The Nyborg Laboratory currently has a number of research projects that focus on the function of the HTLV-1-encoded Tax protein and its role in transcriptional activation and leukemic transformation.
Tax is a potent transcriptional activator that stimulates HTLV-I viral gene expression, which leads to high-level virion production in the infected T-cell. Three 21 base pair repeat enhancer elements, located in the HTLV-I transcriptional control region, are critical to Tax-activated transcription. These elements, which are referred to as viral CREs, carry a central binding site for CREB (and other bZIP proteins), flanked by highly conserved GC-rich DNA sequences. Tax associates with the HTLV-I promoter through protein-protein interactions, and protein-DNA interactions with the minor groove GC-rich sequences. The formation of this Tax-containing promoter-bound complex appears critical in the recruitment of the multifunctional cellular coactivator CBP/p300. While extensively studied, the molecular mechanisms of Tax transcription function are not fully understood.
Projects
ChIP Analysis of Transcription Factor Binding at the Chromosomally Integrated HTLV-I Promoter
We are exploring the transcriptional regulation of the natural, chromosomally-integrated provirus in HTLV-I infected T-cells. These studies investigate the dynamic interaction between Tax, cellular transcription factors, coactivators, and corepressors, at the HTLV-I promoter both in vivo and in vitro. 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 histone modifications at the LTR. Unexpectedly, Tax transactivation leads to a reduction in nucleosomes at the LTR and associated coding region. Nucleosome depletion correlates with RNA polymerase II recruitment and SWI/SNF loss. The M47 Tax mutant, deficient in transcriptional activation, is defective for nucleosome depletion. Although this mutant forms complexes with CREB and p300 at the HTLV-1 promoter in vivo, it is unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results correlate RNA polymerase II recruitment with nucleosome depletion.
Transcriptional Activation by Tax in a Chromatin Context
Tax utilizes the cellular coactivator CBP to mediate its potent transcriptional activation of HTLV-I. The primary molecular event that leads to coactivator recruitment by Tax is the formation of a stable, promoter-bound complex containing Tax and the cellular transcription factor CREB. This complex serves as a high-affinity binding site for direct, DNA-dependent recruitment of the coactivator CBP to the HTLV-I promoter resulting in strong stimulation of transcription. Since CBP carries intrinsic histone acetylation activity, it is believed to activate transcription, at least in part, through histone modification. However, several additional activities have been attributed to CBP, including transcription factor modification as well as recruitment and modification of the general transcription machinery.
We are investigating the mechanisms of Tax-dependent, CBP/p300-mediated activation of HTLV-I transcription using fully recombinant chromatin-assembled transcription templates in vitro. This approach provides a tractable means to examine activator-coactivator interaction in the context of the physiologically relevant chromatin-assembled promoter templates. Recent studies in our laboratory have demonstrated the KIX domain is utilized, perhaps exclusive, in the recruitment of CBP/p300 to the HTLV-I promoter. This is accomplished via direct binding of KIX to the viral CRE-bound Tax/CREB complex. Once recruited, several studies have shown that CBP/p300 mediate Tax-transactivation via the acetyltransferase activity of the coactivators. Recent data in our laboratory demonstrates that the acetyltransferase activity is not directed at the histone amino terminal tails. Instead, we find that non-histone proteins, including Tax and CREB, are the direct acetylation targets of p300. Significantly, Tax/CREB acetylation by p300 is dramatically stimulated when both proteins are bound to the viral CRE DNA.
Biophysical Characterization of Tax
We are also interested in the biochemical characterization and of the Tax/CREB complex. We know that Tax makes protein-protein contacts with the cellular transcription factor CREB and protein-DNA contacts with the highly conserved GC-rich sequences in the viral CRE enhancer elements. We also know that this complex serves as a high affinity-binding site for interaction with KIX. Because of these recent advances, we are pursuing biophysical and structural studies in collaboration with Dr. Karolin Luger, an Associate Professor in the Department of Biochemistry and Molecular Biology.
Laboratory Publications (since 1995)
Gieger, T.R., Sharma, N., Kim, Y.-M., and Nyborg, J.K. The HTLV-1 Tax Protein Confers CBP/p300 Recruitment and Transcriptional Activation Properties to Phosphorylated CREB. Mol. Cell. Biol. in press (2007).
Ramírez, J.A. and Nyborg, J.K. Molecular Characterization of the HTLV-1 Tax Interaction with the KIX Domain of CBP/p300. J. Mol. Biol. 372, 958-969 (2007).
Lopez, D.I., Mick, J.E., and Nyborg, J.K. Purification of CREB to Apparent Homogeneity: Removal of Truncation Products and Contaminating Nucleic Acid. Protein Expression and Purification 55, 406-418 (2007).
