Molecular, Cellular and Physiological Mechanisms of the Mammalian Circadian Clock

描述（由申请人提供）：生物钟被认为协调哺乳动物生理学的各个方面，例如新陈代谢和睡眠/觉醒周期。昼夜节律振荡器的遗传和分子表征表明，这些节奏的基础是互锁的转录/翻译反馈回路。最近的研究结果加强了这样一个概念，即初级反馈回路在生物钟功能中起着至关重要的作用。在该环中，两个bHLH-PAS反式激活因子Clock和Bmall使两个有效的转录抑制因子隐花色素1和隐花色素2异二聚化并刺激其昼夜节律表达。一旦翻译，Cry蛋白然后与Period和酪蛋白激酶1蛋白形成复合物，并易位到细胞核，在那里它们有效地抑制Clock/Bmall转录。这导致Cry基因转录的关闭，这最终导致Clock/BmaM复合物的“去阻遏”和约24小时后转录循环的重新启动。虽然已经观察到染色质重塑和组蛋白乙酰化和甲基化的变化伴随Cry蛋白抑制活性，但Cry蛋白抑制Clock/Bmall复合物的分子机制仍有待阐明。在这里，我们提出了分子，细胞和生理表征的机制，包括时钟功能的Cry抑制。此外，我们提出了一种小鼠模型，该模型与Cry介导的反馈抑制（昼夜节律钟敲除）解偶联，并使用该模型来研究昼夜节律振荡，而不是特定的时钟因子Clock和Bmall，是维持正常代谢功能所必需的假设。最后，我们提出了一种基于机制的策略，以确定和表征小分子的能力，通过对负反馈回路的影响，扰动振荡器功能。因此，该研究的成功完成将为生理学中的生物钟功能提供重要的动物模型，并为基于生物钟的小分子扰动机制奠定基础。

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.