Role of Phosphorylation in Determining Circadian Period Length and Temperature Compensation

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PROJECT SUMMARY The 24-hour cycles of endogenous regulation of physiology, known as circadian rhythms, align organisms to the inherent rotation of the earth. Circadian rhythms influence the regulation of many essential processes, including sleep regulation. Sleep is regulated by circadian rhythms in combination with sleep homeostatic mechanisms in the two-component sleep model. This proposal aims to further our understanding of sleep regulation by investigating key gaps that remain in the molecular mechanism of circadian rhythms, so that we may understand the underlying mechanisms of circadian-based sleep disorders. Filling these gaps will therefore enable us to develop therapies for sleep disorders. Circadian rhythms are defined in part by a ~24-hour period length, and their ability to maintain a consistent period across changes in ambient temperature (temperature compensation – TC). Despite the fundamentality of these properties, questions remain regarding their underlying mechanisms. We will investigate how the feedback loop powering the molecular clock completes a cycle (determining period length) in Aim 1, and the role for kinases and specific phosphorylation events in the TC mechanism in Aim 2. Current evidence suggests that both of these properties are ultimately regulated by phosphorylation. We hypothesize that the mechanism underlying period determination is conserved from Neurospora to mammals, and hence the feedback loop of the mammalian clock is closed by hyperphosphorylation of negative elements, rather than degradation, matching what our lab found in Neurospora. Aim 1 tests our hypothesis that phosphorylation status and not stability of the clock protein PER2 determines period length in mammalian cells. We hypothesize that TC involves precise and dynamic phosphorylation of key clock components by Casein Kinase I and II. Aim 2 uses a combination of phosphoproteomics and epistasis experiments with novel Neurospora strains to establish an integrative TC model that we will then test in mammalian cells in culture, again expecting mechanistic conservation. Overall, this project will shed light on fundamental aspects of circadian rhythms yet to be elucidated, ultimately establishing new models for both period determination and TC. Therefore, successful completion of this work will inform the understanding of all circadian-based disorders, including disorders such as Familial Advanced Sleep Phase Syndrome (FASPS). This fellowship will enable me to fill gaps in my training to become an independent academic scientist. Training will include participation in international conferences, mass spectrometry and modeling courses, meetings with my advisors, thesis committee, and collaborators, and training in teaching techniques, among others. I will take advantage of the rigorous environment at Dartmouth through departmental seminars and be supported in my technical training by our excellent core facilities. My immediate research environment will be the Dunlap/Loros lab that has for decades been a center for studying the molecular basis of circadian rhythms.