Investigation of TOC1 function in the Arabidopsis Circadian Clock

描述（由申请人提供）：大多数经历日常光/暗或温度循环的生物体都有一个称为昼夜节律钟的分子振荡器。生物钟对于协调每天特定时间的生理活动和过程非常重要。重要的是，生物钟在正常运作时提供了健康优势。生物时钟的分子和生化机制尚不清楚，但很明显，生物时钟的基本结构与真核生物相似。本研究的目的是利用拟南芥作为研究真核生物生物钟的模型。具体来说，中心时钟组件CAB表达1 （TOC1）的生化作用仍然是拟南芥生物钟领域的一个悬而未决的问题。在本提案中，最近发现的与TOC1相互作用的转录因子CCA1启动子远足探险1 （CHE1）将用于确定TOC1的生化作用。这项工作将连接拟南芥生物钟的核心组成部分，并将增加我们对真核生物时钟结构的理解。具体目标确定TOC1和ZTL在CHE调控中的作用。TOC1与CHE的相互作用是TOC1与CCA1表达控制之间的第一个生化联系。生物化学、瞬时转录测定和遗传学将用于确定TOC1和ZTL在CHE功能调节中的作用。2. 调控LHY和CCA1表达的转录复合物的发现。Kay实验室已经建立了拟南芥所有转录因子的文库。该文库将通过酵母双杂交筛选，寻找与TOC1或ZTL相互作用的转录因子。一套简化的体内生化和分子技术将用于优先排序和分类与TOC1/ZTL相互作用但也调节LHY和CCA1表达的转录因子。公共卫生相关性：众所周知，生物钟缺陷会导致一系列与人类健康相关的疾病，生物钟成分与癌症进展有关，癌症会减弱生物钟功能。这项研究的结果将增加我们对真核生物生物钟结构的理解，从而与生物钟相关疾病的研究和治疗有关。

DESCRIPTION (provided by applicant): Most organisms that experience daily light/dark or temperature cycles have a molecular oscillator called the circadian clock. The circadian clock is important for coordinating daily physiological activities and processes with specific times of day. Importantly, the circadian clock provides fitness advantage when it functions correctly. The molecular and biochemical mechanisms of the circadian clock are not well understood, but it is clear that the basic architecture of circadian clocks is similar in eukaryotes. The objective of this proposal is to use Arabidopsis as a model to study eukaryotic circadian clocks. Specifically, the biochemical role of the central clock component, TIMING OF CAB EXPRESSION 1 (TOC1), remains an outstanding question in the Arabidopsis circadian clock field. In this proposal, the recently discovered TOC1-interacting transcription factor, CCA1 PROMOTER HIKING EXPEDITION 1 (CHE1) will be used to determine the biochemical role of TOC1. This work will connect the core components of the Arabidopsis circadian clock and will increase our understanding of eukaryotic clock architecture. Specific Aims 1. Determine the roles of TOC1 and ZTL in CHE regulation. The interaction of TOC1 and CHE is the first biochemical link between TOC1 and control of CCA1 expression. Biochemistry, transient transcription assays, and genetics will be used to determine the role of TOC1 and ZTL in regulation of CHE function. 2. Discovery of transcriptional complexes that regulate LHY and CCA1 expression. A library of all transcription factors from Arabidopsis has been created in the Kay lab. This library will be screened by yeast two-hybrid to find transcription factors that interact with TOC1 or ZTL. A streamlined set of in vivo biochemical and molecular techniques will be used to prioritize and categorize the transcription factors that interact with TOC1/ZTL but also regulate LHY and CCA1 expression. Public Health Relevance: Circadian clock defects are known to result in a range of human health related disorders, and clock components are involved in cancer progression and cancer attenuates clock function. The results of this study will increase our understanding of circadian clock architecture in eukaryotes and thus will be relevant to the study and treatment of clock related diseases.