Regulation and function of the circadian factor Period2

昼夜节律因子的调节和功能

• 批准号: 8862098
• 负责人: Seung-Hee Yoo
• 金额: $ 29.65万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8862098
• 关键词: AKT1 gene; Address; Alleles; Behavior; Binding Sites; Biological; Biological Assay; Bioluminescence; Cells; ChIP-seq; Chimeric Proteins; Chromosomes; Circadian Rhythms; Clock protein; Data; Development; Diet; Disease; EMSA; Electroporation; Energy Metabolism; Epidemic; Event; Exhibits; Fatty acid glycerol esters; Feedback; Gene Expression; Gene Targeting; Genetic; Genetic Transcription; Insulin Resistance; Investigation; Knock-in Mouse; Knowledge; Lead; Liver; Mediating; Metabolic; Metabolic Diseases; Metabolic syndrome; Metabolism; MicroRNAs; Molecular; Molecular Conformation; Mus; Mutate; Mutation; Obesity; Personal Satisfaction; Physiological; Physiology; Play; Poly A; Preventive; Proteins; Regulation; Regulatory Pathway; Relative (related person); Reporter; Resistance; Role; Sampling; Signal Transduction; Simian virus 40; Site; System; Therapeutic; Therapeutic Intervention; Time; Transcript; Transcriptional Activation; Transgenic Mice; Translational Regulation; Translations; Untranslated Regions; base; circadian pacemaker; combat; design; embryonic stem cell; feeding; gene repression; human FRAP1 protein; human disease; in vivo; innovation; insight; insulin signaling; interest; mRNA Stability; mutant; novel; overexpression; prevent; promoter; public health relevance; upstream kinase; vector

 DESCRIPTION (provided by applicant): Our daily rhythms in gene expression and metabolism are driven by the circadian oscillator, a biological timer composed of auto-regulatory transcriptional/translational feedback loops. However, molecular regulation and function of core clock components, in particular Period2 (Per2), are not fully understood. Two reporter mouse lines were previously generated, both expressing PER2:LUC fusion proteins from the endogenous Per2 promoter. Whereas the endogenous Per2 3'-UTR remains intact in Per2:Luc mice, it was replaced by an SV40 poly(A) signal in Per2:LucSV mice. Intriguingly, the latter exhibited significantly enhanced circadian amplitude and peak levels of PER2:LUC protein and bioluminescence. Further analysis identified a miR-24 binding site in the 3'-UTR, suggesting an important role of miR-24 in PER2 translation. Furthermore, robust induction of Per2 and Bmal1 transcript levels were observed in Per2:LucSV mice relative to Per2:Luc, suggesting a positive activation role of PER2 in its own transcription. Consistent with the predominant role of the clock in metabolic regulation, preliminary data also illustrated activation of several metabolic regulators in Per2:LucSV mice. Based on these interesting findings, it is hypothesized that PER2 protein levels are controlled by miR-24, and PER2 plays a positive role in Per2 transcription and circadian metabolic function. Aim 1. Determine the pivotal role of miR-24 in PER2 translational regulation and mouse circadian behavior. The 3'-UTR miR-24 binding site in the Per2:Luc knock-in vector was mutated, and candidate targeted ES cell clones were obtained following electroporation. Mutant differentiated cells (Aim 1A) and knock-in mice (Aim 1B) will be derived to examine reporter rhythms, molecular clock and circadian behavior. Aim 2. Delineate the molecular mechanism underlying the positive role of PER2 in Per2 auto- regulation. Based on previous ChIP-seq studies showing Per2 promoter recruitment of both positive (CBP) and negative (REV-ERBs) regulators, molecular studies will be conducted to investigate whether PER2 functions to relieve REV-ERB-dependent Per2 transcriptional repression (Aim 2A), and/or to potentiate CBP- mediated transcriptional activation (Aim 2B). Aim 3. Determine the molecular function of PER2 in energy metabolism. Genetic disruption of the clock leads to insulin resistance and metabolic deficits. To address the reciprocal hypothesis whether enhanced PER2 and circadian rhythms confers metabolic protection, molecular and physiological studies will be conducted to determine whether insulin signaling, a central regulatory pathway for energy metabolism, is activated in Per2:LucSV mice (Aim 3A), and whether these mice are resistant to high-fat diet induced circadian and metabolic abnormalities (Aim 3B).

 描述（由适用提供）：我们在基因表达和代谢方面的每日节奏是由昼夜节振荡器驱动的，这是一个由自动调节的转录/翻译反馈回路组成的生物计时器。但是，尚未完全了解核心时钟组件的分子调节和核心时钟组件的功能。先前产生了两个报告基因的小鼠系，都表达了内源性PER2启动子的PER2：Luc Fusion蛋白。尽管在PER2中，内源性PER2 3'-UTR保持完整：Luc小鼠，但在PER2：LUCSV小鼠中被SV40 poly（a）信号取代。有趣的是，后来的暴露显着增强了昼夜节律放大器和PER2的峰值水平：luc蛋白和生物发光。进一步的分析确定了3'-UTR中的miR-24结合位点，表明miR-24在PER2翻译中的重要作用。此外，在PER2：LUCSV小鼠相对于PER2：LUC中，观察到PER2和BMAL1转录水平的鲁棒诱导，这表明PER2在其自身的转录中具有阳性激活作用。与时钟的主要作用一致 在代谢调节中，初步数据还说明了PER2：LUCSV小鼠中几个代谢调节剂的激活。基于这些有趣的发现，假设PER2蛋白水平受miR-24的控制，并且PER2在PER2转录和昼夜节律代谢功能中起积极作用。目标1。确定miR-24在PER2平移调节和小鼠昼夜节律行为中的关键作用。 PER2中的3'-UTR miR-24结合位点突变为突变，并在电穿孔后获得候选靶向的ES细胞克隆。突变分化的细胞（AIM 1A）和敲入小鼠（AIM 1B）将得出以检查记者节奏，分子时钟和昼夜节律行为。 AIM 2。描述PER2在PER2自动调节中的积极作用的基础分子机制。基于先前的芯片seq研究表明，PER2启动子募集正（CBP）和阴性（REV-ERB）调节剂，将进行分子研究，以调查PER2函数是否为挽救REV-ERB依赖性PER2转录表达（AIM 2A）（AIM 2A）和/或以进行CBP介导的CBP介导的转录转录激活（AIM 2B）。 AIM 3。确定PER2在能量代谢中的分子功能。时钟的遗传破坏会导致胰岛素抵抗和代谢定义。为了解决相互假设，是否将进行代谢保护的PER2和昼夜节律是否传达，将进行分子和物理研究，以确定胰岛素信号传导是否在PER2：LUCSV小鼠（AIM 3A）中激活了能量代谢的中心调节途径（AIM 3A），以及这些小鼠对高脂饮食的抗性和抗性（Abs）是否抗性（Absorant）抗性（Abs）和Metabic civeArcIans（Absorancian）。