Molecular Mechanisms in the Arabidopsis Circadian Clock

描述(申请人提供)：生物钟是一种内源性分子振荡器，周期约为24小时，几乎普遍存在于细菌、真菌、植物和动物物种中。外部刺激，如光或温度，是确保昼夜节律振荡器与当地环境精确共振的夹带线索。自我维持的节律由时钟网络体系结构通过多个互锁的转录反馈环和广泛的翻译后调节来维持。强大的昼夜节律网络根据环境节奏协调生化、生理和行为反应，以优化资源分配和增强健康。我们建议采用遗传学、生物化学、功能基因组学和计算方法的组合来确定构成多层时钟网络的组件和分子机制。通过这项拨款建议的工作，我们将获得直接洞察的领域包括环境输入时钟的基本感觉路径、昼夜网络动态以及输出的机械控制。通过筛选具有核心时钟启动子的全面转录因子文库，我们已经确定了介导对温度输入时钟的感知和转录反应的假定元件。我们将探索它们在与昼夜节律振荡器有关的温度相关现象(夹带、门控和补偿)中的作用。此外，最近在实验室的工作已经确定了关键转录因子TOC1和LUX的生化特性，这为我们理解它们在时钟中的作用提供了重要的进展。我们建议通过生化和分子方法进一步表征它们活动的机制基础，并在基因组水平上探索它们的功能，以了解它们在时钟输出控制中的作用。最后，虽然网络中的健壮性部分建立在冗余的基础上 这种冗余性阻碍了我们通过基因扰动识别新因素并了解其功能的能力。为了绕过这一障碍，我们开发了一种新的计算方法，通过挖掘微阵列数据来识别多基因家族的功能特异性。我们建议将该方法扩展到所有拟南芥转录因子家族，并在影响昼夜节律网络的初步候选基因上验证该方法。这一新工具可以广泛应用于任何具有微阵列表达数据的生物体，以识别可以分离密切相关的同源物或多基因家族成员的功能的扰动。继续努力，如这里提出的工作和我们实验室正在进行的研究，以阐明植物生物钟的分子机制，将补充其他系统中的类似分析，最终转化为我们对生物钟生物学的理解，以影响人类生物钟疾病的治疗。

DESCRIPTION (provided by applicant): The circadian clock is an endogenous molecular oscillator with a period of approximately 24 hours that is present nearly ubiquitously in bacterial fungal, plant, and animal species. External stimuli, such as light or temperature, are entrainment cues that ensure that the circadian oscillator is in precise resonance with the local environment. Self-sustained rhythms are maintained by clock network architecture through multiple, interlocked transcriptional feedback loops and extensive post-translational regulation. The robust circadian network coordinates biochemical, physiological, and behavioral responses with environmental rhythms to optimize resource allocation and increase fitness. We propose to deploy a combination of genetics, biochemistry, functional genomics, and computational approaches to identify the components and molecular mechanisms that underlie the multilayered clock network. Areas that we will gain direct insight into through the work proposed in this grant include the fundamental sensory pathways for environmental input into the clock, circadian network dynamics, and mechanistic control of outputs. By screening a comprehensive transcription factor library with core clock promoters, we have identified putative elements that mediate the perception and transcriptional responses to temperature inputs into the clock. We will explore their roles in temperature- associated phenomenon (entrainment, gating, and compensation) in relation to the circadian oscillator. Also, recent work in the laboratory has identified biochemical properties of key transcription factors, TOC1 and LUX, which provide important advances in our understanding of their roles within the clock. We propose to further characterize the mechanistic underpinnings of their activities through biochemical and molecular approaches, as well as explore their function on a genomic level to understand their role in the control of clock outputs. Finally, while robustness in networks is partially built on redundancy of components, this redundancy hinders our ability to identify new factors and understand their function through genetic perturbations. To circumvent this obstacle, we have developed a new computational approach for identifying functional specificity in multi-gene families by mining microarray data. We propose to expand this approach on all Arabidopsis transcription factor families and validate the approach on preliminary candidates affecting the circadian network. This new tool can be broadly applied to any organism with microarray expression data to identify perturbations that can separate the function of closely related homologs or members of a multi-gene family. Continued efforts such as the work proposed here and the on-going research in our laboratory to elucidate the molecular mechanisms of the plant circadian clock will complement similar analyses in other systems, ultimately translating our understanding of circadian biology to impact the treatment of human circadian disorders.