stimulation of protein SUMO-1-ylation in myocytes and fibroblasts.

Nucleosome eviction and activated transcription require p300 acetylation of histone H3 lysine 14.

Histone posttranslational modifications and chromatin dynamics are inextricably linked to eukaryotic gene expression. Among the many modifications that have been characterized, histone tail acetylation is most strongly correlated with transcriptional activation. In Metazoa, promoters of transcriptionally active genes are generally devoid of physically repressive nucleosomes, consistent with the contemporaneous binding of the large RNA polymerase II transcription machinery. The histone acetyltransferase p300 is also detected at active gene promoters, flanked by regions of histone hyperacetylation. Although the correlation between histone tail acetylation and gene activation is firmly established, the mechanisms by which acetylation facilitates this fundamental biological process remain poorly understood. To explore the role of acetylation in nucleosome dynamics, we utilized an immobilized template carrying a natural promoter reconstituted with various combinations of wild-type and mutant histones. We find that the histone H3 N-terminal tail is indispensable for activator, p300, and acetyl-CoA-dependent nucleosome eviction mediated by the histone chaperone Nap1. Significantly, we identify H3 lysine 14 as the essential p300 acetylation substrate required for dissociation of the histone octamer from the promoter DNA. Together, a total of 11 unique mutant octamer sets corroborated these observations and revealed a striking correlation between nucleosome eviction and strong activator and acetyl-CoA-dependent transcriptional activation. These novel findings uncover an exclusive role for H3 lysine 14 acetylation in facilitating the ATP-independent and transcription-independent disassembly of promoter nucleosomes by Nap1. Furthermore, these studies directly couple nucleosome disassembly with strong, activator-dependent transcription.

Activator-dependent p300 acetylation of chromatin in vitro: enhancement of transcription by disruption of repressive nucleosome-nucleosome interactions.

Condensation of chromatin into higher order structures is mediated by intra- and interfiber nucleosome-nucleosome interactions. Our goals in this study were to determine the impact specific activator-dependent histone acetylation had on chromatin condensation and to ascertain whether acetylation-induced changes in chromatin condensation were related to changes in RNA polymerase II (RNAPII) activity. To accomplish this, an in vitro model system was constructed in which the purified transcriptional activators, Tax and phosphorylated CREB (cAMP-response element-binding protein), recruited the p300 histone acetyltransferase to nucleosomal templates containing the human T-cell leukemia virus type-1 promoter sequences. We find that activator-dependent p300 histone acetylation disrupted both inter- and intrafiber nucleosome-nucleosome interactions and simultaneously led to enhanced RNAPII transcription from the decondensed model chromatin. p300 histone acetyltransferase activity had two distinct components: non-targeted, ubiquitous activity in the absence of activators and activator-dependent activity targeted primarily to promoter-proximal nucleosomes. Mass spectrometry identified several unique p300 acetylation sites on nucleosomal histone H3 (H3K9, H3K27, H3K36, and H3K37). Collectively, our data have important implications for understanding both the mechanism of RNAPII transcriptional regulation by chromatin and the molecular determinants of higher order chromatin structure.

Histone chaperones, histone acetylation, and the fluidity of the chromogenome.

The "chromogenome" is defined as the structural and functional status of the genome at any given moment within a eukaryotic cell. This article focuses on recently uncovered relationships between histone chaperones, post-translational acetylation of histones, and modulation of the chromogenome. We emphasize those chaperones that function in a replication-independent manner, and for which three-dimensional structural information has been obtained. The emerging links between histone acetylation and chaperone function in both yeast and higher metazoans are discussed, including the importance of nucleosome-free regions. We close by posing many questions pertaining to how the coupled action of histone chaperones and acetylation influences chromogenome structure and function.

The HTLV-1 Tax protein: revealing mechanisms of transcriptional activation through histone acetylation and nucleosome disassembly.

