Exploring the structural basis for 24-hour timekeeping in mammals

探索哺乳动物 24 小时计时的结构基础

• 批准号: 9753257
• 负责人: Carrie L Partch
• 金额: $ 47.07万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9753257
• 关键词: ARNTL gene; Acute; Address; Architecture; Back; Behavior; Binding; Biochemical; Biological Clocks; Biophysics; Brain; Cardiovascular Diseases; Cell Line; Cell model; Cells; ChIP-seq; Chronic; Circadian Rhythms; Circadian desynchrony; Clock protein; Complement; Complex; Cryoelectron Microscopy; Data; Development; Electron Microscopy; Exposure to; Feedback; Funding; Gene Activation; Genetic; Genetic Transcription; Health; Health Promotion; Hour; Human; In Vitro; Jet Lag Syndrome; Kinetics; Light; Malignant Neoplasms; Mammals; Messenger RNA; Metabolic Diseases; Modeling; Molecular; Mutation Analysis; Negative Staining; Pathway interactions; Phase; Physiology; Point Mutation; Process; Proteins; Regulation; Risk; Role; Stimulus; Structure; Study models; System; Testing; Therapeutic; Time; Translations; Work; base; circadian; cryptochrome; feeding; gene repression; insight; knockout gene; molecular clock; mutant; optogenetics; protein complex; response

Circadian rhythms are generated by molecular clocks that are universally used to synchronize behavior and physiology with the 24-hour solar cycle. This proposal seeks to understand the biochemical basis of circadian timing and photoentrainment, the process by which molecular clocks are aligned to the external light/dark cycle. In mammals, circadian rhythms arise from set of interlocked transcription feedback loops involving dedicated clock proteins: at the center of this network, CLOCK:BMAL1 activates transcription of its repressors PERIOD (PER) and CRYPTOCHROME (CRY), which ultimately feed back to complete a ~24-hour long cycle of gene activation and repression. Photoentrainment is important to keep molecular clocks on track with environmental light cycles. Chronic circadian misalignment (i.e. jetlag) leads to increased risk for metabolic disorders, cardiovascular disease and cancer due to disruption of the systemic control of physiology by circadian rhythms. While much of the photoentrainment pathway has been laid out from ocular photoreception to its acute induction of Per mRNA in the master clock of the brain, crucially, the final biochemical steps that execute entrainment on the molecular level remain completely unknown. Exposure to light before dawn leads to phase advances of the molecular clock, while light after dusk delays the clock, both of which keep CLOCK:BMAL1 activity aligned with the day. How does this plasticity in phase shifting arise from the same light-dependent induction of Per mRNA that occurs at dusk and dawn? ChIP-seq studies provide evidence for distinct repressive complexes that assemble in the evening, an `early' complex of PER and CRY proteins that assembles at dusk on CLOCK:BMAL1 and a `late' complex of CRY1 bound alone to CLOCK:BMAL1 at dawn. Our central hypothesis is that differences in the composition of early and late repressive complexes are exploited for entrainment, leading to differential regulation by light-induced PER2 at dusk and dawn. We will test this with three specific aims. First, the molecular basis for differences in regulation of CLOCK:BMAL1 by CRY1, CRY2 and PER2 will be defined with biochemical, biophysical, and cellular studies. Second, structures of early and late repressive complexes will be determined by cryo-electron microscopy to identify overall changes in molecular architecture of the core clock proteins that occur throughout the evening. Third, the development of a new optogenetic model for the study of cellular clocks will allow the identification of biochemical determinants by which clocks are entrained to external stimuli. Collectively, these lines of study will address how core clock proteins interact throughout the evening in distinct regulatory complexes to generate circadian timekeeping and respond to external stimuli.

昼夜节律是由分子时钟产生的，分子时钟普遍用于同步行为和 与24小时太阳周期有关的生理学。这项提议试图了解昼夜节律的生物化学基础。 计时和光夹带，分子时钟与外部光/暗周期对齐的过程。 在哺乳动物中，昼夜节律产生于一组相互关联的转录反馈环，涉及专门的 CLOCK蛋白：在这个网络的中心，CLOCK：BMal1激活其抑制物阶段的转录 (PER)和隐花色素(CRY)，它们最终反馈完成一个长达24小时的基因循环 激活和压抑。光夹带对于使分子时钟与环境保持一致很重要 光循环。慢性昼夜节律失调(即时差)会增加代谢紊乱的风险， 心血管疾病和癌症，由于昼夜节律扰乱了系统的生理控制。 虽然大部分的光携带途径是从眼睛的光接收到它的急性诱导 在大脑的主时钟中，至关重要的是，执行携带的最终生化步骤 分子水平仍然完全未知。黎明前暴露在光线下会导致 分子时钟，而黄昏后的灯光会延迟时钟，两者都保持时钟：BMal1活性与 这一天。这种相移的可塑性是如何从同样的光依赖诱导的PER-mRNA产生的？ 发生在黄昏和黎明之间？CHIP-SEQ研究提供了不同的抑制复合体的证据 在晚上组装，在黄昏时钟组装的PER和CREAT蛋白的‘早’复合体：BMal1 以及一个单独与时钟相结合的CRY1‘Late’复合体：黎明时分的BMal1。我们的中心假设是，差异 在早期和晚期的组成中，抑制复合体被用于夹带，导致差异 在黄昏和黎明，光诱导的PER2的调节。我们将用三个具体目标来测试这一点。首先，分子 时钟调节差异的依据：CRY1、CRY2和PER2的BMal1将用生化来定义， 生物物理学和细胞学研究。第二，将确定早期和晚期抑制复合体的结构 通过冷冻电子显微镜来确定核心时钟蛋白分子结构的整体变化 整个晚上都会发生。第三，为研究细胞时钟开发了一种新的光遗传模型 将允许识别生物化学决定因素，通过这些决定因素，时钟将受到外部刺激的影响。 总的来说，这些研究路线将解决核心时钟蛋白如何在整个晚上以不同的方式相互作用 调节复合体，以产生昼夜计时和对外部刺激的反应。