Biochemical and structural analyses of TBL1: insights into the function of a transcriptional regulator
Dimitrova, Yoana Nantcheva
:
2010-10-29
Abstract
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.