The goal of this proposal is to understand how the HIV-1 Tat protein regulates transcription from the HIV-1 LTR. Tat interacts directly with Cyclin T1, the regulatory subunit of CDK9 in the cellular P-TEFb (positive transcription elongation factor) complex. Tat and CycT1 1 bind co-operatively to the viral TAR RNA to recruit P-TEFb to the HIV-1 promoter and facilitate CDK9-mediated phosphorylation of the C-terminal domain (CTD) of the RNAPII large subunit. We recently identified additional proteins that stimulate Tat activity: SKIP (ski-interacting protein), and the GU-alpha RNA helicase II enzyme, which associates with Tat in an RNA-dependent manner in extracts Both proteins enhance HIV-I Tat transcription in vitro and may function during RNAPII elongation. To integrate these and other elongation factors into the overall mechanism of Tat transactivation, we developed a Tat-inducible cell line containing an integrated HIV-1 LTR-eGFP reporter gene that can be used for chromatin immunoprecipitation experiments to examine the precise kinetics and pattern of recruitment of Tat and associated elongation factors to the HIV-1 LTR in vivo.
Five specific aims are proposed here: 1) Identify the protein composition and post-translational modifications of purified native Tat:P-TEFb:TAR complexes. 2) Examine the role of SKIP as a positive effector of HIV-1 transcription by identifying the functional domains required for transcription in vivo and in vitro and identifying cellular proteins that interact with SKIP to stimulate elongation. 3) Examine the role of GU-alpha and other RNA helicases on Tat activity in vivo and in vitro, and effects of RNA helicases on the disassembly of the Tat:P-TEFb:TAR complex during transcription. 4) Characterize additional factors and RNA processing enzymes that bind directly to Tat and/or CycT1. 5) Analyze the Tat-directed recruitment of cellular elongation factors, RNA processing factors, and chromatin remodeling activities at the integrated HIV- 1 LTR promoter by chromatin immunoprecipitation experiments. ? ?
| Chen, Yupeng; Zhang, Lirong; Estarás, Conchi et al. (2014) A gene-specific role for the Ssu72 RNAPII CTD phosphatase in HIV-1 Tat transactivation. Genes Dev 28:2261-75 |
| Brès, Vanessa; Yoshida, Tomonori; Pickle, Loni et al. (2009) SKIP interacts with c-Myc and Menin to promote HIV-1 Tat transactivation. Mol Cell 36:75-87 |
| Yoh, Sunnie M; Lucas, Joseph S; Jones, Katherine A (2008) The Iws1:Spt6:CTD complex controls cotranscriptional mRNA biosynthesis and HYPB/Setd2-mediated histone H3K36 methylation. Genes Dev 22:3422-34 |
| Bres, Vanessa; Yoh, Sunnie M; Jones, Katherine A (2008) The multi-tasking P-TEFb complex. Curr Opin Cell Biol 20:334-40 |
| Yoh, Sunnie M; Cho, Helen; Pickle, Loni et al. (2007) The Spt6 SH2 domain binds Ser2-P RNAPII to direct Iws1-dependent mRNA splicing and export. Genes Dev 21:160-74 |
| Garber, M E; Jones, K A (1999) HIV-1 Tat: coping with negative elongation factors. Curr Opin Immunol 11:460-5 |
| Garber, M E; Wei, P; Jones, K A (1998) HIV-1 Tat interacts with cyclin T1 to direct the P-TEFb CTD kinase complex to TAR RNA. Cold Spring Harb Symp Quant Biol 63:371-80 |
