Date of Award
2006
Degree Type
Thesis
Degree Name
Doctor of Philosophy
Program
Physiology and Pharmacology
Supervisor
Dr. Joseph Torchia
Second Advisor
Dr. Marc Tini
Third Advisor
Dr. Chris Brandi
Abstract
Gene expression is dependent on the ability of sequence specific DNA-binding proteins to regulate the transcription of target genes in a coordinated fashion. This is accomplished, in part, through transcription factor-mediated recruitment of coregulators that activate or repress transcription through multiple mechanisms and are referred to as coactivators or corepressors, respectively. I have employed biochemical approaches in an attempt to gain insights into the function of a classical corepressor protein, N-CoR, as well as a zinc finger protein, ZNF217, which is amplified in multiple cancers and of unknown function. I have demonstrated that N-CoR copurifies with the coactivator CBP. The association of N-CoR and CBP was confirmed in vivo through indirect immunfluorescence and may be direct as demonstrated through protein-protein interaction assays in vitro. This demonstration of an association between the corepressor N-CoR and coactivator CBP is the first report of such an interaction and provides further support to a growing body of evidence that coactivators and corepressors associate in vivo. Additionally, I have demonstrated that N- CoR contains two functionally distinct domains, with the first domain consisting of a CBP interaction domain and the second domain capable of attenuating CBP HAT activity and containing a putative histone interaction domain. Therefore, I have proposed that the first domain functions to sequester CBP through direct binding while the second domain silences CBP HAT activity through a mechanism that may involve direct binding of N-CoR to histones. 111 I have also provided the first biochemical characterization of the ZNF217 repressor complex that consists of the corepressor proteins HDAC2, CoREST, LSD1, and CtBP. This work includes the first mapping of ZNF217 repression domains using ZNF217-Gal4 fusion proteins and a Gal4 luciferase reporter gene. The work reported in this thesis is the first evidence demonstrating that ZNF217 is a DNA binding protein through the identification of a consensus recognition sequence (CRS). Finally, I have demonstrated the E-cadherin promoter contains a ZNF217 CRS and is negatively regulated by ZNF217. These results establish the ZNF217 complex as a novel transcriptional repressor of the E-cadherin gene promoter.
Recommended Citation
Cowger, Jeffery J.M., "BIOCHEMICAL AND FUNCTIONAL ANALYSIS OF TRANSCRIPTIONAL COREPRESSOR COMPLEXES" (2006). Digitized Theses. 4698.
https://ir.lib.uwo.ca/digitizedtheses/4698