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。在具体目标1中，我们将对脉孢子菌的昼夜节律输出网络进行全局分析和描述，使用RNA测序、染色质免疫沉淀和生物信息学来描述细胞中转录的昼夜调控的调控层次。然后，利用脉孢菌特殊的生化遗传学背景，我们将跟踪由钟控基因编码的蛋白质进行的代谢活动，随着钟控活动焦点的出现，我们将开始在脉孢菌的代谢图上铺设钟控过程的频谱，以了解时钟如何调节新陈代谢和生理。在具体目标2中，我们将利用最近对光感受域的原子级结构分析来确定光循环动力学的生物学意义。我们将把光生物学的分析扩展到重要的真菌病原体烟曲霉，在那里我们可以设想一种方法，利用我们对真菌光生物学的理解，使数十万曲霉病患者能够获得新的治疗方法。在具体目标3中，我们将使用RNA测序来确定wt和RIP140基因敲除细胞中脂肪细胞中时钟控制基因的昼夜节律，并将使用RIP140在脂肪细胞中的染色质免疫沉淀来开始剖析这种共激活/共抑制因子在这一重要细胞类型的昼夜生物学中的作用。我们将寻找RIP140与已知的时钟蛋白和参与昼夜节律输出的转录因子的物理联系，并将产生白色脂肪组织特异性的时钟敲除，以探索昼夜节律调节对小鼠脂肪代谢的意义，希望对糖尿病和代谢综合征有更深入的了解。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. In Specific Aim 1, we will carry out 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 Specific Aim 2, we will exploit recent atomic level structure analysis of the photoreceptive domain to determine the biological significance of photocycle kinetics. We will extend analysis of photobiology to the important fungal pathogen Aspergillus fumigatus where we can envision a way to exploit our understanding of fungal photobiology to enable a new treatment for hundreds of thousands of patients with aspergillosis. In Specific Aim 3, we will use RNA sequencing to determine the circadian profile of clock-controlled genes in adipocytes of wt and RIP140 knockout cells, and will use chromatin immunoprecipitation of RIP140 in adipocytes to begin to dissect the role of this co-activator/co-repressor in the circadian biology of this important cell type. We will look for physical association of RIP140 with known clock proteins and transcription factors involved with circadian output, and will generate white adipose tissue-specific knockouts of the clock to probe the significance of circadian regulation to fat metabolism in the mouse, hoping to gain insights into diabetes and metabolic syndrome. 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.