Sharma, N., Lopez, D.I. and Nyborg, J.K. DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase Inducible Domain of CREB: Implications for the Mechanism of CREB Transcription Function. J. Biol. Chem. 282, 19872-19883 (2007).
Kim, Y.-M., Ramírez, J.A., Mick, J.E., Giebler, H.A., Yan, J.-P. and Nyborg, J.K. Molecular Characterization of the Tax-containing HTLV-1 Enhancer Complex Reveals a Prominent Role for CREB Phosphorylation in Tax Transactivation. J. Biol. Chem. 282, 18750-18757 (2007).
Lemasson, I., Lewis, M., Hivin, P., Cavanagh, M-H., Polakowski, N., Thébault, S., Barbeau, B., Nyborg, J.K. and Mesnard, J.M. HTLV-1 bZIP protein interacts with the cellular transcription factor CREB to inhibit HTLV-1 transcription. J. Virology 81, 1543-1553 (2007).
Konesky, K.L., Nyborg, J.K. and Laybourn, P.J. Tax Abolishes Histone H1 Repression of p300 Acetyltransferase Activity at the HTLV-I Promoter. J. Virology, 80, 10542-10553 (2006).
Lemasson, I., Polakowski, N., Laybourn, P.J. and Nyborg, J.K. Tax-Dependent Displacement of Nucleosomes During transcriptional Activation of Human T-cell Leukemia Virus Type 1. J. Biol. Chem. 281,13075-13082 (2006).
Nyborg, J.K. and Peersen, O.B. That Zincing Feeling: The Effects of EDTA on the Behavior of Zinc-Binding Transcriptional Regulators. Biochem. J. 381, e3-e4 (2004).
Lemasson, I., Polakowski, N.J., Laybourn, P.J., and Nyborg, J.K. Transcription Regulatory Complexes Bind the Human T-cell Leukemia Virus 5’ and 3’ Long Terminal Repeats to Control Gene Expression. Mol. Cell. Biol. 24, 6117-6126 (2004).
Livengood, J.A. and Nyborg, J.K. The High Affinity Sp1 Binding Site in the HTLV-1 Promoter Contributes to Tax-Independent Basal Expression. Nuc. Acids Res. 32, 2829-2837 (2004).
Livengood, J.A., Fechter, E.J., Dervan, P.E., and Nyborg, J.K. Paradoxical Effects of DNA Binding Polyamides on HTLV-I Transcription. Frontiers in Bioscience 9, 3058-3067 (2004).
McBryant, S.J., Park, Y.-J., Abernathy, S.M., Laybourn, P.J., Nyborg, J.K. and Luger, K. Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino terminal histone tails. J. Biol. Chem. 278, 44574-44583 (2003).
Georges, S.A., Giebler, H.A., Cole, P.A., Luger, K., Laybourn, P.J., and Nyborg, J.K. Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity that is Chromatin-Dependent and Histone Tail-Independent. Mol. Cell. Biol. 23, 3392-3404 (2003).
Lemasson, I, Polakowski, N.J., Laybourn, P.J. and Nyborg, J.K. Transcription Factor Binding and Histone Modifications on the Integrated Proviral Promoter in HTLV‑I‑Infected T‑Cells. J. Biol. Chem. 177, 49459-49465 (2002).
Livengood, J.A., Scoggin, K.E.S., Van Orden, K., Edayathumangalam, R. McBryant, S.J., Laybourn, P.J. and Nyborg. J.K. p53 Interaction with the SRC-Interacting Domain of CBP/p300 Mediates Coactivator-Dependent Transcriptional Activity. J. Biol. Chem. 277, 9054-9061 (2002).
Georges, S. Kraus, W.L., Luger, K. Nyborg, J.K., and Laybourn, P.J. p300-Mediated HTLV-I Tax Transactivation on Recombinant Chromatin Templates: Histone Tail Deletion Mimics Coactivator Function. Mol. Cell. Biol. 22, 127-137 (2002).
Scoggin, K. E. S., Ulloa, A. and Nyborg, J.K. The Oncoprotein Tax Binds the SRC-Interacting Domain of CBP/p300 to Mediate Transcriptional Activation. Mol. Cell. Biol. 16, 5520-5530 (2001).
Lemasson, I. and Nyborg, J.K. HTLV-I Tax Repression of p73b is Mediated Through Competition for the C/H1 domain of CBP. J. Biol. Chem. 276, 15720-15727 (2001).
Mick, J.E., Colgin, M.A. Brauweiler, A. and Nyborg, J. K. Analysis of CREB mutants in Tax complex formation and transactivation. AIDS Research and Human Retroviruses 16, 1597-1601 (2000).
Van Orden, K. and Nyborg, J.K. Insight into CBP function through the viral oncoprotein Tax. Gene Expression. 9, 29-36 (2000).
