Katherine A. Jones
e-mail: jones@salk.edu |
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We are interested in understanding the molecular events that lead to induction of HIV-1 transcription from integrated provirus in activated T cells. Overall, HIV-1 transcription is tightly regulated by cellular enhancer-binding proteins, which determine the level of RNA initiation, and by the virus-encoded transcription factor, Tat, which controls the efficiency of elongation through its recognition of TAR, an RNA element located in the leader region of all viral transcripts. Transcription of the integrated HIV-1 provirus is also regulated by a dynamic chromatin structure that changes upon activation of T cells, coincident with the induction of viral transcription. Our goal is to identify the cellular transcription factors that establish the chromatin structure observed for integrated provirus in vivo and to examine the role of individual transcription factors in establishing the local chromatin structure at the HIV-1 promoter and controlling viral transcription in a chromatin environment.
Fine-structure chromatin mapping studies carried out by Dr. E. Verdin have previously localized five translationally-positioned nucleosomes in the integrated HIV-1 promoter region, one of which (called nuc-1) directly overlaps the RNA initiation site as well as TAR, the region that mediates trans-activation by Tat. Interestingly, activation of T cells leads to a specific rearrangement or repositioning of the nuc-1 nucleosome, a process that is thought to be controlled by histone acetylation. These positioned nucleosomes define two short nucleosome-free zones, through which most of the cellular regulatory proteins must function to control viral transcription. We have previously demonstrated that the upstream nucleosome-free zone is recognized by the lymphoid-specific proteins, LEF-1 and ETS-1, which function synergistically with NF-kB proteins in the enhancer. Purification and sequencing of the LEF-1 protein revealed that it is a sequence-specific HMG protein, and we demonstrated that it activates transcription from the HIV-1 enhancer and from the alpha-chain of the T cell receptor (TCRa enhancer) in a highly context-sensitive manner. In collaboration with Dr. J. Kadonaga (UCSD) and his colleagues, we recently demonstrated that LEF-1 and ETS-1 are strong activators of viral transcription with chromatin templates, but are unable to activate transcription from naked DNA. We further demonstrated that the NF-kB protein acts together with Sp1 in the promoter to induce hypersentive site formation and is sufficient to position the regulatory nuc-1 nucleosome in the promoter. These findings provide support for previous studies that suggested that the distal region of the HIV-1 enhancer is more important for expression of the integrated HIV-1 provirus than it is for viral enhancer activity in non-chromatin environments. Efforts are currently underway to map the domains of LEF-1 that are critical for activation of HIV-1 transcription on chromatin templates, and to define the mechanism of activation by LEF-1 and ETS-1.
Inspection of the structure of integrated provirus suggests that the regulatory region immediately downstream of the promoter may be more extensive than previously thought. A strong region of DNase I hypersensitivity is located approximately 200 bp downstream of the RNA start site. We are currently analyzing the proteins that bind to this downstream region and plan to examine their effects on chromatin structure and transcriptional activation in vitro. In addition, we are studying in the proteins that mediate transcriptional activation by the HIV-1 Tat protein, including factors that bind specifically to the TAR RNA structures of HIV-1 and HIV-2. Finally, we plan to extend this system by examining the effects of chromatin on transcriptional elongation and Tat activation in vitro. These further studies should help to clarify the important effects of chromatin on the regulation of HIV-1 transcription in vivo.
Pazin, M.J., Sheridan, P.L., Cannon, K., Cao, Z., Keck, J.G., Kadonaga, J.T. and Jones, K.A. (1996). NF-kB-mediated chromatin reconfiguration and transcriptional activation of the HIV-1 enhancer in vitro. Genes & Dev. 10: 37-49.
Sheridan, P.L., Sheline, C.T., Cannon, K., Voz, M.L., Pazin, M.J., Kadonaga, J.T. and Jones, K.A. (1995). Activation of the HIV-1 enhancer by the LEF-1 HMG protein on nucleosome-assembled DNA in vitro. Genes & Dev. 9: 2090-2104.
Jones, K.A. and Peterlin, B.M. (1994). Control of RNA initiation and elongation at the HIV-1 promoter. Annu. Rev. Biochem. 63: 717-743.
Carlsson, P., Waterman, M.L. and
Jones, K.A. (1993). The thymus-enriched HMG protein, hLEF, contains
a context-dependent activation domain required to activate the TCRa
enhancer in T cells. Genes & Dev.7: 2418-2430.
Katherine Jones received her Ph.D. in Biochemistry from UC Riverside and was a postdoctoral fellow at UC Berkeley. She is the recipient of a PEW Scholarship in the Biomedical Sciences. Dr. Jones is an NIH ARR-C Study Section Member and a Review Panel Member of the Howard Hughes Medical Institute for research training fellowships.
