Protein interactions involved in human gene regulation

grantor: University of TorontoProtein-protein interactions are important both for basal transcription and for the regulation of transcription. The core transcription machinery consists of a set of proteins, the general transcription factors, plus the multisubunit enzyme RNA polymerase II. The general transcription factors interact with each other and with RNA polymerase II to enable the polymerase to produce mRNAs. This process is regulated in cells by activator and repressor proteins that increase or decrease respectively the rate of transcription. Activators and repressors have been shown to function through interactions with various other proteins involved in transcription such as the general transcription factors and transcriptional cofactors. In order to increase our understanding of how the core transcription machinery works and how it is regulated, protein interactions among proteins involved in transcription must be identified and characterized. Chapters 2 and 3 of this thesis describe the identification of proteins that interact with the Wilms' tumour suppressor protein, WT1. WT1 is a transcriptional regulator that is able to activate and repress transcription. In chapter 2, a yeast two-hybrid screen is used to screen for cDNAs encoding proteins that interact with amino acids 182 to 295 of WT1. This region of WT1 has been implicated in both activation and repression of transcription. One interacting protein was found: the 90 kD heat shock protein, hsp90. Hsp90 was also able to bind to this region of WT1 'in vitro'. In chapter 3, protein affinity chromatography is used to identify an interaction between amino acids 182 to 295 of WT1 and the general transcription factor IIB. Both alternatively spliced isoforms of WT1 bind TFIIB. In 'in vitro' transcription reactions, addition of amino acids 182 to 295 of WT1 results in inhibition of transcription, which is reversed by the addition of recombinant TFIIB. Using gel mobility shift assays to examine the formation of the pre-initiation complex, WT1 was found to inhibit formation of a TFIIF-TFIIB-TBP-promoter complex, but not a TFIIB-TBP-promoter complex. This result suggests that the interaction between WT1 and TFIIB may be involved in transcriptional repression by WT1. Chapter 4 examines the interactions of two elongation factors, TFIIS and Elongin A, with the RNA polymerase II holoenzyme. An interaction was identified between each of these elongation factors and the general transcription factor IIB. The amino-terminus of each factor, which is sufficient for the interaction with RNA polymerase II holoenzyme, was also sufficient to bind TFIIB. The site of interaction on TFIIB was mapped to amino acids 100 to 240. TFIIB was found to compete with TFIIS for binding to the RNA polymerase II holoenzyme. This suggests that TFIIB can mediate the interaction between these elongation factors and holoenzyme.Ph.D