Eukaryotic organisms employ a variety of important, but poorly understood mechanisms to control the expression of their genes. An understanding of these mechanisms is an important issue since regulation of gene expression plays a pivotal role in the development and differentiation of functionally distinct cell types in a precise spatial manner. In addition, regulation of transcription reflects the ability of cells to respond to extracellular signals and environmental stress. The long term objectives of this research proposal are to understand the molecular mechanisms of inducible gene expression in higher eukaryotes using the interferon-beta (IFN-beta) gene as a model system. The IFN- beta gene is ordinarily tightly repressed, but is expressed at high levels when cells are infected with viruses or treated with double stranded RNA. Previous studies revealed a key role for the High Mobility Group protein HMG I(Y) in virus induction of the IFN-beta gene. HMG I(Y) is not a transcriptional activator on its own but functions in concert with other transcription factors. The highly specific activation of the IFN-beta gene in response to virus infection requires the assembly of a higher order nucleoprotein transcription enhancer complex, the enhanceosome. HMG I functions by promoting the assembly of the enhanceosome by mediating protein-DNA and protein-protein interactions. The overall goals of this proposal are to elucidate the molecular mechanisms by which HMG I(Y) promotes the assembly of the enhanceosome and how the enhanceosome stimulates transcription synergistically. First, a detailed structural and functional characterization of the HMG I(Y) proteins will be carried out to identify domains involved in DNA binding, protein-protein interactions and regions required for the assembly and the function of the IFN-beta enhanceosome. Second, in vivo assays and in vitro transcription systems will be used to determine the mechanisms by which the components of the IFN-beta gene enhanceosome function synergistically in the activation of transcription. If successful, the proposed experiments will provide significant new information regarding the structure and function of transcriptional regulatory proteins and sequences, as well as the mechanisms involved in signal transduction.
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