Transcriptional output pathways of the circadian clock

描述(申请人提供)：人体的生物钟确保睡眠/清醒周期、体温、荷尔蒙分泌、心血管功能、呼吸和新陈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异常高度的保守性，我们目前对人类生物钟功能的大部分了解都是基于对果蝇等模式生物中时钟的基因和分子电路的研究。负责果蝇生物钟自主起搏功能的转录反馈电路已经被详细识别，但关于这些电路与生理和行为中公开节律的通路知之甚少。这一建议是为了验证这样一个假设，即果蝇时钟电路的CLK/CyC转录因子在果蝇大脑中具有直接的转录靶标，从而产生行为节律。具体目标：(1)通过使用一组转基因果蝇来鉴定针对昼夜节律转录因子CLK/CyC的转录靶标，这些转基因果蝇允许在时钟承载细胞中单独诱导每一种转录因子的表达。(2)通过荧光素酶报告实验和染色质免疫沉淀实验在果蝇S2细胞中鉴定CLK/CyC的直接转录靶点。(3)将使用转基因果蝇来确定与大脑中初级转录时钟输出相关的运动和羽化行为表型。意义：拟议中的研究提供了对动物内部计时机制的见解，不仅与与这些机制中断相关的疾病(睡眠障碍、癌症、糖尿病)有关，而且更广泛地涉及到涉及身体功能的诊断和治疗程序，这些程序显示出规律性的每日变化。

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.