A joint project of the Graduate School, Peabody College, and the Jean & Alexander Heard Library

Title page for ETD etd-10282010-173834

Type of Document Dissertation
Author Dimitrova, Yoana Nantcheva
URN etd-10282010-173834
Title Biochemical and structural analyses of TBL1: insights into the function of a transcriptional regulator
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Walter J. Chazin Committee Chair
Brandt Eichman Committee Member
Brenda Schulman Committee Member
Charles Sanders Committee Member
Jennifer Pietenpol Committee Member
Scott Hiebert Committee Member
  • transcriptional regulation
  • ubiquitination
  • beta-catenin
  • TBL1
Date of Defense 2010-10-13
Availability unrestricted
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.

  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  Dimitrova.pdf 5.75 Mb 00:26:37 00:13:41 00:11:59 00:05:59 00:00:30

Browse All Available ETDs by ( Author | Department )

If you have more questions or technical problems, please Contact LITS.