Regulation and function of the circadian factor Period2

昼夜节律因子的调节和功能

• 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
• 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

 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).