Structure-function analysis of TBP in vivo: mechanisms regulating gene expression and proliferation – TBPprolif

The TATA-binding protein (TBP) is one of the central factors in transcription initiation. TBP has a bipartite structure with a highly conserved C-terminal core domain that folds as a molecular saddle responsible for binding DNA via the concave underside and interaction with general transcription factors (GTFs) and a host of other regulatory proteins via the solvent exposed convex surface. TBP forms several distinct complexes with multiple TBP-associated factors (TAFs) to direct transcription by RNA polymerases I, II and III. Further genetic evidence indicates an important role for TBP in regulating cell proliferation. The ability of TBP to engage in multiple interactions with TAFs and other GTFs has prompted many studies aiming to identify the surfaces involved. Systematic mutagenesis of all the solvent exposed residues of the core domain has identified many of these interaction surfaces and the properties of the mutant TBPs have been evaluated, in vitro using pol II and pol III promoters and by transfection in mammalian cells. While this approach has provided considerable insight into the structure-function relationships of TBP, their scope has been limited to in vitro assays or transfections with artificial promoters that do not reproduce the complexity of the real in vivo situation with the diversity of promoters in the cell. - We have developed a unique approach to study TBP function in vivo using mouse embryonic fibroblasts where one allele of the TBP gene has been inactivated and the other has been floxed such that endogenous TBP can be inactivated by Cre-recombinase. Upon inactivation of TBP, the tbp-/- cells die, but expression of wild-type TBP following retrovirus infection restores cell viability. We have verified the ability of several mutant Flag-HA-tagged-(F-H-)TBPs to complement the loss of endogenous TBP and established clonal cell populations expressing mutant F-H-TBPs . Many mutants rescue viablity, but often show slow growth phenotypes. An in depth analysis of one mutant line shows that the expression of only a small number of genes is affected. - We request funding for an extended study where we will generate cell lines with additional F-H-TBP mutants. These lines will be used to analyse a series of mutant F-H-TBPs : 1) for their ability to rescue cell viability and normal proliferation, 2) to evaluate their affect on gene expression by microarray analysis, 3) to assess their recruitment to promoters by chromatin immunoprecipitation (ChIP)-on-chip, 4) to evaluate the ability of each mutant to facilitate recruitment of other GTFs and TBP-partner proteins to target promoters by gene-specific ChiP and ChIP-on-chip, 5) to couple tandem affinity purification and mass-spectrometry to asses the effect of F-H-TBP mutations on interaction with known partners and to identify novel ones, 6) to replace endogenous TBP with wild type and mutant GFP-TBP to evaluate TBP mobility in real time in living cells, and in cells where one or other TBP partners has been inactivated by siRNA. - We believe that this is a novel and ambitious approach to dissecting TBP function in vivo in mammalian cells. The integration of information from the transcriptome, proteome, ChIP and imaging assays, will bring novel information on how TBP orchestrates transcription initiation and regulates cell proliferation. There is a clear synergy between the participating groups. We are in a unique position as Partner 1 is the only group in the world to have developped genetically modified cells allowing this type of study. Partner 2 is one of the leading facilities for mass-spectrometry analysis in France and partner 3 has extensive experience in ChIP analysis, live imaging techniques and mass-spectrometry. Partners 1 and 3 have proven experience in the biochemistry and molecular biology techniques and an long standing history of interest in TBP, its partners and its function in mammalian cells. Partners 1 and 3 have collaborated previo...