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

项目概要/摘要： 生物钟具有计时功能，可以预测环境中的每日变化， 地球上几乎所有的真核生物。~24小时的昼夜节律周期长度对波动有缓冲作用 在外部条件下，包括温度和营养水平，在一个普遍存在的现象， 赔偿周期补偿背后的分子机制在任何一个领域都没有得到很好的理解。 真核生物本项目的长期目标是了解温度补偿（TC）， 营养补偿（NC），以及补偿效应物与核心之间的分子相互作用 时钟网络使用的模式真核生物粗糙脉孢菌。目前关于昼夜节律的知识 生物学来自脉孢菌，因为其核心时钟网络在功能上与哺乳动物同源。 时钟酪蛋白激酶活性是真菌和动物细胞中正常TC所必需的，但额外的调节剂 TC的磷酸化作用和CK磷酸化的特异性靶点尚未得到系统研究。的 生物钟的周期也对不同浓度的葡萄糖进行补偿，这涉及到转录 脉孢菌中的阻遏物机制。目前尚不清楚TC和NC调节途径是否不同，或者 在环境条件中存在波动的情况下，类似地修改核心时钟。我建议1） 通过利用基因敲除收集系统地筛选TC和NC缺陷突变体， 链孢霉模型系统; 2）使用化学遗传学和质谱技术鉴定TC期间的酪蛋白激酶2靶标 光谱方法; 3）核心时钟网络和补偿效应器之间的模型相互作用 以确定计算扰动是否与昼夜节律周期长度的实验数据相匹配。的目标 这项工作是为了表征补偿，真核生物昼夜节律钟的定义特征。人类睡眠 紊乱或轮班工作，或持续经历时差，患癌症的风险更高， 心血管疾病或代谢综合征，由于它们的细胞之间的慢性不同步， 分子钟循环和环境光：暗循环。因此，一个更好的机械理解， 时钟如何缓冲环境变化将具有诊断和治疗效用。

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.