A Functional Genomics Approach to Identify Regulators of Arabidopsis Clock Genes

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DESCRIPTION (provided by applicant): The circadian clock is an internal timekeeper found in all organisms studied to date regulating many aspects of their behavior, metabolism and physiology. For example, processes such as the sleep-wake cycle and metabolism in humans to photosynthesis and flowering in plants are all controlled by the circadian clock. Although genetic components of the circadian clock are known to differ across organisms, the basic architecture consisting of multiple interlocking feedback loops is conserved across species. The long-term goal of this proposal is to understand how the circadian clock network is constructed in eukaryotes. To achieve this, the experiments proposed here will be conducted in the plant model Arabidopsls. As in humans, the circadian clock controls a variety of biological processes in higher plants, and furthermore, -10% of the Arabidopsis gene content is known to be circadian regulated. However, the direct regulatory relationship between clock components at the transcription level is unknown suggesting that new clock genes are yet to be identified. Though numerous genetic screens to identify new clock genes have been performed, isolation of multiple alleles in the known clock genes indicate that these screens are likely saturated. Therefore, building on the current model in Arabidopsis, the specific aim of this study is to identify and characterize the genes involved in positive regulation (activation) of two core clock genes TOCl and LUX using a functional genomics approach. Briefly, a high throughput yeast assay will be performed to screen for putative regulators of TOCl and LUX, using a recently available library representing -2100 predicted Arabidopsis transcription factors (TFs), to identify components that bind to the promoter region of these two genes. Next, a battery of experiments involving mutational screens and circadian output assays will be conducted to establish the role of these regulators in the clock network. This strategy has recently been used to successfully identify a novel transcription factor in Arabidopsis providing a long awaited link between the morning and evening loops of the circadian clock in Arabidopsis. In addition to the controlling many important biological processes, the clock also controls -10% of the genes in humans. Several disorders in humans such as asthma and irregular sleep-wake patterns are linked to abnormal biological rhythms. Along with adding to our knowledge of the clock network in Arabidopsis, the study proposed here could contribute to our understanding of the circadian clock in humans and possibly treatment for related disorders.