The long term objectives of this research proposal are to understand the mechanisms of tissue-specific and inducible gene expression in higher eukaryotes. The model systems chosen for this study are the fat body- specific expression of the Drosophila alcohol dehydrogenase (Adh) gene, and the induction of Drosophila immune response genes in the fat body. Studies of the Adh gene will focus on a well characterized fat body- specific transcriptional enhancer designated the Adh Adult Enhancer (AAE), which is comprised of several distinct regulatory sequences that interact synergistically to achieve normal levels of Adh expression in the fat body. Transcriptional activators and repressors that bind to the AAE have been identified and cloned, and their functional roles in Adh gene expression will be determined. The functional studies will involve the characterization of protein-protein interactions in vitro, and the analysis of site directed mutations on these interactions and on transcription in vivo. Cotransfection analyses with cultured Drosophila cells, and Drosophila P-element transformation experiments will be employed to study transcription in vivo. The ultimate goal of these studies is to understand how the multicomponent Adh enhancer complex is assembled in a tissue-specific manner, and how it activates transcription. The second major goal of the proposed research is to investigate the role of a Drosophila rel family transcription factor, Dif, in the regulation of Drosophila immune response gene expression. Experiments will be carried out to determine the mechanisms by which Dif is activated by bacterial infection, a process that likely requires the inactivation of an inhibitory protein, cactus, which sequesters Dif in the cytoplasm of uninfected fat body cells. Studies will be carried out to determine whether the ubiquitin-proteasome pathway of protein degradation is required for the inactivation of cactus. This objective will be accomplished by examining the effects of specific proteasome inhibitors and Drosophila proteasome mutants on Dif activation, and the induction of immune response genes. Studies will also be carried out to determine whether the transcriptional activity of Dif is controlled positively and negatively by two distinct high mobility group (HMG) proteins, HMG D1 and DSP1, respectively, and to elucidate the mechanisms involved in HMG coactivation and corepression. These objectives will be achieved by analyzing protein-DNA and protean-protein interactions in vitro, by cotransfection studies with cultured Drosophila cells, and by Drosophila P-element transformation experiments. There are striking parallels between the induction of Drosophila immune response genes in the fat body and the induction of acute phase response genes in the mammalian liver. Thus, an understanding of the mechanisms involved in these processes in Drosophila should significantly advance our understanding of the regulation of gene expression in the immune and inflammatory responses in man.

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National Institute of General Medical Sciences (NIGMS)
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Molecular Biology Study Section (MBY)
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Harvard University
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