Transcriptional output pathways of the circadian clock

描述（由申请人提供）：人体内的昼夜节律钟确保诸如睡眠/觉醒周期、体温、激素分泌、心血管功能、呼吸和代谢等功能正确定时。哺乳动物和昆虫的生物钟之间有着非常高的保守性，我们目前对人类生物钟功能的理解大部分是基于对模式生物（如果蝇）中生物钟的基因和分子电路的研究。负责果蝇时钟自主起搏器功能的转录反馈电路已经被详细确定，但对将这些电路连接到生理和行为中的明显节奏的途径知之甚少。这个建议的目的是测试的假设，果蝇时钟电路的CLK/CYC转录因子有直接的转录靶点在果蝇大脑中产生的行为节奏。具体目标：（1）将使用一组转基因果蝇来鉴定对昼夜节律转录因子CLK/CYC特异的转录靶标，所述转基因果蝇允许在携带时钟的细胞中单独诱导这些转录因子中的每一种的表达。(2)CLK/CYC的直接转录靶标将通过在果蝇S2细胞中进行的荧光素酶报告基因测定和染色质免疫沉淀实验来鉴定。(3)将使用转基因果蝇确定与脑中初级转录时钟输出相关的运动和羽化行为表型。重要性：拟议的研究提供了对动物内部时间保持机制的洞察力，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，而且更普遍地涉及身体功能的诊断和治疗程序，显示出规律的日常变化。

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.