Paul J. Laybourn

Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA

H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A–H2B dimer to the (H3–H4)2 tetramer; in addition, the C‐terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd‐NCP), and found that Bbd‐NCP had a more relaxed structure in which only 118±2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd‐NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain‐swap experiments. Bbd‐containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd‐NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.

Preferential Binding of the Histone (H3-H4) 2 Tetramer by NAP1 Is Mediated by the Amino-terminal Histone Tails*

The yeast nucleosome assembly protein 1 (yNAP1) participates in many diverse activities, such as the assembly of newly synthesized DNA into chromatin and the rearrangement of nucleosomes during transcriptional activation. yNAP1 does not require ATP hydrolysis to perform these functions and is a valuable tool for in vitro chromatin assembly. Using recombinant histone complexes, we show that yNAP1 has a preference for binding the (H3-H4)2 tetramer over the (H2A-H2B) dimer. We find that the loss of the histone tails abrogates this preference for H3 and H4, and we demonstrate a direct interaction between yNAP1 and the amino-terminal tails of H3 and H4. yNAP1 binds to one histone fold domain, thus specifying the stoichiometry of the complexes formed with the histone dimer and tetramer. Finally, we provide evidence that the acidic carboxyl-terminal region of yNAP1, although dispensable for nucleosome assembly in vitro, contributes to binding via structure-independent electrostatic interactions. Our results are consistent with recent mechanistic investigations of NAP1 and expand our understanding of the histone chaperone family of assembly factors.

Transcription Factor Binding and Histone Modifications on the Integrated Proviral Promoter in Human T-cell Leukemia Virus-I-infected T-cells

The human T-cell leukemia virus (HTLV-I)-encoded Tax protein is a potent transcriptional activator that stimulates expression of the integrated provirus. Biochemical studies indicate that Tax, together with cellular transcription factors, interacts with viral cAMP-response element enhancer elements to recruit the pleiotropic coactivators CREB-binding protein and p300. Histone acetylation by these coactivators has been shown to play a major role in activating HTLV-I transcription from chromatin templates in vitro. However, the extent of histone modification and the precise identity of the cellular regulatory proteins bound at the HTLV-I promoter in vivo is not known. Chromatin immunoprecipitation analysis was used to investigate factor binding and histone modification at the integrated HTLV-I provirus in infected T-cells (SLB-1). These studies reveal the presence of Tax, a variety of ATF/CREB and AP-1 family members (CREB, CREB-2, ATF-1, ATF-2, c-Fos, and c-Jun), and both p300 and CREB-binding protein at the HTLV-I promoter. Consistent with the binding of these coactivators, we observed histone H3 and H4 acetylation at three regions within the proviral genome. Histone deacetylases were also present at the viral promoter and, following their inhibition, we observe an increase in histone H4 acetylation on the HTLV-I promoter and a concomitant increase in viral RNA. Together, these results suggest that a variety of transcriptional activators, coactivators, and histone deacetylases participate in the regulation of HTLV-I transcription in infected T-cells.

p300-Mediated Tax Transactivation from Recombinant Chromatin: Histone Tail Deletion Mimics Coactivator Function

ABSTRACT Efficient transcription of the human T-cell leukemia virus type 1 (HTLV-1) genome requires Tax, a virally encoded oncogenic transcription factor, in complex with the cellular transcription factor CREB and the coactivators p300/CBP. To examine Tax transactivation in vitro, we used a chromatin assembly system that included recombinant core histones. The addition of Tax, CREB, and p300 to the HTLV-1 promoter assembled into chromatin activated transcription several hundredfold. Chromatin templates selectively lacking amino-terminal histone tails demonstrated enhanced transcriptional activation by Tax and CREB, with significantly reduced dependence on p300 and acetyl coenzyme A (acetyl-CoA). Interestingly, Tax/CREB activation from the tailless chromatin templates retained a substantial requirement for acetyl-CoA, indicating a role for acetyl-CoA beyond histone acetylation. These data indicate that during Tax transcriptional activation, the amino-terminal histone tails are the major targets of p300 and that tail deletion and acetylation are functionally equivalent.

