A Functional Genomics Approach to Identify Regulators of Arabidopsis Clock Genes

描述（由申请人提供）：生物钟是迄今为止研究的所有生物体中发现的内部计时器，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，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生物体中存在差异，但由多个互锁反馈环组成的基本结构在物种中是保守的。这项提案的长期目标是了解生物钟网络是如何在真核生物中构建的。为了实现这一点，这里提出的实验将在植物模型中进行。与人类一样，生物钟控制着高等植物中的多种生物过程，此外，已知约10%的拟南芥基因内容受昼夜节律调节。然而，在转录水平上的时钟组件之间的直接调控关系是未知的，这表明新的时钟基因尚未被确定。尽管已经进行了许多遗传筛选以鉴定新的时钟基因，但已知时钟基因中的多个等位基因的分离表明这些筛选可能是饱和的。因此，建立在目前的模式，在拟南芥中，本研究的具体目的是确定和表征的基因参与正调控（激活）的两个核心时钟基因TOCl和LUX使用功能基因组学方法。简言之，将进行高通量酵母测定以筛选TOCl和LUX的推定调节子，使用最近可获得的代表约2100个预测的拟南芥转录因子（TF）的文库，以鉴定结合这两个基因的启动子区的组分。接下来，将进行一系列涉及突变筛选和昼夜节律输出测定的实验，以确定这些调节因子在时钟网络中的作用。这种策略最近已被用来成功地确定一种新的转录因子在拟南芥提供了期待已久的联系之间的昼夜节律钟在拟南芥的早晨和晚上的循环。除了控制许多重要的生物过程外，生物钟还控制着人类约10%的基因。人类的几种疾病，如哮喘和不规则的睡眠-觉醒模式与异常的生物节律有关。沿着我们对拟南芥生物钟网络的了解，这里提出的研究可能有助于我们理解人类的生物钟，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.