Regulation and function of the circadian factor Period2

昼夜节律因子的调节和功能

基本信息

批准号：
9251300
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
项目类别：
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/9251300
关键词：
AKT1 gene ARNTL gene Address Alleles Alpha Cell Behavior Binding Sites Biological Biological Assay Bioluminescence Cell Differentiation process Cells ChIP-seq Chimeric Proteins Circadian Rhythms Clock protein Clone Cells Data Development Disease EMSA Electroporation Energy Metabolism Energy Metabolism Pathway Epidemic Event Exhibits FOXO1A gene FRAP1 gene Feedback Gene Expression Gene Targeting Genetic Genetic Transcription High Fat Diet Homeostasis Insulin Resistance Investigation Knock-in Knock-in Mouse Knowledge Liver Mediating Metabolic Metabolic Diseases Metabolic syndrome Metabolism MicroRNAs Molecular Mus Mutate Mutation Obesity Periodicity Pharmacology Physiological Physiology Play Poly A Preventive Proteins Recruitment Activity Regulation Regulatory Pathway Reporter Resistance Role Sampling Signal Transduction Simian virus 40 Site System Therapeutic Therapeutic Intervention Time Transcript Transcriptional Activation Transgenic Mice Translational Regulation Translations base chromosome conformation capture circadian pacemaker combat design embryonic stem cell feeding gene repression human disease in vivo innovation insight insulin signaling mRNA Stability mutant novel overexpression prevent promoter public health relevance targeted treatment upstream kinase vector

项目摘要

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（由申请人提供）：我们的基因表达和代谢的日常节律是由昼夜节律振荡器驱动的，这是一种由自动调节转录/翻译反馈环路组成的生物计时器，然而，核心时钟组件的分子调节和功能，特别是周期2。 (Per2)，尚未完全了解。之前生成了两个报告小鼠系，均表达来自内源 Per2 启动子的 PER2:LUC 融合蛋白。 Per2:Luc 小鼠中的 3'-UTR 保持完整，而 Per2:LucSV 小鼠中的 3'-UTR 被 SV40 poly(A) 信号取代。有趣的是，后者表现出 PER2:LUC 蛋白和生物发光的昼夜节律振幅和峰值水平显着增强。分析确定了 3'-UTR 中的 miR-24 结合位点，表明 miR-24 在 PER2 翻译中发挥重要作用，此外，Per2 和 Bmal1 转录物的强诱导作用。在 Per2:LucSV 小鼠中观察到相对于 Per2:Luc 的水平，表明 PER2 在其自身转录中的积极激活作用与时钟的主要作用一致。在代谢调节方面，初步数据还表明 Per2:LucSV 小鼠中多个代谢调节因子的激活。基于这些有趣的发现，人们重新认识到 PER2 蛋白水平受 miR-24 控制，并且 PER2 在 Per2 转录和昼夜节律中发挥积极作用。目标 1. 确定 miR-24 在 PER2 翻译调节和小鼠昼夜节律行为中的关键作用 miR-24 的 3'-UTR 结合位点。 Per2:Luc 敲入载体发生突变，电穿孔后获得候选靶向 ES 细胞克隆。突变分化细胞 (Aim 1A) 和敲入小鼠 (Aim 1B) 将衍生用于检查报告基因节律、分子时钟和昼夜节律行为。目标 2. 根据之前显示 Per2 启动子招募正 (CBP) 和负的 ChIP-seq 研究，描述 PER2 在 Per2 自动调节中发挥积极作用的分子机制。 (REV-ERB) 调节剂，将进行分子研究以调查 PER2 是否具有缓解 REV-ERB 依赖性 Per2 转录抑制 (目标 2A) 和/或增强 CBP 介导的转录激活 (目标 2B) 的功能。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缺陷。昼夜节律赋予代谢保护，将进行分子和生理学研究，以确定胰岛素信号传导（能量代谢的中央调节途径）是否在 Per2:LucSV 小鼠中被激活（目标 3A），以及这些小鼠是否对高脂肪饮食具有抵抗力诱发昼夜节律和代谢异常（目标 3B）。