The mechanism controlling the switch between gene activation and repression is critically important for understanding the process of transcriptional regulation. Gene expression is highly controlled through a dynamic exchange between co-activators and co-repressors from DNA bound transcription factors. TBL1 is an essential multi-domain scaffolding protein that appears to mediate the switch between transcriptional activation and repression of β-catenin and nuclear hormone receptors (NHRs). The mechanism of TBL1 as a transcriptional regulator has been the main focus of this dissertation.
The role of TBL1 was first investigated in the context of the SCF(TBL1) complex in the poly-ubiquitination and proteasomal degradation of β-catenin under UV-induced genotoxic stress. Over-expression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of β-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to poly-ubiquitinate β-catenin. Moreover, TBL1 was found to protect β-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Both Siah-1 and TBL1 bind to the armadillo repeat domain of β-catenin, suggesting poly-ubiquitination of β-catenin is regulated by competition between Siah-1 and TBL1.
To gain insight into the function of TBL1 within multi-protein complexes that regulate the transcriptional activity of β-catenin and NHRs, this work pursued the structural and biochemical characterization of TBL1. Expression, purification and analysis of full length TBL1 and different domain constructs revealed that it forms a stable tetramer through the N-terminal LisH domain. The structural architecture of full length TBL1 and the spatial organization of the domains were characterized by small angle x-ray scattering (SAXS) and analytical ultracentrifugation (AUC). An ab initio model of TBL1 was generated, revealing an extended anti-parallel dimer of dimers. The structure of TBL1 has a large surface area that can accommodate multiple binding partners, suggesting a role for TBL1 tetramerization in facilitating the formation of multi-protein assemblies. TBL1 mutants that form only dimers were designed and validated to enable future functional studies of the mechanism of TBL1 in regulating the transcriptional activity of β-catenin and NHRs.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-10282010-173834 |
| Date | 29 October 2010 |
| Creators | Dimitrova, Yoana Nantcheva |
| Contributors | Scott Hiebert, Brandt Eichman, Walter J. Chazin, Brenda Schulman, Jennifer Pietenpol, Charles Sanders |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-10282010-173834/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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