Identification and Analysis of Circadian Clock-Controlled Genes

生物钟控制基因的鉴定和分析

基本信息

批准号：
8236609
负责人：
JENNIFER J. LOROS
金额：
$ 70.71万
依托单位：
DARTMOUTH COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-09-01 至 2015-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8236609
关键词：
Adipocytes Adipose tissue Aspergillosis Aspergillus fumigatus Biochemical Genetics Biochemistry Bioinformatics Biological Biological Assay Biological Clocks Biological Models Biology Cell Line Cell physiology Cells ChIP-seq Circadian Rhythms Clock protein Diabetes Mellitus Disease Environment Epitopes Eukaryota Eukaryotic Cell Feedback Foundations Gene Expression Gene Expression Profile Genes Genetic Goals Grant Human Human body Informatics Kinetics Knock-out Language Life Light Mammalian Cell Maps Mental disorders Metabolic Metabolic syndrome Metabolism Molecular Genetics Motion Mouse Cell Line Mus Mycoses NRIP1 gene Neurospora Nuclear Receptors Organism Output Pathway interactions Patients Photobiology Photoreceptors Physiology Process Proteins Protocols documentation RNA RNA Sequences Reagent Regulation Reporter Role Structure System Techniques Time Transcriptional Regulation Work cell behavior cell type chromatin immunoprecipitation circadian pacemaker empowered fungus human NRIP1 protein insight lipid metabolism next generation pathogen response skills tool transcription factor

项目摘要

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.

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周期和途径——它们如何工作，它们如何与环境同步，以及时间信息如何产生它们用于调节细胞的行为。该提案重点关注神经孢菌模型系统以及小鼠和哺乳动物细胞系，以了解细胞生理学和代谢的昼夜节律控制的基础范例。我们还继续长期努力，旨在了解脉孢菌的昼夜节律光感受和光生物学，并利用这一点在显着的真菌病原体上开辟新的领域。在具体目标 1 中，我们将对脉孢菌属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昼夜节律调节的调控层次。然后，利用脉孢菌中可用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，并且当时钟控制活动的焦点出现时，我们将开始奠定时钟谱-控制脉孢菌代谢图上的过程，以了解生物钟如何调节新陈代谢和生理机能。在具体目标 2 中，我们将利用最新的光感受域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，我们可以设想一种方法，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。在具体目标 3 中，我们将使用 RNA 测序来确定 wt 和 RIP140 敲除细胞脂肪细胞中时钟控制基因的昼夜节律图谱，并将使用脂肪细胞中 RIP140 的染色质免疫沉淀来开始剖析这种共激活剂/的作用。这种重要细胞类型的昼夜节律生物学中的共阻遏物。我们将寻找RIP140与已知的时钟蛋白和涉及昼夜节律输出的转录因子的物理关联，并将产生白色脂肪组织特异性的时钟敲除，以探讨昼夜节律调节对小鼠脂肪代谢的重要性，希望获得深入的了解糖尿病和代谢综合征。这些项目是互补且相互丰富的，因为它们各自依赖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时间的变化。