Genetic and Molecular Dissection of Regulatory Mechanisms Underlying Temperature and Nutritional Compensation of the Circadian Clock in Neurospora crassa

项目摘要/摘要： 生物钟提供计时功能，以预测环境的日常变化，并被使用 地球上几乎所有的真核生物。~24小时的昼夜节律周期长度针对波动进行了缓冲 在外部条件下，包括温度和营养水平，在一种普遍存在的现象中称为 补偿。周期补偿背后的分子机制还不是很清楚。 真核生物。该项目的长期目标是了解温度补偿(TC)， 营养补偿(NC)以及补偿效应器和核心之间的分子相互作用 时钟网络使用了真核生物粗糙脉孢菌的模型。目前关于昼夜节律的许多知识 生物学来自脉孢子虫，因为它的核心时钟网络在功能上与哺乳动物同源 钟。在真菌和动物细胞中，酪蛋白激酶活性是正常TC所必需的，但额外的调节因子 对于TC和CKS磷酸化的特定靶点还没有进行系统的研究。这个 时钟的周期也被不同浓度的葡萄糖补偿，这涉及到转录 脉孢子虫的抑制器机械。目前尚不清楚TC和NC的调控途径是否不同，或者 在环境条件存在波动的情况下，核心时钟也会进行类似的修改。我建议1) 通过利用基因敲除集合系统地筛选在TC和NC中有缺陷的突变 脉孢菌模型系统；2)利用化学遗传学和质谱法鉴定TC过程中酪蛋白激酶2的靶点 光谱学方法；以及3)核心时钟网络和补偿效应器之间的相互作用的模型 以确定计算扰动是否与昼夜周期长度的实验数据相匹配。的目标是 这项工作是为了描述补偿，这是真核生物生物钟的一个定义特征。有睡眠的人类 疾病或轮班工作暴露，或不断经历时差，患癌症的风险更高， 心血管疾病，或代谢综合征，由于它们基于细胞的慢性不同步 分子时钟周期和环境光：暗周期。因此，改进了对 如何缓冲时钟对环境变化的影响将具有诊断和治疗作用。

Project Summary/Abstract: The circadian clock serves a time-keeping function to anticipate daily changes in the environment and is used by almost every eukaryote on the planet. The ~24-hour circadian period length is buffered against fluctuations in external conditions, including temperature and nutrient levels, in a ubiquitous phenomenon called compensation. The molecular mechanism underlying period compensation is not well understood in any eukaryotic organism. The long-term goal of this project is to understand temperature compensation (TC), nutritional compensation (NC), and the molecular interactions between compensation effectors and the core clock network using the model eukaryote Neurospora crassa. Much of the current knowledge on circadian biology has come from Neurospora, as its core clock network is functionally homologous to the mammalian clock. Casein kinase activity is required for normal TC in both fungal and animal cells, but additional regulators of TC and the specific targets for phosphorylation by CKs have not been systematically investigated. The clock’s period is also compensated to different concentrations of glucose, which involves transcriptional repressor machinery in Neurospora. It is currently unknown if TC and NC regulatory pathways are distinct, or if the core clock is modified similarly in the presence of fluctuations in environmental conditions. I propose to 1) systematically screen for mutants with defects in TC and NC by leveraging the knockout collection of the Neurospora model system; 2) identify Casein Kinase 2 targets during TC using a chemical genetics and mass spectrometry approach; and 3) model interactions between the core clock network and compensation effectors to determine if computational perturbations match experimental data on circadian period length. The goal of this work is to characterize compensation, a defining feature of eukaryotic circadian clocks. Humans with sleep disorders or shift work exposure, or who continually experience jet-lag, have a higher risk to develop cancer, cardiovascular disease, or metabolic syndrome due to chronic dyssynchrony between their cell-based molecular clocks cycling and environmental light:dark cycles. Thus, an improved mechanistic understanding of how the clock is buffered against environmental changes will have diagnostic and therapeutic utility.