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EXAMINING THE CONTRIBUTION OF CHROMATIN AND CHROMATIN ASSOCIATED FACTORS TO TRANSCRIPTION REGULATION

Hainer, Sarah (2012) EXAMINING THE CONTRIBUTION OF CHROMATIN AND CHROMATIN ASSOCIATED FACTORS TO TRANSCRIPTION REGULATION. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Regulation of gene expression is complex, involving the coordinated effect of a large number of proteins with a wide range of activities that control the recruitment and activity of RNA polymerases. Eukaryotic genomes are packaged into chromatin, where positioned nucleosomes are used to control transcriptional output. More recently, transcription of ncDNA has been shown to be pervasive and act as a regulator of gene expression as well.
Previous studies in Saccharomyces cerevisiae described a gene repression mechanism whereby transcription of intergenic ncDNA (SRG1) over the promoter of the adjacent SER3 gene interferes with the binding of transcription factors. In this work, I contributed evidence to support a mechanism whereby SRG1 transcription represses SER3 by controlling chromatin. In the presence of serine, transcription of SRG1 ncDNA is initiated upstream of the adjacent SER3 gene and extends across the SER3 promoter. As RNA pol II transcribes SRG1, the accompanying Spt6 and Spt16 histone chaperones reassemble nucleosomes over the SER3 promoter, which then interfere with transcription factor binding resulting in SER3 repression. In response to serine starvation, SRG1 transcription is reduced, causing nucleosome depletion over the SER3 promoter, which in turn allows transcription factors to bind and activate SER3 transcription.
I then use this system of gene regulation to identify and characterize mutant versions of the Spt16 histone chaperone and histone H3 and H4 proteins that are defective for transcription-coupled nucleosome assembly. These studies identify single amino acid substitutions in these proteins that cause a loss of nucleosome occupancy specifically over highly transcribed regions, revealing regions of the proteins that play critical roles in orchestrating transcription-coupled nucleosome assembly.
Furthermore, I provide evidence that a subset of the amino acids in histone H3 function to control chromatin dynamics, and may function as a binding site for histone chaperones Spt6 and Spt16. Therefore, when the residues are mutated, these factors can no longer bind chromatin, resulting in slowed nucleosome reassembly over transcribed regions. Together, my work further elucidates the mechanism of SER3 regulation by transcription of SRG1 and reveals a set of novel residues working to regulate this mechanism and transcription-coupled nucleosome dynamics in general.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Hainer, Sarahsjh13@pitt.eduSJH13
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairMartens, Josephmartens@pitt.eduMARTENS
Committee MemberArndt, Karenarndt@pitt.eduARNDT
Committee MemberBrodsky, Jeffjbrodsky@pitt.eduJBRODSKY
Committee MemberHildebrand, Jeffjeffh@pitt.eduJEFFH
Committee MemberJohn, Binojohn@johnlab.org
Date: 26 September 2012
Date Type: Publication
Defense Date: 19 July 2012
Approval Date: 26 September 2012
Submission Date: 31 July 2012
Access Restriction: 3 year -- Restrict access to University of Pittsburgh for a period of 3 years.
Number of Pages: 325
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: Transcription, nucleosome, chromatin, ncRNA, SRG1/SER3
Date Deposited: 27 Sep 2012 01:27
Last Modified: 15 Nov 2016 14:01
URI: http://d-scholarship.pitt.edu/id/eprint/13326

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