A Dual Transcriptional and High Content Assay for Cryptochrome

隐花色素的双重转录和高内涵测定

• 批准号: 7170097
• 负责人: JOHN B HOGENESCH
• 金额: $ 19.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7170097
• 关键词: NIH Roadmap Initiative tag; chemical registry /resource; chromophore; circadian rhythms; genetic transcription; high throughput technology; messenger RNA; protein localization; protein structure function; sleep disorders; small molecule; technology /technique development; transcription factor

DESCRIPTION (provided by applicant): In mammals, the circadian clock (or core oscillator) regulates biological rhythms such as temperature, blood pressure, hormone secretion, metabolism, and sleep wake behavior to a precise twenty four hour cycle. Circadian sleep disorders, such as advanced sleep phase syndrome (ASPS) severely disrupt the sleep wake cycle and dependent physiology. Genetic, biochemical, and cellular evidence have demonstrated that these biological rhythms are governed by the mammalian circadian clock, which is composed of a cellular transcriptional/translational feedback loop. In the core loop, a heterodimeric complex of Clock and Bmal transactivators regulate expression of a battery of output genes including the Cryptochromes, Cry1 and Cry2. The Cryptochrome molecules subsequently translocate to the nucleus and potently repress the activity of the Clock/Bmal complex, thereby inhibiting their own mRNA expression. This process takes approximately twenty-four hours and comprises the primary feedback loop of the clock. Cryptochrome deficient mice display arrhythmic locomotor activity behavior, establishing their critical role in clock function. In plants and bacteria, Cryptochromes have also been shown to bind chromophores and act as photoreceptors that communicate light information to the circadian clock. However, conclusive experiments to establish a photoreceptor role for Cryptochrome in mammalian clock function have been complicated by the arrhythmic behavior of the knockout mice. What is needed is a method to transiently inactivate Cryptochrome's repression function. Small molecule tool compounds could address this question, but as yet, specific inhibitors of Cryptochrome have not yet been developed. To address this limitation, we propose to identify small molecule modulators of Cryptochrome that transiently and reversibly inactivate its transcriptional function. The approach we propose takes advantage of its potent repression of Clock and Bmal in cell based assays, and furthermore has the ability to inform the mechanism of this perturbation by following Cryptochrome protein levels and intracellular localization. In addition, these compounds would provide proof of concept in modulation of important circadian clock parameters by small molecules. Thus, successful completion of the proposed research would provide a tool to address an important scientific question, as well as lay the groundwork for mechanism-based perturbation of the clock.

描述(申请人提供)：在哺乳动物中，生物钟(或核心振荡器)调节生物节律，如温度、血压、激素分泌、新陈代谢和睡眠唤醒行为，精确到24小时周期。昼夜节律睡眠障碍，如晚期睡眠相综合征(ASPS)，严重扰乱睡眠觉醒周期和依赖的生理。遗传、生化和细胞证据表明，这些生物节律是由哺乳动物的生物钟控制的，而生物钟由细胞转录/翻译反馈环组成。在核心环中，时钟和BMAL反式激活子的异源二聚体复合体调节一系列输出基因的表达，包括隐花色素、Cry1和Cry2。随后，隐色素分子转移到细胞核，并有效地抑制Clock/BMAL复合体的活性，从而抑制其自身的mRNA表达。这一过程大约需要24小时，并包括时钟的主要反馈回路。隐色素缺陷小鼠表现出心律失常的运动行为，确立了它们在时钟功能中的关键作用。在植物和细菌中，隐花色素也被证明可以结合生色团，并作为光感受器向生物钟传递光信息。然而，确定隐花色素在哺乳动物时钟功能中的光感受器作用的结论性实验因基因敲除小鼠的心律失常行为而变得复杂。现在需要的是一种暂时使隐染色质抑制功能失效的方法。小分子工具化合物可以解决这个问题，但到目前为止，还没有开发出特定的隐色素抑制剂。为了解决这一局限性，我们建议识别小分子的隐色素调节剂，它可以瞬时和可逆地使其转录功能失活。我们提出的方法利用了它在基于细胞的分析中对时钟和BMAL的有效抑制，并且能够通过跟踪隐花色素蛋白水平和细胞内定位来告知这种扰动的机制。此外，这些化合物将为小分子调节重要的生物钟参数提供概念上的证据。因此，拟议研究的成功完成将提供一个工具来解决一个重要的科学问题，并为时钟的基于机制的扰动奠定基础。