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Browsing by Author "Nyborg, Jennifer K., advisor"

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    Integrating p300 functions in HTLV-1 transcription initiation
    (Colorado State University. Libraries, 2014) Luebben, Whitney R., author; Nyborg, Jennifer K., advisor; Stargell, Laurie, committee member; Laybourn, Paul, committee member; Prenni, Jessica, committee member; Quackenbush, Sandra, committee member
    The HTLV-1 provirus overcomes a repressive chromatin environment for efficient transcription of its genome. This is accomplished by the robust recruitment of the coactivator protein, p300, to the viral enhancer sites through interactions with DNA bound pCREB and the viral transactivating protein, Tax. Recruitment of p300 to the HTLV-1 promoter results in histone acetylation and nucleosome depletion from the promoter region in the presence of the histone chaperone, Nap1. To study the histone acetylation requirements for Nap1-dependent nucleosome disassembly, we utilized immobilized in vitro assembled chromatin templates containing site specific K→R mutations within the N-terminal tails of the histones. Through these studies, we identified histone H3, lysine 14 as the functionally relevant acetylation site for Nap1-dependent nucleosome disassembly. Additionally, we found a significant correlation between nucleosome disassembly from the HTLV-1 promoter and acetylation-dependent transcription activation. These studies suggest that nucleosome disassembly is a prerequisite for transcription activation, as nucleosome disassembly creates a nucleosome free region within the HTLV-1 promoter, allowing for the subsequent recruitment of Pol II and general transcription machinery for activation of transcription. The identification of a single and specific acetyl-lysine residue led us to the hypothesis that the p300 acetyl-lysine binding domain (bromodomain) was involved in HTLV-1 transcription activation through recognition of H3K14ac. To test this hypothesis, we utilized a p300 bromodomain deletion mutant and a CBP/p300 specific bromodomain inhibitor, (SGC-CBP30), to investigate the involvement of the p300 bromodomain in HTLV-1 transcription activation. Importantly, we found that the p300 bromodomain is not involved in the initial recruitment of the coactivator to the chromatin template as previously proposed, rather the bromodomain functions after recruitment to the promoter and following acetylation of the histone tails. These findings are consistent with a role for the p300 bromodomain in nucleosome disassembly and uncover a novel function for the bromodomain in gene activation.
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    Mutational analysis of the human histone chaperone, Nap1, in nucleosome disassembly at the HTLV-1 promoter
    (Colorado State University. Libraries, 2010) Kolean, Jennica Laura, author; Nyborg, Jennifer K., advisor; Stargell, Laurie A., committee member; Bailey, Susan M., committee member
    The human genome is packaged to fit within the confines of the nucleus through the interaction with four core histone proteins, H2A, H2B, H3 and H4. These proteins organize the genetic material, however they also make it difficult for the cells to access the information stored within the DNA sequence for processes such as transcription and replication. One of the mechanisms by which the genetic information can be accessed is post-translational modifications of the histone tails. Post-translational modifications, such as acetylation, act to neutralize charges on the histone tails and also serve to create new binding sites for other proteins. These modifications have been associated with decompaction of condensed chromatin, alteration of nucleosome structure, and partial or complete disassembly of the histone octamer. Our laboratory uses human T-cell leukemia virus, type 1 (HTLV-1) as a model for studying eukaryotic transcription activation and gene regulation. Previous studies using chromatin immunoprecipitation to look at the HTLV-1 promoter have correlated transcription activation with a decrease in post-translational modifications that are traditionally associated with gene activation. This decrease in activating modifications was due to a decrease in histone occupancy at the promoter in vivo. To recapitulate the results observed in vivo, we developed an immobilized template assay using the biotin labeled HTLV-1 promoter fragment bound to a magnetic streptavidin coupled bead. Nucleosome disassembly at the HTLV-1 promoter is dependent on the presence of the virally encoded Tax protein, as well as the phosphorylated form of the cellular protein cyclic-AMP response element binding protein (pCREB), cellular coactivators CREB binding protein (CBP)/p300, acetyl coenzyme A (acetyl CoA), acceptor DNA and nucleosome assembly protein (Nap1). Tax and pCREB recruit the histone acetyltransferase, CBP/p300, which acetylates histone tails prior to disassembly of the octamer. Nap1 is unique in this reaction because this was the first example of a histone chaperone supporting disassembly of the entire octamer in an acetyl CoA dependent fashion, independent of ATP consumption or the presence of chromatin remodeling complexes. In this study we examined the domains of Nap1 required for in vitro nucleosome disassembly at the HTLV-1 promoter template through a series of rationally designed deletion mutants. Crystal structures of yeast Nap1 and SET/TAF-Iβ; were used as models for designing mutations in the human Nap1 protein. Our results show that the minimal domain of human Nap1 able to support nucleosome disassembly is contained within amino acid residues 196-290. Using histone binding assays, we also found that the ability to disassemble nucleosomes is independent of histone interaction in vitro. Removal of the β-hairpin that is required for Nap1 oligomerization renders the protein unable to support disassembly. This suggests that the oligomeric form of Nap1 is required for nucleosome disassembly at the HTLV-1 promoter.
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    Tax deregulation of host-cell proteins
    (Colorado State University. Libraries, 2008) Kim, Young-Mi, author; Nyborg, Jennifer K., advisor
    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus etiologically linked to an aggressive and generally fatal malignancy called adult T-cell leukemia (ATL) and to a chronic inflammatory neurological disease. Only a small percentage of infected individuals develop ATL following a prolonged latency period of up to 30 years post infection. The dominant mechanism of virus transmission in an infected individual is through clonal expansion of HTLV-1 infected cells. The HTLV-1-encoded protein Tax is the prominent player in promoting mitotic replication. Tax is also directly linked to malignant transformation and the etiology of ATL. Tax is a potent transcriptional activator that stimulates HTLV-1 viral gene expression. Three 21 base pair repeat enhancer elements called viral cyclic AMP response elements (vCREs), located in the HTLV-1 transcriptional control region, are critical to Tax-activated transcription. Tax associates with the vCREs through protein-DNA interactions and through protein-protein interaction with the cellular transcription factor cAMP response element binding (CREB) protein. Together this complex recruits the cellular coactivators CBP/p300. The role of Ser133 phosphorylated CREB in mediating Tax function in HTLV-1 transcription has long been controversial. Our data reveal that CREB phosphorylation is absolutely required for viral Tax transactivation. Consistent with this, Tax induces constitutively elevated levels of phosphorylated CREB in vivo and in vitro. We further investigated the mechanism of Tax-mediated CREB phosphorylation and uncovered a novel function of Tax: stimulation of CREB phosphorylation via the Ca2+/Calmodulin (CaM)-dependent protein kinase (CaM kinase) pathway to promote viral transcription. In addition to Tax-dependent CREB phosphorylation, we found that Tax upregulates B-cell lymphatic leukemia protein 3 (Bcl-3) and cyclin D1 expression, two key determinants of cell fate. Furthermore, Tax interacts with Bcl-3 in vivo and in vitro. Deregulation of these key host-cell proteins by Tax may contribute to the transformation of T-cells.