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: 10208370
• 负责人: Akhilesh Basi Reddy
• 金额: $ 47.29万
• 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/10208370
• 关键词: ARNTL gene; Address; Affect; Automobile Driving; Behavior; Behavioral; Binding; Biochemical; Cardiovascular Diseases; Cell physiology; Cells; ChIP-seq; Circadian Dysregulation; Circadian Rhythms; Clock protein; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Diabetes Mellitus; Disease; ETS Family Protein; Erythrocytes; Family; Feedback; Fibroblasts; Future; Genes; Genetic Transcription; Genome; Genomics; Glucose; Glycolysis; Goals; Health; Heart Diseases; Hour; Human; Immunoprecipitation; Investigation; Knock-out; Knockout Mice; Life; Liver; Lung Inflammation; Maintenance; Malignant Neoplasms; Mammals; Maps; Mass Spectrum Analysis; Measures; Mediating; Mental disorders; Metabolic; Metabolic Diseases; Metabolism; Methods; Modeling; Molecular; Mus; Nuclear; Organism; Output; Oxidation-Reduction; Pathway interactions; Pattern; Pentosephosphate Pathway; Periodicity; Phase; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Process; Proteins; Proteome; Proteomics; Regulation; Reporting; Research; Role; Skin; Societies; System; Testing; Therapeutic Intervention; Time; Tissue Sample; Tissues; Transcript; Transcription Process; Translations; Vascular Diseases; base; circadian; circadian pacemaker; circadian regulation; desthiobiotin; experimental study; human disease; insight; molecular clock; molecular modeling; novel; peroxiredoxin; programs; recruit; shift work; transcription factor; transcriptome; virtual

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.

项目总结 生物钟被认为存在于生活的几乎所有层面，并在 维持与昼夜循环一致的生理和行为过程。这个 在分子水平上描述昼夜节律的传统模型围绕着 转录/翻译反馈环(TTFL)。在这些模型中，BMAL1被认为是一个 这是守时系统不可或缺的组成部分。然而，我们发现了普遍存在的分子 BMal1-/-小鼠转录组和蛋白质组的振荡。将进行一项研究计划 为了从机制上全面理解这些“非典范”的昼夜节律 BMal1-/-小鼠。该项目将主要侧重于理解转录(目标1)和后转录。 转录(目标2)BMal1-/-小鼠细胞和组织的功能。(Aim1)我们的初步数据 这表明我们看到的新的昼夜节律可能是由ETS家族的招募所支撑的 转录因子进入发条装置。我们将通过敲击Key Ets来从功能上测试他们的角色 使用CRISPR的蛋白质。我们还将进行芯片序列实验，以寻找驱动基因的靶点 我们看到的有节奏的文字记录。此外，我们将进行核蛋白质组学来阐明新的 可能调节节律转录的转录因子。此外，我们将执行蛋白质 免疫沉淀质谱联用分析ETS蛋白与蛋白质的相互作用 氧化还原蛋白可能在物理上相互作用。(目标2)我们在BMal1基因敲除细胞中发现了氧化还原振荡， 这意味着这些基因在BMal1-/-小鼠的发条过程中发挥了作用。因此，我们将调查小说 利用我们拥有的一种新的氧化还原蛋白质组学工作流程在BMal1基因敲除细胞中进行氧化还原振荡 发展起来的。此外，我们还将描述细胞的节律性磷酸蛋白质组和动态组，其中 我们发现，BMal1的缺失可能会对其产生深远的影响。最后，我们将确定是否 代谢昼夜节律振荡发生在BMal1基因敲除中。获得新的分子洞察力 昼夜节律将指导未来的治疗干预，以缓解与 昼夜节律紊乱，这在当代社会非常普遍。