Molecular Mechanisms in the Arabidopsis Circadian Clock

描述（由申请人提供）：本提案的长期目标是了解生物钟网络如何在真核细胞中构建。已知生物钟调节广泛分布在包括人类在内的生物系统中的许多基本细胞过程。在高等植物中，生物钟网络调节着从光合作用到细胞伸长再到控制开花时间的各种过程。我们选择拟南芥作为模式生物，并已确定了几个时钟基因的遗传筛选。这些时钟组件之间的相互调节定义了一个初始的分子反馈回路，形成了高等植物多回路时钟网络的详细模型的基础。我们在之前的所有具体目标上都取得了实质性进展，包括几个新的时钟基因的鉴定和表征。该提案中的实验旨在通过继续识别参与调节已知关键时钟组件（如CCA 1和TOC 1）转录的时钟因子，建立当前的时钟模型。为此，我们创建了一个独特的功能基因组学资源，包括200多个循环转录因子的文库，可用于检测DNA结合以及蛋白质-蛋白质相互作用。这导致了一种新的转录因子TCP 21的发现，结合到CCA 1启动子。该提案旨在表征TCP 21并在核心网络中识别其他转录调节因子。我们还确定了一种新的时钟转录因子LUX，其特征在于它的DNA靶基因和相互作用的合作伙伴。我们建议广泛表征时间依赖性蛋白质-蛋白质相互作用和翻译后机制，这些机制为时钟的转录组分增加了关键的额外控制层。最后，我们计划使用系统生物学方法来表征时钟输出网络的逻辑。我们希望探索植物和动物时钟网络之间系统架构的相似性。鉴于昼夜节律调节生理学的普遍性，模式生物中昼夜节律系统的表征将影响我们对与人类福祉的许多已知特征相关的起搏器机制和故障的理解。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand how circadian clock networks are constructed in eukaryotic cells. Circadian clocks are known to regulate many essential cellular processes widely distributed across biological systems, including humans. In higher plants, the clock network regulates diverse processes ranging from photosynthesis to cell elongation to the control of flowering time. We have chosen Arabidopsis as a model organism and have identified several clock genes from genetic screens. The reciprocal regulation amongst these clock components defined an initial molecular feedback loop which forms the basis for elaborated models of multiple loop clock networks in higher plants. We have made substantial progress on all of the previous specific aims, including the identification and characterization of several new clock genes. The experiments in this proposal aim to build on the current clock models by continuing to identify clock factors involved in regulating the transcription of known key clock components such as CCA1 and TOC1. To this end, we have created a unique functional genomics resource consisting of a library of more than 200 cycling transcription factors that can be used to detect DNA binding as well as protein-protein interactions on a spectrum of targets. This has led to the discovery of a novel transcription factor TCP21 that binds to the CCA1 promoter. This proposal aims to characterize TCP21 and identify additional transcriptional regulators within the core network. We have also identified a novel clock transcription factor LUX that will be characterized in terms of its DNA target genes and interaction partners. We propose to extensively characterize the time dependent protein-protein interactions and post- translational mechanisms that add critical additional layers of control to the transcriptional components of the clock. Finally, we plan to use a systems biology approach to characterize the logic underlying the output networks of the clock. We wish to explore the similarities in system architecture between plant and animal clock networks. Given the ubiquity of circadian-regulated physiology, characterization of circadian systems in model organisms will impact our understanding of the pacemaker mechanisms and malfunctions associated with many known features of human well-being.