The human T-cell leukemia virus, type-1 (HTLV-1)-encoded Tax protein is required for high-level transcription of the virus. Tax function is strictly dependent upon the phosphorylated form of the cellular transcription factor CREB (pCREB), and together they bind novel cAMP response elements located within the viral promoter. The DNA-bound Tax/pCREB complex recruits the cellular coactivators CBP/p300, which are essential for viral gene expression. The coactivators, via their histone acetyltransferase activity, function to promote changes in chromatin architecture that are permissive to transcriptional activation. Tax expression in vivo recruits p300 to the HTLV-1 promoter and correlates with depletion of nucleosomes from the integrated provirus. We recently developed a novel in vitro, chromatin-based experimental system that recapitulates the eviction of nucleosomes from the HTLV-1 promoter observed in vivo. These assays establish the essential function of Tax/pCREB recruitment of CBP/p300, and concomitant histone acetylation, in the nucleosome disassembly process. These observations are of particular significance, as Tax mediates disassembly of the full nucleosome octamer independent of transcriptional activity and ATP utilization. Instead, nucleosome eviction is absolutely dependent upon acetyl CoA and the histone chaperone Nap1. In this review, we will discuss HTLV-1, Tax transactivation, and our recent findings that uncover the critical role of Tax in promoting chromatin transitions that accompany activation of viral transcription. We will describe the phenomenon of acetylation-dependent promoter nucleosome disassembly and the emerging view that the formation of nucleosome-free promoter regions may represent a general prerequisite for transcriptional activation in eukaryotes.

RGS13 acts as a nuclear repressor of CREB.

Cyclic AMP-induced phosphorylation of the transcription factor CREB elicits expression of genes mediating diverse biological functions. In lymphoid organs, the neurotransmitter norepinephrine stimulates beta(2)-adrenergic receptors on B lymphocytes to promote CREB-dependent expression of genes like the B cell Oct 2 coactivator (OCA-B). Although CREB phosphorylation recruits cofactors such as CBP/p300 to stimulate transcription, bona fide endogenous inhibitors of CREB-coactivator or CREB-DNA interactions have not emerged. Here, we identified RGS13, a member of the Regulator of G protein Signaling (RGS) protein family, as a nuclear factor that suppresses CREB-mediated gene expression. cAMP or Ca(2+) signaling promoted RGS13 accumulation in the nucleus, where it formed a complex with phosphorylated CREB and CBP/p300. RGS13 reduced the apparent affinity of pCREB for both the CRE and CBP. B lymphocytes from Rgs13(-/-) mice had more beta(2)-agonist-induced OCA-B expression. Thus, RGS13 inhibits CREB-dependent transcription of target genes through disruption of complexes formed at the promoter.

The proto-oncogene Bcl3, induced by Tax, represses Tax-mediated transcription via p300 displacement from the human T-cell leukemia virus type 1 promoter.

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-kappaB pathway. An intronic NF-kappaB binding site within the Bcl3 gene served as the primary target of Tax-induced NF-kappaB 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.

The coactivators CBP/p300 and the histone chaperone NAP1 promote transcription-independent nucleosome eviction at the HTLV-1 promoter.

The human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma. The multifunctional virally encoded oncoprotein Tax is responsible for malignant transformation and potent activation of HTLV-1 transcription. Tax, in complex with phosphorylated cAMP response element binding protein (pCREB), strongly recruits the cellular coactivators CREB binding protein (CBP)/p300 to the viral promoter concomitant with transcriptional activation. Although the mechanism of activator/coactivator-mediated transcriptional activation is poorly understood, the recruitment of CBP/p300 by regulatory factors appears to function, in part, by promoting changes in chromatin architecture that are permissive to transcriptional activation. Here, we show that CBP/p300 recruitment promotes histone acetylation and eviction of the histone octamer from the chromatin-assembled HTLV-1 promoter template in vitro. Nucleosome disassembly is strictly acetyl-CoA dependent and is not linked to ATP utilization. We find that the histone chaperone, nucleosome assembly protein 1 (NAP1), cooperates with CBP/p300 in eviction of the acetylated histones from the chromatin template. These findings reveal a unique mechanism in which the DNA-bound Tax/pCREB complex recruits CBP/p300, and together with NAP1, the coactivators cooperate to dramatically reduce nucleosome occupancy at the viral promoter in an acetylation-dependent and transcription-independent fashion.

The human T-cell leukemia virus type 1 tax protein confers CBP/p300 recruitment and transcriptional activation properties to phosphorylated CREB.

