Regulation and function of the circadian factor Period2

 描述（由申请人提供）：我们的基因表达和代谢的日常节律由昼夜节律振荡器驱动，昼夜节律振荡器是一种由自动调节转录/翻译反馈环组成的生物计时器。然而，核心时钟组件的分子调控和功能，特别是Period 2（Per 2），尚未完全理解。先前产生了两个报告小鼠系，两者均从内源性Per 2启动子表达PER 2：LUC融合蛋白。尽管内源性Per 2 3 '-UTR在Per 2：Luc小鼠中保持完整，但在Per 2：LucSV小鼠中被SV 40 poly（A）信号取代。有趣的是，后者表现出显著增强的昼夜节律振幅和峰值水平的PER 2：LUC蛋白和生物发光。进一步的分析鉴定了3 '-UTR中的miR-24结合位点，表明miR-24在PER 2翻译中的重要作用。此外，相对于Per 2：Luc，在Per 2：LucSV小鼠中观察到Per 2和Bmal 1转录水平的稳健诱导，表明PER 2在其自身转录中的积极激活作用。与时钟的主导作用相一致 在代谢调节中，初步数据还说明了Per 2：LucSV小鼠中几种代谢调节剂的激活。基于这些有趣的发现，推测PER 2蛋白水平受miR-24控制，并且PER 2在Per 2转录和昼夜代谢功能中起积极作用。 目标1。确定miR-24在PER 2翻译调控和小鼠昼夜节律行为中的关键作用。突变Per 2：Luc敲入载体中的3 '-UTR miR-24结合位点，并在电穿孔后获得候选靶向ES细胞克隆。将衍生突变分化细胞（Aim 1A）和敲入小鼠（Aim 1B），以检查报告基因节律、分子钟和昼夜节律行为。 目标二。阐明PER 2在PER 2自动调节中的积极作用的分子机制。基于先前的ChIP-seq研究显示Per 2启动子募集阳性（CBP）和阴性（REV-ERB）调节剂，将进行分子研究以研究PER 2是否具有缓解REV-ERB依赖性Per 2转录抑制（Aim 2A）和/或增强CBP介导的转录激活（Aim 2B）的功能。 目标3：确定PER 2在能量代谢中的分子功能。生物钟的遗传破坏导致胰岛素抵抗和代谢缺陷。为了解决增强的PER 2和昼夜节律是否赋予代谢保护的相互假设，将进行分子和生理学研究以确定胰岛素信号传导（能量代谢的中心调节途径）是否在Per 2：LucSV小鼠中被激活（Aim 3A），以及这些小鼠是否对高脂肪饮食诱导的昼夜节律和代谢异常具有抗性（Aim 3B）。

 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).