Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
David H. Price
In humans, RNA polymerase II is the sole source of messenger RNAs that are ultimately translated into proteins and its transcriptional activity is highly regulated.
Mechanisms have evolved to control which, when, and to what degree genes are transcribed. Because most cells have the same genome, control of transcription is essential in maintaining cellular identity. Misregulation of Pol II transcription is a hallmark of both cancer and retroviral infection. This research investigates the regulation of Pol II transcription and related co-transcriptional mRNA capping.
Chromatin immunoprecipitation experiments were used to characterize the composition of nucleosomes and Pol II, DSIF and NELF occupancies at bidirectional promoters and enhancers. In collaboration with Alberto Bosque and Vicente Planelles, sequencing experiments were performed in a primary T cell model of HIV latency and a role for sequence-specific recruitment of STAT5 was established in HIV reactivation. In contrast, analysis of Myc binding in vitro and in cells demonstrated that transcription machinery played a major role in recruiting Myc to genomic sites. A precise method was also developed to detect polymerase-associated nascent transcripts in nuclei.
The roles of Cdk7, a subunit of TFIIH that phosphorylates Pol II during initiation, were characterized by treatment of nuclear extracts and cells with THZ1, a recently developed covalent inhibitor with anti-cancer properties. Inhibition of Cdk7 was demonstrated to cause defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. Capping of nascent RNAs was found to be spatially and temporally regulated in part by a previously undescribed THZ1-sensitive factor present in nuclear extract. THZ1 impacted pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to misloading of DSIF.
In vitro and sequencing methods were used to describe an extremely rapid and global transcriptional response to hydrogen peroxide. During periods of oxidative stress, termination was likely inhibited and Pol II accumulated at promoters and enhancers after as few as two minutes, and clearance of these polymerases required P-TEFb. In the presence of flavopiridol, a potent P-TEFb inhibitor, non-productive elongation was observed and a potential role for P-TEFb in termination was proposed.
ChIP-Seq, mRNA Capping, Pol II, P-TEFb, THZ1
xi, 160 pages
Includes bibliographical references (pages 135-160).
Copyright © 2016 Kyle Andrew Nilson