Investigation of TOC1 function in the Arabidopsis Circadian Clock

描述(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分子振荡器，称为生物钟。生物钟对于协调日常生理活动和过程与一天中特定的时间是很重要的。重要的是，当生物钟正常工作时，它会提供健身优势。生物钟的分子和生化机制还不是很清楚，但很明显，生物钟的基本结构在真核生物中是相似的。这项建议的目的是利用拟南芥作为模型来研究真核生物的生物钟。具体地说，中央时钟组分的生化作用，CAB表达的定时1(TOC1)，在拟南芥昼夜节律时钟领域仍然是一个悬而未决的问题。在这个提案中，最近发现的与TOC1相互作用的转录因子CCA1启动子徒步旅行1(CHE1)将被用来确定TOC1的生化作用。这项工作将连接拟南芥昼夜节律时钟的核心组件，并将增加我们对真核生物时钟结构的理解。具体目的1.确定TOC1和ZTL在胆碱酯酶调节中的作用。TOC1与胆碱酯酶的相互作用是TOC1与CCA1表达调控之间的第一个生化环节。生物化学、瞬时转录试验和遗传学将被用来确定TOC1和ZTL在胆碱酯酶功能调节中的作用。2.发现了调控LHY和CCA1表达的转录复合体。Kay实验室已经创建了一个包含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，以寻找与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对与TOC1/ZTL相互作用但也调节LHY和CCA1表达的转录因子进行优先排序和分类。公共卫生相关性：生物钟缺陷已知会导致一系列与人类健康相关的疾病，而时钟成分与癌症的进展有关，癌症会削弱时钟功能。这项研究的结果将加深我们对真核生物生物钟结构的理解，从而对时钟相关疾病的研究和治疗具有重要意义。

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.