dc.contributor.author | Brown, Alexandra | |
dc.date.accessioned | 2023-08-16T18:43:38Z | |
dc.date.available | 2023-08-16T18:43:38Z | |
dc.date.issued | 2023-08-11 | |
dc.identifier.uri | http://hdl.handle.net/10222/82791 | |
dc.description.abstract | CREB-binding protein (CBP) and its homolog p300 (CBP/p300) are sequestered by transcription factors to thousands of promoters and enhancers throughout the human genome, where they modify chromatin structure and enhance gene expression. CBP/p300 also play an important role in the development and progression of human diseases such as cancer, where they stimulate the expression and activity of oncogenic proteins. Utilizing a combination of structural, biophysical, and functional assays I investigate the molecular interactions between CBP/p300 and two oncogenic transcription regulators, Wnt-signaling protein β-catenin and the microphthalmia- associated transcription factor (MITF). With the use of protein pulldown assays, isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy (NMR) I determine that the N-terminal and C-terminal transactivation domains (TAD1 and TAD2, respectively) of MITF and β-catenin are intrinsically disordered and bind with variable affinities to both the transcription adapter zinc finger domains (TAZ1 and TAZ2) of CBP/p300. Using NMR-based chemical-shift perturbation studies I show that the β-catenin TAD1 interacts with TAZ2 at a specific binding interface between helices ⍺-1-⍺-2-⍺-3, and that β-catenin TAD2 binds to TAZ2 with extended contacts over ⍺-1-⍺-4 and to TAZ1 at a hydrophobic groove between ⍺-1-⍺-2. Mutagenesis studies revealed regions Asp58-Leu103 and Asp750-Leu781 are critical for β-catenin: CBP/p300 binding and for β-catenin transcriptional activation using luciferase- reporter gene assays. I provide the first ever NMR-derived structure of the MITF TAD1 in complex with TAZ2, which was found to form a dynamic ensemble with the hydrophobic TAZ2 surface. I also identify two acidic motifs Arg110-Ala14, Asp334- Asp345, and one serine-rich motif from Ser372-Asp381 integral to maintain MITF: CBP/p300 binding and necessary for MITF transcriptional activity. Furthermore, I develop a peptide inhibitor derived from adenoviral E1A protein that displaces these TAZ2 interactions from MITF and β-catenin in vitro and functionally impedes their transcriptional function in cells. Finally, I propose three models in which both β- catenin and MITF TADs may cooperate to recruit CBP/p300 to target gene promoters. These findings provide the high-resolution structural information that would be required to develop targeted therapeutics strategies against CBP/p300 and its interactions in the future. | en_US |
dc.language.iso | en | en_US |
dc.subject | Transcription Regulation | en_US |
dc.subject | Melanoma Skin Cancer | en_US |
dc.subject | Structural Biology | en_US |
dc.subject | Protein Interactions | en_US |
dc.subject | NMR Spectroscopy | en_US |
dc.title | Investigating the Molecular Mechanisms of CBP/p300 Recruitment by the Transcriptional Regulators MITF and β-catenin | en_US |
dc.date.defence | 2023-07-20 | |
dc.contributor.department | Department of Biochemistry & Molecular Biology | en_US |
dc.contributor.degree | Doctor of Philosophy | en_US |
dc.contributor.external-examiner | Dr. Katherine Borden | en_US |
dc.contributor.graduate-coordinator | Dr. Jamie Kramer | en_US |
dc.contributor.thesis-reader | Dr. Denis Dupré | en_US |
dc.contributor.thesis-reader | Dr. Jan Rainey | en_US |
dc.contributor.thesis-reader | Dr. Paul Liu | en_US |
dc.contributor.thesis-supervisor | Dr. David Langelaan | en_US |
dc.contributor.ethics-approval | Not Applicable | en_US |
dc.contributor.manuscripts | Yes | en_US |
dc.contributor.copyright-release | No | en_US |