Molecular, Cellular and Physiological Mechanisms of the Mammalian Circadian Clock

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DESCRIPTION (provided by applicant): The circadian clock is thought to coordinate aspects of mammalian physiology such as metabolism and the sleep wake cycle. Genetic and molecular characterization of the circadian oscillator shows that interlocked transcriptional/translational feedback loops underlie these rhythms. Recent findings have reinforced the notion that the primary feedback loop plays a vital role in circadian clock function. In this loop, two bHLH-PAS transactivators, Clock and Bmall, heterodimerize and stimulate circadian expression of two potent transcriptional repressors, Cryptochrome 1 and Cryptochrome 2. Once translated, Cry proteins then form a complex with the Period and casein kinase 1 proteins, and translocate to the nucleus where they potently represses Clock/Bmall transcription. This results in the shut down of Cry gene transcription, which eventually results in 'de-repression' of the Clock/BmaM complex and re-initiation of the transcriptional cycle after -24 hours. Although changes in chromatin remodeling and histone acetylation and methylation have been observed to accompany Cry protein repression activity, the molecular mechanism by which Cry proteins act to repress the Clock/Bmall complex remains to be elucidated. Here we propose molecular, cellular, and physiological characterization of the mechanisms underlying clock function including Cry repression. Furthermore, we propose generation of a mouse model that is uncoupled from Cry-mediated feedback repression (a circadian clock knockout) and use this model to investigate the hypothesis that circadian oscillation, rather than the specific clock factors Clock and Bmall, is required to maintain normal metabolic function. Finally, we propose a mechanism-based strategy to identify and characterize small molecules with the capacity to perturb oscillator function via effects on the negative feedback loop. Thus successful completion of the proposed research would result in an important animal model for ascribing circadian clock function in physiology, as well as lay the foundation for mechanism based small molecule perturbation of the clock.