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Investigation into the recruitment and functions of Paf1C

Ellison, Mitchell (2020) Investigation into the recruitment and functions of Paf1C. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

My work focuses on how transcription, the first step in gene expression, is affected by accessory factors and epigenetic modifications. Because gene expression defects are the cause of many diseases it is critical that we understand this fundamental process. Regulation of RNA polymerase II (Pol II) transcription requires accessory factors such as the Polymerase Associated Factor 1 complex or Paf1C. Paf1C facilitates a suite of co-transcriptional histone modifications, regulates transcription elongation efficiency, and performs many other functions. Paf1C must be tethered to the active Pol II elongation complex to function correctly. The Cdc73 and Rtf1 subunits of Paf1C are critical to this interaction. The Rtf1-Spt5 interaction is understood, but interactions governing Cdc73 attachment are unclear. Using an in vivo cross-linking strategy, interactions between the C-domain of Cdc73 and both Spt6, an essential elongation factor, and the Pol II C- terminal domain were identified. Cross-linking and mass spectrometry analysis data supported by mutational analysis, revealed that the C-domain of Cdc73 interacts with multiple domains of Spt6. In vitro protein binding assays support a direct interaction between recombinant Spt6 and Cdc73. Acute depletion of Spt6 followed by ChIP-seq demonstrates that genome-wide Paf1 occupancy requires Spt6, thus revealing an entirely new mechanism for Paf1C recruitment. Once properly recruited to Pol II, Paf1C regulates the transcriptome; however, its effects on non-coding RNAs (ncRNAs) and pervasive transcription are poorly understood. Using a genetic background that enriches for unstable transcripts, I demonstrated that deletion of PAF1 affects all classes of Pol II transcripts including antisense transcripts and other classes of ncRNAs. Further analysis revealed that H3K36me3 defects in a paf1∆ strain are likely causing much of the de-repression we observe for both coding and ncRNAs. Taken together these results identify Spt6 as a key Paf1C recruitment factor and demonstrate that Paf1C function is critical to maintain proper transcript levels.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Ellison, Mitchellmae92@pitt.edumae920000-0002-5682-096X
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairArndt, Karen M.arndt@pitt.eduarndt
Committee MemberLee, Miler T.miler@pitt.edumiler
Committee MemberBrodsky, Jeffery L.jbrodsky@pitt.edujbrodsky
Committee MemberVanDemark, Andrew P.andyv@pitt.eduandyv
Committee MemberPugh, B. Franklin
Date: 28 July 2020
Defense Date: 16 July 2020
Approval Date: 16 September 2020
Submission Date: 26 July 2020
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 332
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: No
Uncontrolled Keywords: Paf1C, Spt6, Cdc73, transcription, epigenetics
Date Deposited: 16 Sep 2020 13:45
Last Modified: 16 Sep 2020 13:45
URI: http://d-scholarship.pitt.edu/id/eprint/39471

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