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的各个方面，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特征表明，互锁的转录/翻译反馈回路是这些节律的基础。最近的研究结果强化了初级反馈回路在生物钟功能中起着至关重要作用的观点。在这个循环中，两个bHLH-PAS转录激活因子Clock和Bmall异源二聚体化并刺激两个有效的转录抑制因子Cryptochrome 1和Cryptochrome 2的昼夜表达。一旦被翻译，Cry蛋白就会与周期蛋白和酪蛋白激酶1蛋白形成复合物，并转运到细胞核，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，最终导致Clock/BmaM复合物的“去抑制”，并在-24小时后重新启动转录周期。虽然已经观察到Cry蛋白抑制活性伴随着染色质重塑和组蛋白乙酰化和甲基化的变化，但Cry蛋白抑制Clock/Bmall复合体的分子机制仍有待阐明。在这里，我们提出了包括哭泣抑制在内的时钟功能机制的分子、细胞和生理特征。此外，我们建议建立一种与哭泣介导的反馈抑制（生物钟敲除）分离的小鼠模型，并使用该模型来研究维持正常代谢功能所需的昼夜节律振荡，而不是特定的生物钟因子clock和Bmall的假设。最后，我们提出了一种基于机制的策略，通过对负反馈回路的影响来识别和表征具有扰动振荡器函数能力的小分子。因此，本研究的成功完成将为生理上解释生物钟功能提供一个重要的动物模型，并为基于生物钟小分子扰动机制的研究奠定基础。

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.