Mechanistic Basis of Circadian Clocks in Bmal1 Knockout Mice

Bmal1 基因敲除小鼠生物钟的机制基础

• 批准号: 10798455
• 负责人: Akhilesh Basi Reddy
• 金额: $ 24.5万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798455
• 关键词: ARNTL gene; Affect; Automobile Driving; Behavioral; Cell physiology; Cells; ChIP-seq; Circadian Dysregulation; Circadian Rhythms; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Diabetes Mellitus; Disease; ETS Family Protein; Family; Feedback; Future; Genes; Genetic Transcription; Genomics; Goals; Health; Heart Diseases; Human; Immunoprecipitation; Knock-out; Knockout Mice; Life; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mental disorders; Metabolic; Metabolic Diseases; Modeling; Molecular; Mus; Nuclear; Oxidation-Reduction; Periodicity; Physiological; Physiology; Play; Process; Proteins; Proteome; Proteomics; Pulmonary Inflammation; Research; Role; Societies; System; Testing; Therapeutic Intervention; Tissues; Transcript; Vascular Diseases; circadian; circadian pacemaker; experimental study; human disease; insight; novel; posttranscriptional; programs; recruit; shift work; transcription factor; transcriptome

PROJECT SUMMARY Circadian clocks are believed to exist at almost all levels of life and play a fundamental role in maintenance of physiological and behavioral processes in accordance with the day-night cycle. The conventional model that describes the circadian clockwork at the molecular level revolves around transcriptional/translational feedback loops (TTFLs). In these models, BMAL1 is believed to act as an indispensable component of the timekeeping system. However, we have found pervasive molecular oscillations in the transcriptome and proteome of Bmal1-/- mice. A research program will be undertaken to obtain a comprehensive mechanistic understanding of these “non-canonical” circadian rhythms in Bmal1-/- mice. The project will broadly focus on understanding transcriptional (Aim 1) and post- transcriptional (Aim 2) functioning of cells and tissues from Bmal1-/- mice. (Aim1) Our preliminary data suggest that the novel circadian rhythms we see might be underpinned by the recruitment of ETS family transcription factors into the clockwork. We will functionally test their role by knocking out key ETS proteins using CRISPR. We will also perform ChIP-seq experiments to find genomic targets driving rhythmic transcripts that we see. In addition, we will perform nuclear proteomics to elucidate novel transcription factors that might mediate rhythmic transcription. Furthermore, we will perform protein interaction analyses using immunoprecipitation mass spectrometry to determine how ETS proteins and redox proteins may physically interact. (Aim 2) We found redox oscillations in Bmal1 knockout cells, implying that these play a role in the clockwork of Bmal1-/- mice. Consequently, we will investigate novel redox oscillations in Bmal1 knockout cells using a novel redox proteomics workflow that we have developed. In addition, we will characterize the rhythmic phospho-proteome and kinome of cells, which we have found could be profoundly affected by deletion of Bmal1. Finally, we will determine whether metabolic circadian oscillations occur in Bmal1 knockouts. Gaining new molecular insights into the circadian clockwork will guide future therapeutic interventions to alleviate the disorders associated with circadian disruption, which are highly prevalent in contemporary society.

项目摘要 据信昼夜节律钟表在几乎所有层面上都存在，并在 根据昼夜周期维护身体和行为过程。这 传统模型描述分子水平的昼夜节律旋转 转录/翻译反馈循环（TTFLS）。在这些模型中，BMAL1被认为是 计时系统必不可少的组件。但是，我们发现了普遍的分子 BMAL1 - / - 小鼠的转录组和蛋白质组的振荡。将进行研究计划 为了获得对这些“非规范”昼夜节律的全面理解 BMAL1 - / - 小鼠。该项目将广泛专注于理解转录（AIM 1）和后 BMAL1 - / - 小鼠的细胞和组织的转录（AIM 2）功能。 （AIM1）我们的初步数据 建议我们看到的新颖的昼夜节律可能是由ETS家族招募的基础 转录因子进入发条。我们将通过淘汰关键ET来在功能上测试他们的角色 使用CRISPR的蛋白质。我们还将执行CHIP-SEQ实验以找到驱动基因组目标 我们看到的有节奏的成绩单。此外，我们将执行核蛋白质组学以阐明新颖 转录因子可能介导节奏转录。此外，我们将执行蛋白质 使用免疫沉淀质谱法进行相互作用分析，以确定ETS蛋白质和 氧化还原蛋白可能在物理上相互作用。 （AIM 2）我们发现BMAL1基因敲除细胞中的氧化还原振荡， 这意味着这些在bmal1 - / - 鼠标的发条中起着作用。因此，我们将研究小说 使用新型的氧化还原蛋白质组学工作流程中，BMAL1基因敲除细胞中的氧化还原振荡我们已有 发达。此外，我们将表征细胞的节奏磷酸化 - 磷酸化蛋白质组和神经组，这些磷酸化蛋白质组 我们发现，BMAL1的删除可能会严重影响。最后，我们将确定是否 BMAL1敲除中发生的代谢昼夜节律振荡。获得新的分子见解 昼夜节律发条将指导未来的治疗干预措施，以减轻与之相关的疾病 昼夜节律中断，在当代社会中非常普遍。