Untapped resources for gene discovery include an explosion of genome sequence information, high-throughput methods for transcript profiling and high-throughput, functional genomics technologies. Experiments are proposed whose long-term goals are to refine and validate new genomics- and functional genomics-based techniques for the identification and analysis of genes that acts in neural development or function. The approaches developed here are generally applicable for gene discovery and expression analysis in the nervous system. The specific experiments proposed, analysis of CREB-dependent pathways of neural plasticity, should serve as a paradigm to guide future studies. A computational screen of a Drosophila genome database for CREB-induced genes involved in neural plasticity will be performed and validated. The screen will establish the viability of promoter sequence-based approaches to studying gene regulation in metazoa; it will also provide: i) a test panel of activity-induced Drosophila genes, and ii) the opportunity to implement new software designed for more mathematically rigorous microarray analysis. A commercially available mammalian microarray will be screened to select mammalian genes strongly induced in human SHSY5Y cultured cells by depolarization and cAMP agonists, treatments known to activate genes involved in long-term plasticity. Once downstream CREB-responsive genes are identified, reporters based on their promoter elements will be engineered in order to embark on a fluorescence-based functional genomics screen for critical regulators of the CREB pathway. First, GFP fusions will be created in which GFP coding sequences are fused downstream of 5' regulatory elements from the genes whose expression behavior correlates with the CREB-response. By a series of genetic engineering steps and FACS selections, cell lines will be generated in which fluorescence serves as a surrogate phenotype for activation of the CREB pathway. A set of such lines, each of which coopts a particular CREB-induced gene, will be developed and used in transdominant genetic experiments to identify modulators of the CREB pathway. Experiments to study the functions of these modulators in vivo will be initiated in rodents and in Drosophila using resources developed as part of this proposal.
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