Transcriptional output pathways of the circadian clock

描述（由申请人提供）：人体中的生物钟确保诸如睡眠/觉醒周期，体温，激素分泌，心血管功能，呼吸和代谢等功能的正确计时。哺乳动物和昆虫的生物钟之间存在着高度的守恒，我们目前对人类生物钟功能的理解，很大程度上是基于对模式生物（如果蝇）生物钟的基因和分子电路的研究。负责果蝇生物钟自主起搏器功能的转录反馈回路已被详细识别，但对这些回路与生理和行为中明显节律之间的联系途径知之甚少。该提议旨在验证果蝇时钟回路的CLK/CYC转录因子在果蝇大脑中产生行为节律的直接转录靶点的假设。具体目的：(1)将使用一组转基因果蝇来鉴定昼夜节律转录因子CLK/CYC的特异性转录靶点，这些转基因果蝇允许在生物钟细胞中单独诱导这些转录因子的表达。(2) CLK/CYC的直接转录靶点将通过荧光素酶报告基因测定和在果蝇S2细胞中进行的染色质免疫沉淀实验来确定。(3)利用转基因果蝇确定与大脑初级转录时钟输出相关的运动和羽化行为表型。意义：拟议的研究提供了对动物内部时间保持机制的见解，不仅与这些机制中断相关的疾病（睡眠障碍，癌症，糖尿病）有关，而且更普遍地涉及日常变化的身体功能的诊断和治疗程序。

DESCRIPTION (provided by applicant): Circadian clocks in the bodies of humans ensure that functions such as the sleep/wake cycle, body temperature, hormonal secretion, cardiovascular function, respiration and metabolism are timed correctly. There is an exceptionally high degree of conservation between the circadian clocks of mammals and insects and much of our current understanding of clock function in humans is based on studies of the genes and the molecular circuits of the clock in model organisms such as Drosophila. Transcriptional feedback circuits responsible for the autonomous pacemaker function of the Drosophila clock have been identified in detail, but much less is known about the pathways connecting these circuits to overt rhythms in physiology and behavior. This proposal is designed to test the hypothesis that the CLK/CYC transcription factor of the Drosophila clock circuits has direct transcriptional targets in the Drosophila brain that generate behavioral rhythms. Specific Aims: (1) Transcriptional targets specific to the circadian transcription factor CLK/CYC will be identified with the use of a set of transgenic flies that allow expression of each of these transcription factors to be induced individually in clock-bearing cells. (2) Direct transcriptional targets for CLK/CYC will be identified by luciferase reporter assays and chromatin immunoprecipitation experiments performed in Drosophila S2 cells. (3) Locomotor and eclosion behavior phenotypes associated with primary transcriptional clock output in the brain will be determined with the use of transgenic flies. Significance: The insight provided by the proposed studies into the internal time keeping mechanisms of animals is not only relevant to diseases associated with disruptions of these mechanisms (sleep disorders, cancer, diabetes), but also more generally to diagnostic and treatment procedures involving bodily functions that show regular daily variations.