Transcription Regulatory Complexes Bind the Human T-Cell Leukemia Virus 5′ and 3′ Long Terminal Repeats To Control Gene Expression

ABSTRACT The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that integrates randomly into the T-cell genome. Two long terminal repeats (LTRs) flank the integrated provirus. The upstream and downstream LTRs carry identical promoter sequences. Studies with other retroviruses suggest that the downstream promoter is silent and that RNA polymerases initiating at the upstream promoter proceed through the 3′ LTR. In this study, we used the chromatin immunoprecipitation assay to compare the binding of transcription regulatory proteins at both the upstream and downstream promoters in HTLV-1-infected cell lines and adult T-cell leukemia-lymphoma cells. Unexpectedly, we detected a nearly equal distribution of activator (Tax, CREB, ATF-1, ATF-2, c-Fos, and c-Jun) and regulatory protein (CBP, p300, TAFII250, and polymerase II) binding at both the upstream and downstream promoters. Consistent with this observation, we found that the downstream promoter was transcriptionally active, suggesting that the two promoters are functionally equivalent. We also detected asymmetrical binding of histone deacetylases (HDAC-1, -2, and -3) at both promoters. All three HDACs strongly repressed Tax transactivation, and this repression correlated with displacement of Tax from the HTLV-1 promoter. These effects were reciprocal, as Tax expression reversed HDAC repression and displaced HDACs from the HTLV-1 promoter. These data suggest that HTLV-1 transcriptional regulation at both the 5′ and 3′ LTRs is mediated, in part, through the mutually exclusive binding of Tax and HDACs at the proviral promoters.

Induction of the rat prodynorphin gene through Gs-coupled receptors may involve phosphorylation-dependent derepression and activation.

Prodynorphin transcription is activated via Gs-coupled receptors through a cyclic AMP (cAMP)-dependent pathway. Four cAMP response elements (CREs) are present within the rat prodynorphin (RD) control region, and all four CREs appear to function in RD regulation. Three CREs located upstream between -1860 and -1504 are critical for receptor-responsive activity, but their function is distance dependent unless they act together with a fourth CRE found in exon 1. Regulation of RD also appears to involve multiple CRE-binding proteins. Both CRE-binding protein (CREB) and activator protein 1 (AP-1) can regulate RD, but their effects are in opposite directions; CREB represses and AP-1 activates RD. CREB-induced repression and AP-1 activation require distinct elements within the control region, but their binding and functions overlap at CRE-3. While CREB repression is dependent on CRE-3, AP-1 activation (and cAMP induction) of RD requires additional CREs (CRE-1, -2, and -4). CREB repression blocks AP-1 activation in unstimulated cells. However, phosphorylation relieves CREB-induced repression and enhances AP-1 activation. Gs-coupled receptor activation of RD may require phosphorylation-dependent derepression and activation steps.

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.

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.

RNA polymerase II and TBP occupy the repressed CYC1 promoter

Saccharomyces cerevisiae CYC1 gene expression has been studied in great detail with regard to the response to oxygen availability and carbon source. In the absence of oxygen and the presence of glucose, the CYC1 gene is completely repressed. Chromatin structure is thought to play an important role in CYC1 gene regulation, as nucleosome depletion results in 94‐fold derepression. In addition, the CYC1 core promoter has been used extensively in hybrid constructs to study activation by heterologous transcription factors. Therefore, we set out to map the chromatin structure of the CYC1 promoter and determine its role in CYC1 gene regulation. We report here that the repressed CYC1 promoter contains no positioned nucleosomes over the core promoter. However, we did find TFIID and RNA polymerase II bound in a complex on the repressed promoter. These results indicate that recruitment of TFIID and RNA polymerase II are not rate‐limiting steps in CYC1 activation.

Human T Lymphotropic Virus Type 1 protein Tax reduces histone levels.

BackgroundHuman T-Lymphotropic Virus Type-1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virally encoded Tax protein is thought to be necessary and sufficient for T-cell leukemogenesis. Tax promotes inappropriate cellular proliferation, represses multiple DNA repair mechanisms, deregulates cell cycle checkpoints, and induces genomic instability. All of these Tax effects are thought to cooperate in the development of ATLL.ResultsIn this study, we demonstrate that histone protein levels are reduced in HTLV-1 infected T-cell lines (HuT102, SLB-1 and C81) relative to uninfected T-cell lines (CEM, Jurkat and Molt4), while the relative amount of DNA per haploid complement is unaffected. In addition, we show that replication-dependent core and linker histone transcript levels are reduced in HTLV-1 infected T-cell lines. Furthermore, we show that Tax expression in Jurkat cells is sufficient for reduction of replication-dependent histone transcript levels.ConclusionThese results demonstrate that Tax disrupts the proper regulation of replication-dependent histone gene expression. Further, our findings suggest that HTLV-1 infection uncouples replication-dependent histone gene expression and DNA replication, allowing the depletion of histone proteins with cell division. Histone proteins are involved in the regulation of all metabolic processes involving DNA including transcription, replication, repair and recombination. This study provides a previously unidentified mechanism by which Tax may directly induce chromosomal instability and deregulate gene expression through reduced histone levels.