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Investigating the role of the DNA entry-exit site of the nucleosome in transcription termination

Hildreth, Ashley Elizabeth (2019) Investigating the role of the DNA entry-exit site of the nucleosome in transcription termination. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Normal cellular function relies on the precise regulation of gene expression. In eukaryotes, DNA is packaged as chromatin, which acts as a barrier between the transcription machinery and genomic material. Chromatin consists of repeating nucleosomes, which contain approximately 147 base pairs of DNA surrounding an octamer of histone proteins H2A, H2B, H3, and H4. Transcription is controlled by factors that remove or modify nucleosomes, allowing RNA polymerase II to contact otherwise occluded DNA. The mechanisms by which nucleosome organization is controlled are well understood in regard to transcription initiation and elongation. Despite a few studies showing that transcription-coupled histone modifications and select chromatin remodelers are important for proper termination, little else is known about the role of chromatin at this step. Therefore, the goal of my dissertation work was to address how nucleosomes contribute to control of transcription termination. I conducted a genetic screen in Saccharomyces cerevisiae for histone mutations that cause termination defects. Interestingly, I found that many histone residues required for termination reside in or near the DNA entry-exit site of the nucleosome. The DNA entry-exit site is critical for nucleosome occupancy, a well-studied transcription-coupled histone modification, and prevention of several other transcription-related phenotypes that I have tested. Genome-wide analysis in mutants reveals altered nucleosome occupancy and transcriptional output. To test the hypothesis that stable nucleosomes within termination regions are required to act as physical roadblocks to the polymerase, I integrated a “superbinder” DNA sequence to position a stable nucleosome at a candidate locus. Nucleosome occupancy increased substantially at the targeted location and suppressed termination read-through of the locus to the level of a wildtype control. Together, these data implicate the DNA entry-exit site as an important player in maintenance of chromatin organization that supports proper transcription, including termination. Further investigation is underway to determine whether the function of the DNA entry-exit site in transcription termination depends on interaction with other chromatin-related factors. These studies will open up exciting new avenues for pursuit by future researchers in the Arndt lab and will help the field to better understand how the critical final step of transcription, termination, is regulated.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Hildreth, Ashley Elizabethaeh77@pitt.eduaeh770000-0003-1182-6759
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairArndt, Karen M.arndt@pitt.eduarndt0000-0003-1320-9957
Committee MemberChapman, Deborah L.dlc7@pitt.edudlc7
Committee MemberBerman, Andrea J.ajb190@pitt.eduajb190
Committee MemberBernstein, Kara A.karab@pitt.edukarab
Committee MemberSchwacha, Anthonyschwacha@pitt.eduschwacha
Date: 25 September 2019
Date Type: Publication
Defense Date: 25 June 2019
Approval Date: 25 September 2019
Submission Date: 9 July 2019
Access Restriction: 3 year -- Restrict access to University of Pittsburgh for a period of 3 years.
Number of Pages: 227
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: Yes
Uncontrolled Keywords: chromatin DNA entry-exit nucleosome Pol II Saccharomyces cerevisiae transcription
Date Deposited: 25 Sep 2019 14:32
Last Modified: 25 Sep 2019 14:32
URI: http://d-scholarship.pitt.edu/id/eprint/37074

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