Enzymatic Disassembly of Promoter Bound 7SK snRNP Drives Transcription Elongation of HIV and Cellular Genes
McNamara, Ryan Philip
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Gene expression of the human immunodeficiency virus (HIV) and cellular primary responsive genes (PRG’s) is regulated at the step of transcription elongation. Shortly after transcription initiation, RNA Polymerase II (Pol II) pauses and it only enters into productive elongation after inducible transcription factors (TF’s) recruit the P-TEFb kinase to phosphorylate Pol II in response to stimuli. To ensure tight regulation of this process, the majority of P-TEFb is held in a catalytically inactive form, reversibly bound to the 7SK small nuclear ribonucleoprotein (snRNP). In the absence of stimuli, the 7SK snRNP resides in both the nucleoplasm and promoter regions. However, an understanding of how TF’s capture P-TEFb from the 7SK snRNP at the promoter and the mechanism and purpose of localizing the 7SK snRNP to promoters has been largely unexplored. It was therefore my goal to biochemically and functionally characterize this pathway through the use of both the HIV encoded TF Tat and cellular TF’s such as nuclear factor kappa b (NF-κB). Detailed throughout this dissertation, I present the novel findings that HIV Tat and NF-κB function to recruit the PPM1G phosphatase to their targeted promoters, which dephosphorylates P-TEFb and triggers its release from the 7SK snRNP. Additionally, this extraction of P-TEFb from the 7SK snRNP occurs at promoters through the transcriptional regulator KAP1, which physically tethers the snRNP to promoters genome wide. Recruitment of the 7SK snRNP complex occurs after transcription initiation, allowing P-TEFb to be directly positioned for rapid extraction by TF’s upon stimuli and transferred onto the paused Pol II. The enzymatic uncoupling of P-TEFb from the promoter bound 7SK snRNP enables rapid Pol II elongation and gene expression in response to stimuli (for PRG’s) or in the presence of Tat (for HIV). Ultimately, these findings indicate that inducible transcription programs can rapidly respond to environmental cues through the localized positioning of elongation factors at promoters. Moreover, these findings illustrate that HIV has evolved to hijack a cellular gene expression program, thus leading to viral takeover of the host and progression of AIDS.