Giebler, H.A., Lemasson, I. and Nyborg, J.K. p53 recruitment of CBP mediated through phosphorylated CREB: A novel pathway for tumor suppressor regulation. Mol. Cell. Biol. 20, 4849-4858 (2000).
Van Orden, K., Giebler, H.A., Lemasson, I., Gonzales, M. and Nyborg, J.K. Binding of p53 to the KIX domain of CBP: A potential link to HTLV-I associated leukemogenesis. J. Biol. Chem. 274, 26321-26328 (1999).
Lenzmeier, B.A., Baird, E.E., Dervan, P.B. and Nyborg, J.K. The Tax Protein-DNA Interaction is Essential for HTLV-I Transactivation in vitro. J. Mol. Biol. 291, 731-744 (1999).
Van Orden, K., Yan, J.-P. Ulloa, A. and Nyborg, J.K. Binding of the Human T-Cell Leukemia Virus Tax Protein to the Coactivator CBP Interferes with CBP-mediated Transcriptional Control. Oncogene. 18, 3766-3772 (1999).
Van Orden, K. and Nyborg, J.K. Binding of HTLV-I Tax to the Coactivator CBP Disrupts the Transcription Activity of c-Jun, in Molecular Pathogenesis of HTLV-I: a current perspective. eds. O. John Semmes and Marie-Louise Hammarskjold, ABI Professional Publications, (USA) (1999).
Lenzmeier, B.A. and Nyborg, J.K. Molecular Mechanisms of Viral Transcription and Cellular Deregulation Associated with the HTLV-I Tax Protein. Gene Ther. Mol. Biol. 3, 327-345 (1999).
Colgin, M.A. and Nyborg, J.K. The HTLV-I Oncoprotein Tax Inhibits the Transcriptional Activity of c-Myb through Competition for CBP. J. Virol. 72, 9396-9399 (1998).
Yan, J.-P., Garrus, J.E., Giebler, H.A., Stargell, L.A. and Nyborg, J.K. Molecular Interactions Between the Human T-Cell Leukemia Virus Tax Protein and the Cellular Coactivator CBP. J. Mol. Biol. 281, 395-400 (1998).
Howard, R.D., McIllwraith, C.W., Trotter, G.W. and Nyborg, J.K. Cloning of equine interleukin 1a receptor antagonist and determination of its full-length cDNA sequence. Am. J. Vet. Res. 59, 712-716 (1998).
Howard, R.D., McIllwraith, C.W., Trotter, G.W. and Nyborg, J.K. Cloning of equine Interleukin-1a and equine Interleukin-1b and determination of their full-length cDNA sequence. Am. J. Vet. Res. 59, 704-711 (1998).
Brooks, P.A., Cockerell, G.L. and Nyborg. J.K. Activation of BLV Transcription by NF-kB and Tax. Virology 243, 94-98 (1998).
Lenzmeier, B.A., Giebler, H.A. and Nyborg, J.K. HTLV-I Tax Requires Direct Access to DNA for Recruitment of CBP. Mol. Cell. Biol. 18, 721-731 (1998).
Giebler, H.A., Loring, J., Van Orden, K., Colgin, M., Garrus, J., Escudero, K., Brauwieler, A. and Nyborg, J.K. Anchoring of CBP to the HTLV-I Promoter: A Molecular mechanism of Tax Transactivation. Mol. Cell. Biol. 17, 5156-5164 (1997).
Lenzmeier, B. and Nyborg, J.K. In vitro Transcription of Human T-cell Leukemia Virus Type I is RNA Polymerase II Dependent. J. Virology 71, 2577-2580 (1997).
Brauweiler, A., Garrus, J.E., Reed, J.C. and Nyborg, J.K. Repression of Bax Gene Expression by the HTLV-I Tax Protein: Implications for Suppression of Apoptosis in Virally-Infected Cells. Virology 231, 135-140 (1997).
Gottesfeld, J.M., Johnson, D.L. and Nyborg, J.K. Transcriptional Activation of RNA Polymerase III-Dependent Genes by the HTLV-I Tax Protein. Mol. Cell. Biol. 16, 1777-1785 (1996).
Uittenbogaard, M.N., Giebler, H.A., Reisman, D. and Nyborg, J.K. Transcriptional Repression of p53 by the Human T-Cell Leukemia Virus type I Tax Protein. J. Biol. Chem. 270, 28503-28506 (1995).
Brauweiler, A., Garl, P., Franklin, A.A., Giebler, H.A. and Nyborg, J.K. A Molecular Mechanism for HTLV-I Latency and Tax Transactivation. J. Biol. Chem. 270, 12814-12822 (1995).
Baranger, A.M., Palmer, C.R., Hamm, M.K., Giebler, H.A., Brauweiler, A., Nyborg, J.K. and Schepartz, A. Mechanism of DNA Binding Enhancement by the HTLV-I Transactivator Tax. Nature 376, 606-608 (1995).