Identification and Analysis of Circadian Clock-Controlled Genes

 描述（由申请人提供）：我们的长期目标是用遗传学和生物化学的语言描述构成细胞内昼夜节律系统的反馈循环和途径-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用于调节细胞的行为。该建议侧重于模型系统脉孢菌，以及小鼠和哺乳动物细胞系，以了解细胞生理和代谢的昼夜节律控制的基本模式。我们还继续长期的努力，旨在了解脉孢菌的昼夜光感受和光生物学，并利用这一点在一个突出的真菌病原体上开辟新的天地。我们的工作有三个重点。一个焦点预计在脉孢菌的昼夜输出网络的全球分析和描述，使用RNA测序，染色质免疫沉淀，和生物信息学描述的调控层次结构，在细胞中的昼夜调节转录。然后，利用脉孢菌独特的生化遗传学背景，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出现，我们将开始奠定基础。 在脉孢菌代谢图上的时钟控制过程的光谱，看看时钟如何调节代谢和生理。在第二个焦点中，我们将把我们对昼夜节律输出途径的了解应用于哺乳动物细胞，使用RNA测序来确定野生型和RIP 140敲除小鼠脂肪细胞和巨噬细胞中时钟控制基因的昼夜节律谱。我们将使用RNA-seq来表征WT和突变细胞的转录组，并使用染色质免疫沉淀来开始剖析这种共激活子/共抑制子在这些重要细胞类型的昼夜节律生物学中的作用。这些实验将探索昼夜节律调节对哺乳动物脂肪代谢和免疫功能的重要性，希望深入了解人类糖尿病，代谢综合征，免疫功能的时间差异的发病率。第三个重点是光生物学。我们将探索光激活主要真菌光感受器的机制，并扩展重要真菌病原体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新的治疗方法。这些项目是互补和相互丰富的，因为它们都依赖于遗传和分子技术来解剖，并最终了解细胞对其环境的反应和真核细胞的组织作为时间的函数。

 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.