Mechanisms of Immediate Early Gene Regulation in Neurons

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DESCRIPTION (provided by applicant): The ability to acquire, store and recall memories is a defining feature of the brain that is conserved from humans to Drosophila. Immediate Early Genes (IEGs) are genes whose expression is stimulated by neuronal activity, and is often transient, a feature that makes them prime candidates for modifying neural plasticity. Yet very little is understood about the regulation and function of IEGs in any organism. Aberrations in neural plasticity, learning and memory can cause severe neurological conditions such as Alzheimer's, dementia, post-traumatic stress and autism, underscoring the need for elucidating molecular mechanisms that underlie memory. We have identified a suite of 288 IEGs in the Drosophila brain using RNASeq analysis after exposure to sensory stimuli of various durations (10, 20, 30, 45 mins), 65% of which have homologs in humans. Based on the kinetics of expression modulation the IEGs cluster into 5 groups suggesting differences in regulatory mechanisms. Using an unbiased bioinformatics approach we were able to identify over-represented DNA sequence motifs in the upstream regions of each cluster of IEGs. The motifs for cluster 1 and 2 are remarkably alike, and match the binding site of the GATA family of transcription factors. Genetic and molecular analysis showed that a GATA factor, grain, was indeed required for expression of IEGs. The goal of this proposal is to test the hypothesis that grain and other GATA factors play a role in activity-dependent modulation of IEG expression and therefore a central role in acquisition and retention of memory. The proposal is highly significant since this represents one of the first major IEG regulatory pathways since CREB and could present an opportunity to understand the link between neuronal activity, IEG expression and memory. In addition, the proposal will use IEG promoters to provide transgenic tools to map activated neural circuits in brains of live behaving flies. The approach for analysis of IEG regulation by GATA factors will be accomplished in two specific aims outlined here. (1) The first aim will validate the role of grain in IEG regulation, identify its genome-wide targets, map changes in its localization in the brain and investigate its role in memory. (2) The second aim will investigate the regulatory DNA sequences of the GATA- dependent IEGs to test for direct regulation. In addition it will develop transgenic flies where the IEG promoter will be used to encode a genetic fluorescent reporter that marks activated neurons. Successful completion of the proposed studies will uncover transcription factors and neuronal circuits underlying fundamental mechanisms of learning and memory and establish tools that will be widely useful for anatomical studies of neural circuits.