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.

昼夜节律是由普遍用于同步行为和的分子时钟产生的 具有24小时太阳周期的生理学。该建议旨在了解昼夜节律的生化基础 时间和光座，分子钟与外部光/暗周期对齐的过程。 在哺乳动物中，昼夜节律是由涉及专用的互锁转录反馈循环产生的 时钟蛋白：在该网络的中心，时钟：BMAL1激活其阻遏物周期的转录 （per）和加密色素（哭泣），最终返回以完成〜24小时长的基因周期 激活和抑制。光登录对于通过环境保持分子时钟很重要 光周期。慢性昼夜节律的未对准（即喷射lag）导致代谢性疾病的风险增加， 由于昼夜节律对全身对生理控制的控制，心血管疾病和癌症。 虽然大部分光学隔离途径已从眼球感受到其急性诱导 在大脑的主时钟中的per mRNA，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。在黎明导致阶段进展之前，暴露在光线之前 分子时钟，黄昏后的光线延迟时钟，这两个都保持时钟：BMAL1活动与 一天。相位转移中的这种可塑性如何来自mRNA的相同的光依赖性诱导 这发生在黄昏和黎明吗？ Chip-seq研究为不同的抑制性复合物提供了证据 晚上组装，每次哭泣的蛋白质的“早期”综合体在钟的黄昏组装：bmal1 一个cry1的“晚期”复合体绑在黎明时时钟：bmal1。我们的中心假设是差异 在早期和晚期抑制性复合物的组成中，被利用用于夹带，导致差异 在黄昏和黎明时通过光诱导的PER2调节。我们将以三个特定目标进行测试。首先，分子 CRY1，CRY2和PER2的时钟调节：BMAL1的调节差异的基础将用生化定义， 生物物理和细胞研究。其次，将确定早期和晚期抑郁症的结构 通过冷冻电子显微镜来确定核心时钟蛋白分子结构的总体变化， 整个晚上发生。第三，开发用于研究细胞时钟的新光遗传学模型 将允许鉴定将时钟夹在外部刺激中的生化决定因素。 总的来说，这些研究线将解决核心时钟蛋白在整个晚上的相互作用 监管复合物产生昼夜节律并应对外部刺激。