The human T-cell leukemia virus-encoded oncoprotein Tax is a potent activator of viral transcription. Tax function is strictly dependent upon the cellular transcription factor CREB, and together they bind cAMP response elements within the viral promoter and mediate high-level viral transcription. Signal-dependent CREB phosphorylation at Ser(133) (pCREB) correlates with the activation of transcription. This activation has been attributed to recruitment of the coactivators CBP/p300 via physical interaction with the KIX domain. Here we show that the promoter-bound Tax/pCREB complex strongly recruits the recombinant, purified full-length coactivators CBP and p300. Additionally, the promoter-bound Tax/pCREB (but not Tax/CREB) complex recruits native p300 and potently activates transcription from chromatin templates. Unexpectedly, pCREB alone failed to detectably recruit the full-length coactivators, despite strong binding to KIX. These observations are in marked contrast to those in published studies that have characterized the physical interaction between KIX and pCREB and extrapolated these results to the full-length proteins. Consistent with our observation that pCREB is deficient for binding of CBP/p300, pCREB alone failed to support transcriptional activation. These data reveal that phosphorylation of CREB is not sufficient for CBP/p300 recruitment and transcriptional activation. The regulation of transcription by pCREB is therefore more complex than is generally recognized, and coregulators, such as Tax, likely play a critical role in the modulation of pCREB function.

Molecular characterization of HTLV-1 Tax interaction with the KIX domain of CBP/p300.

The viral oncoprotein Tax mediates transcriptional activation of human T-cell leukemia virus type 1 (HTLV-1). Both Tax and the cellular transcription factor CREB bind to viral cyclic AMP response elements (vCREs) located in the viral promoter. Tax and serine 133 phosphorylated CREB (pCREB) bound to the HTLV-1 promoter facilitate viral transcription via the recruitment of the large cellular coactivators CBP/p300. While the interaction between the phosphorylated kinase inducible domain (pKID) of pCREB and the KIX domain of CBP/p300 has been well characterized, the molecular interactions between KIX, full-length Tax, and pCREB have not been examined. Here we biochemically characterized the interaction between Tax and KIX in a physiologically relevant complex containing pCREB and vCRE DNA. Our data show that Tax and pCREB simultaneously and independently bind two distinct surfaces on the KIX domain: Tax binds KIX at the previously characterized mixed-lineage leukemia (MLL) protein interaction surface while pCREB binds KIX at the pKID-KIX interface. These results provide evidence for a model in which Tax and pCREB bind distinct surfaces of KIX for effective CBP/p300 recruitment to the HTLV-1 promoter. We also show that MLL competes with Tax for KIX binding, suggesting a novel mechanism of Tax oncogenesis in which normal MLL function is disrupted by Tax.

Purification of CREB to apparent homogeneity: removal of truncation products and contaminating nucleic acid.

The cAMP response element binding protein (CREB) is a mammalian transcription factor which regulates the expression of many cellular genes. CREB is commonly expressed in Escherichia coli and purified by heat-extraction followed by affinity chromatography. We have discovered that although this purification yields a reasonably pure product which is active in DNA-binding and functional assays, it contains a large amount of nucleic acid as well as CREB truncation products and other polypeptides. Consequently, this CREB is inadequate for use in biophysical studies including crystallography, and spectroscopic analysis such as analytical ultracentrifugation, FRET, and circular dichroism. We revised the purification protocol to incorporate expression in the Rosetta host strain, nuclease treatment, and denaturing/high salt size-exclusion chromatography. We typically obtain 10mg of CREB per liter of culture media that is 99% homogenous, free of nucleic acid, and amenable to biophysical studies. Comparison of CREB from the original and revised protocols shows similar affinities for the cAMP response element (CRE) but small differences in their secondary structures when assayed by limited proteolysis and circular dichroism.

DNA binding and phosphorylation induce conformational alterations in the kinase-inducible domain of CREB. Implications for the mechanism of transcription function.

