The human transcription elongation factor P-TEFb, consisting of a CDK9/cyclin T heterodimer, promotes both general and HIV-specific elongation by phosphorylating RNA polymerase II and overcoming pausing by negative elongation factors. It is also a co-factor for the HIV-1 Tat protein, whose recruitment of P-TEFb to the nascent viral mRNA is essential for HIV replication. The PI's laboratory has recently identified the 7SK snRNA and hexamethylene bisacetamide (HMBA)-inducible protein 1 (HEXlM1) as two P-TEFb-associated nuclear factors. Their interactions with P-TEFb require the phosphorylation of P-TEFb on possibly the conserved CDK9 T-loop. P-TEFb loses its kinase and transcriptional activities when associated with 7SK and HEXlM1 to form the 7SK snRNP. With 7SK bridging the HEXlM1:P-TEFb interaction, HEXIM1 inhibits the P-TEFb kinase. Both 7SK and HEXIM1 are dissociated from P-TEFb when cells respond to stresses caused by a global inhibition of transcription. Consistent with its inhibition of PTEFb, HEXlM1 strongly suppresses HIV-1 transcription in vivo. Our data support a model in which the phosphorylated P-TEFb is targeted for inhibition by the coordinated actions of HEXlM1 and 7SK. This proposal seeks to elucidate the mechanism by which HEXlM1/7SK inhibit the P-TEFb kinase through structure-function analyses of the 7SK snRNP. Experiments will also be performed to investigate whether 7SK plays a direct, active role in the inhibitory action beyond its mediation of the HEXlM1:P-TEFb binding. In addition, the effect of reducing HEXlM1 expression by RNAi on CTD phosphorylation and HIV-1 transcription will be analyzed. Because HEXlM1 is induced by HMBA, a potent inducer of cell differentiation, the roles of HEXlM1 and P-TEFb in controlling this key process will be examined. The preliminary data so far implicate a role of protein phosphatase-1 (PP-1) in activating HIV-1 transcription and dissociating 7SK/HEXIM1 from P-TEFb in vitro. A major effort will be devoted to the investigation of a possible role of PP-1 in regulating the stress-induced disruption of the 7SK snRNP and activation of P-TEFb in vivo. Our ultimate goal is to elucidate the molecular mechanisms controlling eukaryotic gene expression at the stage of elongation. Investigation of how the activity of P-TEFb, a key elongation factor and Tat co-factor, is regulated by HEXlM1/7SK and how this regulation affects HIV-1 transcription and cellular gene expression will be very informative in this regard.
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