The overall goal of this research project is to understand some of the detailed molecular mechanisms involved in regulation of transcription in higher eukaryotic cells. Attention will focus on several Drosophila proteins that were initially identified by genetic methods as being important in controlling pattern formation in early embryogenesis, and which are now known to be transcriptional regulators. For the purposes of this project, these proteins will be studied as prototypical regulators that illustrate general mechanisms of gene control. That is, the aim is not to understand detailed pathways of developmental regulation in the Drosophila embryo, but rather to understand underlying biochemical mechanisms. Specifically, ongoing studies dealing with three specific regulators, or types of regulators, will be pursued. All the experiments will involve transfection assays in cultured cells, protein-protein interaction studies and in vitro transcription experiments with purified components. 1) Mechanisms of transcriptional activation will be studied using several previously described homeodomain activators. Recent experiments have focused on understanding how the glutamine-rich activation region from the Fushi tarazu protein functions (in the context of a GAL4-ftz fusion), and have provided evidence that a novel, direct interaction with the general transcription factor TFIIB is essential. Experiments to investigate further the mechanism by which this very strong, non-acidic activator functions will be performed. Using the GAL4-ftz activator together with others (e.g., GAL4-VP16), possible mechanisms of transcriptional synergy will also be examined. 2) Mechanisms of transcriptional repression will be investigated, using the homeodomain proteins Even-skipped (Eve) and Engraved (En). Both proteins are among the best characterized transcriptional repressors, and available data suggest both, especially Eve, function as direct, or active, repressors that interfere with or prevent assembly of the preinitiation complex. Recent experiments indicating that Eve repression which involves a direct, functional interaction with the TATA binding protein, TBP, will be extended to understand the details of this interaction, as well as how it leads to transcriptional repression. A possible role of phosphorylation in modulating activity will also be investigated. 3) Mechanisms of transcription factor regulation will be investigated by continuing studies on Dorsal (D1), a rel/NF-kappaB family member. Previous work has indicated that regulated nuclear transport of D1 can be reproduced in transfected Schneider cells, and that the activities of at least two genetically defined regulators of D1, Toll and tube, can be detected and analyzed. Those studies led to a model involving activation of D1 by direct phosphorylation, which both induces nuclear transport and enhances the transcriptional activating potential of D1, with recent studies indicating that the tube protein functions as both a chaperon and coactivator of D1. Experiments to examine further the mechanisms by which D1 nuclear transport is regulated, and its activity modulated, will be performed.
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