This proposal focusses on the function of the singleminded (Sim) gene and its dimerization partner, Darnt, during nervous system development. Sim encodes a transcription factor of the bHLH-PAS family. It is expressed in the midline cells and has been shown in previous work of the investigator to directly activate transcription of several midline specific genes, such as slit and Toll. The investigator has identified the protein encoded by human ARNT, another bHLH-PAS protein, as a potential dimerization partner of Sim. Furthermore, he has identified the Drosophila homolog of ARNT, called Darnt. The first goal of this proposal is to study the function of Darnt by generating mutations, as well as using Darnt dominant negative constructs targeted to specific tissues. Mutations will be generated following standard genetic screens, including an EMS mutagenesis (using a deficiency shown to uncover the Darnt gene) and a P-element transposition mutagenesis. Mutations will be analyzed phenotypically as homozygotes, over a deficiency and in heteroallelic combinations. Since the investigator has established the expressions pattern of Darnt in his preliminary results, he has made specific prediction as to the embryonic tissues which might be affected. A list of markers allowing him to study these tissues is provided. It is further proposed to generate germline clones, using the FLP/FRT technique, since Darnt is expressed at high levels in preblastoderm embryos. Using the Gal4-UAS system, dominant negative forms of Darnt (lacking the parts of the basic region required for DNA binding) will be targeted to different organs, such as the embryonic tracheae and the eye. The second goal is the analysis of the subcellular distribution of Darnt and Sim. A soluble Darnt fusion protein was used to generate antibodies. These will be used to study Darnt expression in embryos, brains and eye discs. Expression of Darnt will be analyzed in the background of mutations in Sim, hsp83, trachealess (trh) and Sima, potential dimerization partners of Darnt. A HA tagged form of Sim will be expressed under UAS control in tissues in which Sim normally does or does not activate transcription. It is hypothesized that nuclear transport of Sim (which could be mediated by Darnt) is required for Sim function. In tissues in which Sim normally does not activate transcription, the nuclear localization factors required for its transport should be missing, and Sim (artificially expressed in such tissues) should remain cytoplasmic. The third goal, to carry out in vitro binding studies of Sim/Darnt protein, synthesized by a baculovirus expression system, to Toll DNA sequences which had been identified as binding sites for Sim. The Sim/Darnt binding sites on the Toll and slit promoter will be mapped by a DNAseI footprinting assay. In another experiment it will be tested whether Sim/Darnt directly represse in the midline, the expression of the gene vnd which normally appears beside the midline. Promoter constructs of vnd will be made which mimic the normal vnd pattern. Sim/Darnt binding sites will be identified and mutated. The result on expression (i.e., does the mutant construct appear in the midline) will be carried out. The fourth goal entails studies of Darnt/Sim binding. In addition to co-immunoprecipitation of baculovirus synthesized Sim/Darnt protein, the yeast two-hybrid system, which has worked very well in the investigator's hands, will be used. Different mutant forms of the two proteins will be tested for binding in these assays. The co-immunoprecipitation assay will be used to establish whether Darnt also binds in vivo to other Drosophila PAS genes (Trh, Per, Sima). The fifth goal addresses spatial specificity of Darnt/Sim action. Studies on another bHLH protein, MyoD, had shown that two adjacent residues in the bHLH region are required for the binding of a muscle specific partner, Mef-2, which is responsible for the muscle specific activation of MyoD. Homologous residues were identified in Sim and Trh. The Gal-4/UAS system will be used to express ectopically forms of Sim and Trh in which these residues were mutated. The effect on transcriptional specificity will be tested.
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