The homeobox is a 180 bp protein-coding sequence found in nearly every eukaryote that has been investigated, spanning a broad evolutionary spectrum from yeast to man. It is conceivable that a common function of all homeobox genes in higher eukaryotes is the control of cell fate during embryonic development. Approximately one-half of the genes that control Drosophila embryogenesis contain a copy of the homeobox. Genetic studies have implicated the homeobox gene activities in the morphogenesis of the worm, C. elegans, while experimental disruptions of homeobox gene activities in frogs and mice suggest an important role in vertebrate development as well. Recent studies have shown that the homeobox protein domain mediates sequence-specific DNA binding activities, suggesting that homeobox genes control development by modulating gene expression at the level of transcription. Direct support for this notion has been obtained in studies employing transient cotransfection systems and in vitro transcription assays. However, it is not clear how homeobox proteins regulate specific target genes since a broad range of divergent homeobox proteins have been found to contain similar DNA binding activities in vitro. Classical genetic studies in Drosophila and C. elegans, as well as molecular analyses done in cell culture systems, suggest that homeobox proteins act cooperatively and competitively to regulate gene expression. The overall goal of the proposed research is to determine how homeobox proteins function as transcription factors to control cell fate in the early Drosophila embryo. This study will include: (i) A detailed dissection of both the cis and trans elements responsible for the autoregulation of the segmentation gene even-skipped; (ii) in vitro assays to determine whether homeobox proteins bind to DNA cooperatively, and/or interact to form multimeric complexes; (iii) an analysis of factors that bind to the even-skipped promoter in Drosophila nuclear in vitro transcription extracts, and characterization of interactions between homeobox proteins and general transcription factors in such extracts; (iv) cotransfection assays in Drosophila tissue culture cells to analyze protein-protein interactions involved in transcriptional activation and repression mediated by combinations of homeobox proteins; and, (v) P- transformation assays to determine the impact of selective mutations in homeobox proteins on development.
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