The long term objective of our work is to understand the molecular mechanisms underlying synergistic gene expression in eukaryotic cells. We will study how a well-characterized eukaryotic transactivator called ZEBRA differentially controls transcription of a family of target genes involved in the lytic cycle of Epstein-Barr virus. Our hypothesis is that the concentration of ZEBRA regulates the timing of target gene expression, and that different promoters respond to specific activator thresholds based on a promoter architecture that guides the cooperative assembly of upstream promoter-activator complexes and the ability of these complexes to recruit limiting components of the RNA polymerase II general transcription machinery to a gene. Our approach will be to correlate biochemical events in purified transcription systems with natural lytic expression patterns to establish a model for how a single activator can singularly control a genetic regulatory circuit.
Our specific aims are to i.) refine our knowledge of ZEBRA-mediated differential gene regulation in vivo and recreate expression of a subset of target genes in vitro; ii) employ purified ZEBRA, Sp-1 and HMG-1 in a study of how promoter architecture regulates assembly of nucleoprotein structures called enhanceosomes on target promoters; iii) investigate the biochemical mechanism by which activators recruit TFIIA, TFIID and TFIIB to a core promoter to nucleate assembly of a preinitiation complex and iv.) further study the biochemical details of a ZEBRA-mediated isomerization event in TFIID, necessary for full levels of gene activation in vitro. The mechanism by which a eukaryotic activator controls gene expression has represented one of the most intensively studied problems in contemporary biology, largely because of the central role aberrant gene expression plays in pathological processes. My laboratory has the technology to combine biochemical and biologic approaches towards understanding the molecular details of this central issue.
