A Functional Genomics Approach to Identify Regulators of Arabidopsis Clock Genes

描述（由申请人提供）：生物钟是一种内部计时器，存在于迄今为止研究的所有生物体中，调节其行为，代谢和生理的许多方面。例如，人类的睡眠-觉醒周期和新陈代谢到植物的光合作用和开花等过程都是由生物钟控制的。虽然生物钟的遗传成分在不同的生物中是不同的，但由多个连锁反馈回路组成的基本结构在不同物种中是保守的。这项提议的长期目标是了解真核生物的生物钟网络是如何构建的。为了实现这一点，这里提出的实验将在拟南芥植物模型中进行。与人类一样，高等植物的生物钟控制着多种生物过程，此外，已知-10%的拟南芥基因含量受昼夜节律调节。然而，在转录水平上，时钟组分之间的直接调控关系尚不清楚，这表明新的时钟基因尚未被发现。尽管已经进行了许多遗传筛选以鉴定新的时钟基因，但已知时钟基因中多个等位基因的分离表明这些筛选可能已经饱和。因此，在拟南芥现有模型的基础上，本研究的具体目的是利用功能基因组学方法鉴定和表征参与两个核心时钟基因TOCl和LUX正调控（激活）的基因。简而言之，高通量酵母试验将用于筛选TOCl和LUX的推定调节因子，使用最近可用的-2100预测拟南芥转录因子（TFs）文库，以确定与这两个基因的启动子区域结合的成分。接下来，将进行一系列涉及突变筛选和昼夜节律输出分析的实验，以确定这些调节因子在生物钟网络中的作用。这一策略最近被用于在拟南芥中成功鉴定一种新的转录因子，为拟南芥生物钟的早晚循环提供了期待已久的联系。除了控制许多重要的生物过程外，生物钟还控制着人类10%的基因。人类的一些疾病，如哮喘和不规则的睡眠-觉醒模式，都与异常的生物节律有关。随着我们对拟南芥生物钟网络的了解的增加，这里提出的研究可能有助于我们对人类生物钟的理解，并可能治疗相关疾病。

DESCRIPTION (provided by applicant): The circadian clock is an internal timekeeper found in all organisms studied to date regulating many aspects of their behavior, metabolism and physiology. For example, processes such as the sleep-wake cycle and metabolism in humans to photosynthesis and flowering in plants are all controlled by the circadian clock. Although genetic components of the circadian clock are known to differ across organisms, the basic architecture consisting of multiple interlocking feedback loops is conserved across species. The long-term goal of this proposal is to understand how the circadian clock network is constructed in eukaryotes. To achieve this, the experiments proposed here will be conducted in the plant model Arabidopsls. As in humans, the circadian clock controls a variety of biological processes in higher plants, and furthermore, -10% of the Arabidopsis gene content is known to be circadian regulated. However, the direct regulatory relationship between clock components at the transcription level is unknown suggesting that new clock genes are yet to be identified. Though numerous genetic screens to identify new clock genes have been performed, isolation of multiple alleles in the known clock genes indicate that these screens are likely saturated. Therefore, building on the current model in Arabidopsis, the specific aim of this study is to identify and characterize the genes involved in positive regulation (activation) of two core clock genes TOCl and LUX using a functional genomics approach. Briefly, a high throughput yeast assay will be performed to screen for putative regulators of TOCl and LUX, using a recently available library representing -2100 predicted Arabidopsis transcription factors (TFs), to identify components that bind to the promoter region of these two genes. Next, a battery of experiments involving mutational screens and circadian output assays will be conducted to establish the role of these regulators in the clock network. This strategy has recently been used to successfully identify a novel transcription factor in Arabidopsis providing a long awaited link between the morning and evening loops of the circadian clock in Arabidopsis. In addition to the controlling many important biological processes, the clock also controls -10% of the genes in humans. Several disorders in humans such as asthma and irregular sleep-wake patterns are linked to abnormal biological rhythms. Along with adding to our knowledge of the clock network in Arabidopsis, the study proposed here could contribute to our understanding of the circadian clock in humans and possibly treatment for related disorders.