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.

昼夜节律由分子钟产生，分子钟普遍用于同步行为和 24小时太阳周期的生理学。该提案旨在了解昼夜节律的生化基础 计时和光夹带，分子钟与外部光/暗周期对齐的过程。 在哺乳动物中，昼夜节律源自一组互锁的转录反馈环路，涉及专用的转录反馈环路。 时钟蛋白：在该网络的中心，CLOCK:BMAL1 激活其阻遏蛋白的转录 PERIOD (PER) 和 CRYPTOCHROME (CRY)，最终反馈完成约 24 小时长的基因周期 激活和抑制。光夹带对于保持分子钟与环境保持正轨非常重要 光周期。慢性昼夜节律失调（即时差）会导致代谢紊乱的风险增加， 由于昼夜节律破坏全身生理控制而导致心血管疾病和癌症。 虽然大部分光夹带途径已经从眼睛光接收到其急性诱导 大脑主时钟中每个 mRNA 的含量，至关重要的是，执行夹带的最终生化步骤 分子水平仍然完全未知。黎明前暴露在光线下会导致相位提前 分子钟，而黄昏后的光延迟了时钟，两者都使 CLOCK:BMAL1 活动与 那天。这种相移的可塑性是如何由 Per mRNA 的相同光依赖性诱导产生的 发生在黄昏和黎明？ ChIP-seq 研究为不同的抑制复合物提供了证据 PER 和 CRY 蛋白的“早期”复合体在晚上组装，在黄昏时在 CLOCK:BMAL1 上组装 以及在黎明时单独与 CLOCK:BMAL1 结合的 CRY1 的“晚期”复合体。我们的中心假设是差异 在早期和晚期压抑情结的组成中被利用来夹带，导致差异 黄昏和黎明时光诱导的 PER2 的调节。我们将通过三个具体目标对此进行测试。首先，分子 CRY1、CRY2 和 PER2 对 CLOCK:BMAL1 的调节差异的基础将通过生化、 生物物理学和细胞研究。其次，将确定早期和晚期压抑情结的结构 通过冷冻电子显微镜来识别核心时钟蛋白分子结构的整体变化 发生在整个晚上。三、开发用于细胞时钟研究的新光遗传学模型 将允许识别时钟被夹带到外部刺激的生化决定因素。 总的来说，这些研究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。 调节复合物产生昼夜节律计时并对外部刺激做出反应。