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Novel Systematic Strategies for Elucidating Cellular Regulatory Circuits (RegulatoryCircuits)
Start date: Jan 1, 2009, End date: Dec 31, 2013 PROJECT  FINISHED 

The precise regulation of gene expression has been the subject of extensive scrutiny. Nonetheless, there is a big gap between genomic characterization of transcriptional responses and our predictions based on known molecular mechanisms and networks and of transcription regulation. In this proposal I argue for an approach to bridge this gap by using a novel experimental strategy that exploits the recent maturation of two technologies: the use of fluorescence reporter techniques to monitor promoter activity and high-throughput genetic manipulations for the construction of combinatorial genetic perturbations. By combining these, we will screen for genes that modulate the transcriptional response of target promoters, use genetic interactions between them to better resolve their functional dependencies, and build detailed quantitative models of transcriptional processes. We will use the budding yeast model organism, which allows for efficient manipulations, to dissect two transcriptional responses that are prototypical of many regulatory networks in living cells: [1] The early response to osmotic stress, which is mediated by at least two signaling pathways and multiple transcription factors, and [2] the central carbon metabolism response to shifts in carbon source, which involves multiple sensing and signaling pathways to maintain homeostasis. Our approach will elucidate mechanisms that are opaque to classical screens and facilitate building detailed predictive models of these responses. These results will lead to understanding of general principles that govern transcriptional networks. This is the first approach to comprehensively characterize the molecular mechanisms that modulate a transcriptional response, and arrange them in a coherent network. It will open many questions for detailed biochemical investigations, as well as set the stage to extend these ideas to use more detailed phenotypic assays and in more complex organisms.
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