Protein interactions in E2F1-mediated transcriptional activation

grantor: University of TorontoTranscriptional activators of RNA polymerase II bind promoter regions of genes and regulate mRNA synthesis through protein interactions with components of the transcription apparatus. In chapter two I describe the first characterization of interactions between transcriptional activators and the general transcription factor TFIIH. In particular, the herpes virion protein VP16 and the tumour suppressor protein p53 are both shown to bind yeast TFIIH. The human transcriptional activator E2F1 regulates gene expression at the G1/S boundary of the cell cycle, and chapters three and four contain an analysis of the E2F1 activation domain. Using protein affinity chromatography, I have shown that the activation domain of E2F1 binds the human and yeast multi-subunit general transcription factors TFIID and TFIIH. E2F1 binds the yeast and human TATA-box binding protein (TBP) of TFIID, and also binds the p62 and p89 subunits of human TFIIH, and the yeast homologue of p62, TFB1. Deletions and single amino acid substitutions of E2F1 were analyzed for their effect on TFIIH- and TFIID-binding. There are two distinct binding sites in E2F1 for TBP (amino acids 386-417 and 415-437), and one for TFIIH (409-437), which overlaps both TBP-binding sites. Mutant proteins were expressed as fusions to the DNA-binding domain of the yeast transcription factor GAL4, and tested for transcriptional activation in tissue culture. Each minimal TBP-binding site activates transcription, while fragments of E2F1 that do not bind TBP cannot activate. A mutation in E2F1 that enhances TFIIH-binding results in an increase in activation, however TFIIH-binding is not required for activation. These results strongly suggest that the interactions between E2F1 and both TBP and TFIIH contribute to transcriptional activation by E2F1. The retinoblastoma protein (Rb) binds E2F1 and inhibits its ability to activate transcription. In chapter five, Rb is shown to compete with both TBP and p62 for binding to E2F1. A tumorigenic form of Rb that is deficient in its ability to inhibit EM-mediated activation is also deficient in competing with TBP for binding to E2F1. This supports a model in which Rb inhibits E2F1-mediated transcriptional activation by blocking interactions between E2F1 and both TFIID and TFIIH.Ph.D