CREB-mediated activation of target gene transcription is stimulated by protein kinase A (PKA) phosphorylation at serine 133. This is followed by recruitment of the coactivators CREB-binding protein (CBP) or p300. Conversely, the decline in expression during the attenuation phase is linked to CREB dephosphorylation by nuclear phosphatases. The CREB bZIP domain, which promotes dimerization and promoter binding, as well as the kinase-inducible domain (KID), which interacts with the KIX domain of CBP/p300, are both largely unstructured in solution and become more structured once bound to their respective ligands. In this study, we biochemically characterize DNA- and phosphorylation-induced conformational alterations in CREB that may play a role in its transcriptionally poised, activated state. We find that sequence-specific DNA binding of pCREB renders the protein resistant to serine 133 dephosphorylation by protein phosphatase 1. Paradoxically, CREB bound to DNA and chromatin is efficiently phosphorylated by PKA, indicating that the KID region exists in a different conformation depending on its phosphorylation state. Consistent with this observation, we find that phosphorylation of DNA-bound CREB promotes an alternate conformation characterized by an apparent increase in the size or asymmetry of the complex and a qualitative change in proteolytic sensitivity. Together, our data indicate that DNA binding promotes a global conformational change in CREB that alters the structure of KID. PKA phosphorylation of KID in the DNA-bound state induces a phosphatase-resistant conformation that may prolong transcriptional activity.

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, Ser(133)-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.

Human T-cell leukemia virus type 1 (HTLV-1) bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription.

The complex human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes several proteins that are unique to the virus within its 3'-end region. Among them, the viral transactivator Tax and posttranscriptional regulator Rex are well characterized, and both positively regulate HTLV-1 viral expression. Less is known about the other regulatory proteins encoded in this region of the provirus, including the recently discovered HBZ protein. HBZ has been shown to negatively regulate basal and Tax-dependent HTLV-1 transcription through its ability to interact with specific basic-leucine zipper (bZIP) proteins. In the present study, we found that HBZ reduces HTLV-1 transcription and virion production. We then characterized the interaction between HBZ and the cellular transcription factor CREB. CREB plays a critical role in Tax-mediated HTLV-1 transcription by forming a complex with Tax that binds to viral cyclic AMP-response elements (CREs) located within the viral promoter. We found that HBZ and CREB interact in vivo and directly in vitro, and this interaction occurs through the bZIP domain of each protein. We also found that CREM-Ia and ATF-1, which share significant homology in their bZIP domains with the bZIP domain of CREB, interact with HBZ-bZIP. The interaction between CREB and HBZ prevents CREB binding to the viral CRE elements in vitro and in vivo, suggesting that the reduction in HTLV-1 transcription by HBZ is partly due to the loss of CREB at the promoter. We also found that HBZ displaces CREB from a cellular CRE, suggesting that HBZ may deregulate CREB-dependent cellular gene expression.

Tax abolishes histone H1 repression of p300 acetyltransferase activity at the human T-cell leukemia virus type 1 promoter.

Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter.

Tax-dependent displacement of nucleosomes during transcriptional activation of human T-cell leukemia virus type 1.

The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in "activating" histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion.

Paradoxical effects of DNA binding polyamides on HTLV-1 transcription.

Human T-cell leukemia virus type-1 (HTLV-1) depends on the virally encoded transcription factor Tax for efficient viral replication and gene expression. In a complex with CREB, Tax contacts the minor groove of the promoter DNA at guanine and cytosine rich sequences that flank three of the off-consensus cyclic-AMP response elements (CREs). In this study, we used six Tax-directed pyrrole-imidazole polyamides specifically designed to block Tax binding to DNA at each GC sequence of the three viral CREs. We found that four of these polyamides disrupt binding of the Tax/CREB complex in vitro, and that these same molecules also inhibit Tax-mediated transcription in vitro on chromatin-assembled templates. However, of these four Tax/CREB-specific polyamides, only one polyamide appears to be uniquely Tax specific. We show that polyamides can enter the nuclei of HTLV-1 infected T-cells, and two of the four polyamides down-regulated virion production in these cells. Together, these data illustrate the importance of studying polyamide inhibition of gene expression in vitro and in vivo, as the function of the polyamides in living cells is not fully understood. Finally, our data indicates that targeted disruption of the Tax/CREB complex, or other complexes which assemble on the HTLV-1 promoter, may provide a novel approach for inhibiting viral replication in vivo.