Regulation of Nuclear Receptor Function during Drosophila Development

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The formation of a complex organism from a single cell, the fertilized egg, requires the expression of different genes at different times and in different cells as the embryo develops. Many of the gene products controlling these early steps in development determine cell fate through their function as regulatory genes, encoding transcription factors or signaling proteins that regulate the expression of downstream cascades of genes encoding products directly involved in growth and differentiation of different cell types. If these regulatory genes are active in the wrong cells at the wrong time, it is catastrophic for the embryo, which will likely die as a result of this mis-expression. The goal of this project is to unravel the mechanisms controlling the activity of embryonic regulatory genes. It will use a well-established animal model, the fruit fly Drosophila melanogaster, for these studies. The focus is on one family of embryonic transcription factors, a nuclear receptor, which is regulated by interactions with protein partners. Findings will provide a fundamental model for studies of other members of the nuclear receptor family, in Drosophila and other species, by contributing to an understanding of cell type specification during embryonic development and elucidating different levels at which the function of transcription factors is regulated to produce unique cell types. This proposal will train students from diverse backgrounds in the laboratory in bench science, critical thinking and scientific presentation. In addition, it will support formal collaborations with a majority minority middle school in Maryland(Wilde Lake Middle School)to bolster the genetics curriculum for 7th graders and with the MARC program at Northeastern Illinois University that will send up to two students/year for summer internships to Northwestern. These efforts are part of ongoing efforts on the part of both the PI and Co-PI to educate the public about science. Nuclear Receptors (NRs) are a large family of transcription factors that control a broad range of biological processes. Although the activity of many NRs is regulated by ligand, a large number of these receptors are orphan NRs that share a common fold with NRs with known ligands. This likely reflects the ancestral state of this protein family. How is the activity of orphan NRs regulated? Drosophila Ftz-F1 is the founding member of the NR5A nuclear receptor family. In previous work, the Principle Investigator and colleagues showed that Drosophila Ftz-F1 interacts with the homeodomain protein Ftz, a classic pair-rule protein expressed in seven stripes in embryos in the primordia of body regions missing in ftz mutant animals. Ftz-F1 and Ftz form a stable complex in vivo and bind to composite DNA sites, synergistically activating target gene expression. Thus, although Ftz-F1 is a strong transcriptional activator alone in vitro, and it is present in the nuclei of all somatic cells of the embryo, its activity is limited to the cells in which Ftz is also expressed (Ftz+ cells). These studies demonstrate that protein-protein interactions modulate Ftz-F1 activity and provide an in vivo model system to dissect the mechanisms underlying this regulation. Here we will test whether Ftz-F1 is regulated: (1) at the level of DNA binding; (2) by a corepressor/coactivator exchange, and/or (3) by Ftz-induced conformational change. This work will combine biochemical and structural methods with molecular genetic approaches in Drosophila embryos to evaluate the contributions of each of these mechanisms to the regulation of NR activity in vivo. It will train postdoctoral fellows, graduate students and undergraduate students in molecular biology, genetics, biochemistry, biophysics and, most importantly, critical thinking: generating, testing and evaluating hypotheses.