Regulation and function of the circadian factor Period2

 描述(申请人提供)：我们基因表达和新陈代谢的日常节律是由昼夜节律振荡器驱动的，昼夜节律振荡器是由自动调节转录/翻译反馈回路组成的生物计时器。然而，核心时钟成分的分子调控和功能，特别是周期2(Perdio2，PER2)，尚不完全清楚。之前已经建立了两个报告鼠系，都表达来自内源性PER2启动子的PER2：Luc融合蛋白。而在PER2：LUC小鼠中，内源性PER2 3‘-UTR保持不变，而在PER2：LucSV小鼠中，它被SV40聚(A)信号所取代。有趣的是，后者表现出显著增强的昼夜节律幅度和PER2：Luc蛋白和生物发光的峰值水平。进一步分析发现，miR-24在3‘-UTR区有一个miR-24结合位点，提示miR-24在PER2翻译中起重要作用。此外，相对于PER2：Luc，在PER2：LucSV小鼠中观察到PER2和BMal1转录水平的强烈诱导，这表明PER2在其自身的转录中具有正的激活作用。与时钟的主导作用一致 在代谢调节方面，初步数据还表明PER2：LucSV小鼠体内的几种代谢调节因子被激活。基于这些有趣的发现，推测PER2蛋白水平受miR-24控制，PER2在PER2转录和昼夜代谢功能中发挥积极作用。目的1.确定miR-24在PER2翻译调控和小鼠昼夜节律行为中的关键作用。PER2：Luc敲入载体中的3‘-UTRmiR-24结合位点发生突变，电穿孔后获得了候选靶向ES细胞克隆。突变的分化细胞(Aim 1A)和敲入鼠(Aim 1B)将被用来检测报告节律、分子时钟和昼夜行为。目的2.阐明PER2在PER2自身调节中积极作用的分子机制。基于先前的CHIP-SEQ研究显示PER2启动子同时招募正(CBP)和负(REV-ERBS)调节因子，将进行分子研究，以探讨PER2是否具有缓解依赖REV-ERB的PER2转录抑制(AIM 2A)和/或增强CBP介导的转录激活(AIM 2B)的功能。目的3.确定PER2在能量代谢中的分子功能。生物钟的基因破坏会导致胰岛素抵抗和代谢缺陷。为了阐明增强的PER2和昼夜节律是否提供代谢保护这一相互作用的假说，将进行分子和生理学研究，以确定PER2：LucSV小鼠(AIM 3A)是否激活了胰岛素信号，以及这些小鼠是否对高脂饮食引起的昼夜节律和代谢异常(AIM 3B)具有抵抗力。

 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).