Identification and Analysis of Circadian Clock-Controlled Genes

 描述(申请人提供)：我们的长期目标是用遗传学和生物化学的语言描述组成细胞内昼夜节律系统的反馈周期和途径-它们如何工作，它们如何与环境同步，以及它们产生的时间信息如何被用来调节细胞的行为。这项建议侧重于模型系统脉孢子虫以及小鼠和哺乳动物细胞系，以了解细胞生理和新陈代谢的昼夜节律控制的范例。我们还继续进行一项长期的努力，旨在了解脉孢子菌的昼夜节律光接收和光生物学，并利用这一点来开辟一种显著的真菌病原体的新领域。我们的工作有三个重点。一个焦点是使用RNA测序、染色质免疫沉淀和生物信息学来描述控制细胞中转录的昼夜节律调节的调控层次，对脉孢子菌的昼夜节律输出网络进行全球分析和描述。然后，利用脉孢菌提供的生化遗传学的特殊背景，我们将跟踪由时钟控制基因编码的蛋白质进行的代谢活动，随着时钟控制活动的焦点出现，我们将开始铺设 脉孢子菌代谢图上时钟控制过程的光谱，以了解时钟如何调节新陈代谢和生理。在第二个焦点中，我们将把我们关于昼夜节律输出途径的知识应用于哺乳动物细胞，使用RNA测序来确定wt和RIP140基因敲除小鼠的脂肪细胞和巨噬细胞中时钟控制基因的昼夜节律分布。我们将使用RNA-SEQ来表征WT和突变细胞的转录本，并使用染色质免疫沉淀来开始剖析这种共激活/共抑制因子在这些重要细胞类型的昼夜生物学中的作用。这些实验将探索昼夜节律对哺乳动物脂肪代谢和免疫功能的意义，以期深入了解人类糖尿病、代谢综合征和免疫功能的时间差异。第三个关注点是光生物学。我们将探索光激活真菌主要光感受器的机制，并扩展对重要真菌病原菌烟曲霉的光生物学分析。我们设想并将测试一种方法，利用我们对真菌光生物学的理解，为数十万曲霉病患者提供一种新的治疗方法。这些项目是互补和相互丰富的，因为它们各自依靠遗传和分子技术来剖析并最终理解细胞对其环境的反应以及真核细胞的组织随着时间的变化。

 DESCRIPTION (provided by applicant): Our long term goal is to describe in the language of genetics and biochemistry the feedback cycles and pathways that comprise intracellular circadian systems - how they work, how they are synchronized with the environment, and how time information generated by them is used to regulate the behavior of cells. This proposal focuses on the model system Neurospora, as well as on the mouse and mammalian cell lines, to understand the paradigms underlying circadian control of cell physiology and metabolism. We also continue a longstanding effort aimed at understanding circadian photoreception and photobiology in Neurospora and use this to break new ground on a salient fungal pathogen. Our work has three foci. One Focus anticipates a global analysis and description of the circadian output network in Neurospora, using RNA sequencing, chromatin immunoprecipitation, and bioinformatics to describe the regulatory hierarchy governing circadian regulation of transcription in a cell. Then, using the exceptional background of biochemical genetics available in Neurospora, we will track the metabolic activities carried out by the proteins encoded by clock-controlled genes, and as foci of clock-controlled activities emerge, we will begin to lay the spectrum of clock-controlled processes on the Neurospora metabolic map to see how the clock regulates metabolism and physiology. In the second Focus, we will apply our knowledge of circadian output pathways to mammalian cells, using RNA sequencing to determine the circadian profile of clock-controlled genes in adipocytes and in macrophages from wt and RIP140 knockout mice. We will use RNA-seq to characterize the transcriptomes of WT and mutant cells and use chromatin immunoprecipitation to begin to dissect the role of this co-activator/co-repressor in the circadian biology of these important cell types. These experiments will probe the significance of circadian regulation to fat metabolism and to immune function in a mammal, with the hope of gaining insights into the incidence in humans of diabetes, metabolic syndrome, time-of-day differences in immune function. A third Focus is in photobiology. We will explore the mechanism whereby light activates the principal fungal photoreceptor, and extend analysis of photobiology in the important fungal pathogen Aspergillus fumigatus. We envision and will test a way to exploit our understanding of fungal photobiology to enable a new treatment for hundreds of thousands of patients with aspergillosis. These projects are complementary and mutually enriching in that they each rely on genetic and molecular techniques to dissect, and ultimately to understand, the response of cells to their environment and the organization of eukaryotic cells as a function of time.