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，在所有被研究的生物体中都存在，调节它们的行为、新陈代谢和生理的许多方面。例如，从人类的睡眠-觉醒周期和新陈代谢到植物的光合作用和开花等过程都受到生物钟的控制。虽然生物钟的遗传成分在不同的生物体中是不同的，但由多个连锁反馈环组成的基本结构在不同物种之间是保守的。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，在高等植物中，生物钟控制着各种生物过程，此外，已知-10%的拟南芥基因含量受昼夜节律调控。然而，在转录水平上时钟成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识别。虽然已经进行了大量的遗传筛选来识别新的时钟基因，但从已知时钟基因中分离出多个等位基因表明，这些筛选可能已经饱和。因此，本研究在现有拟南芥模型的基础上，利用功能基因组学方法对两个核心时钟基因TOCL和LUX的正向调控(激活)相关基因进行了鉴定和鉴定。简而言之，将使用最近可用的代表-2100预测的拟南芥转录因子(TF)的文库，进行高通量酵母试验以筛选TOCL和LUX的可能调节因子，以确定与这两个基因的启动子区域结合的组件。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，以确定这些调节器在时钟网络中的作用。这一策略最近已被成功地用于在拟南芥中发现一种新的转录因子，在拟南芥的生物钟的早晚之间提供了期待已久的联系。除了控制许多重要的生物过程外，时钟还控制着人类10%的基因。人类的一些疾病，如哮喘和不规律的睡眠-觉醒模式，与异常的生物节律有关。除了增加我们对拟南芥生物钟网络的了解外，这里提出的这项研究还可能有助于我们理解人类的生物钟，并可能帮助我们治疗相关疾病。

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.