Spatiotemporal Organization of Key Circadian Clock Regulators by PML

PML 关键昼夜节律时钟调节器的时空组织

基本信息

批准号：
8735969
负责人：
CHENG CHI LEE
金额：
$ 28.72万
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：
2013
资助国家：
美国
起止时间：
2013-09-20 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8735969
关键词：
Abnormal Cell Acetylation Acute Promyelocytic Leukemia Affect Animal Model Behavior Binding Biological Assay Biological Clocks Biological Process Cell Line Cell Nucleus Cell physiology Circadian Rhythms Clock protein Co-Immunoprecipitations Collection Complex Crying DNA Damage Deacetylation Embryo Epitopes Fibroblasts Foundations Genes Growth Health Hormonal Human Immunofluorescence Immunologic In Vitro Investigation Laboratories Lead Life Liver Lysine Maps Metabolic Methods Modeling Mus Mutation Names Normal Cell Nuclear Nuclear Protein Nuclear Proteins Output Pathway interactions Pattern Peripheral Physiological Play Process Protein Acetylation Proteins Recombinant Proteins Recombinants Recruitment Activity Regulation Regulator Genes Reporter Role Signal Transduction Sleep Sleep Wake Cycle Tissues Tumor Suppression Tumor Suppressor Proteins Visual Work awake cell growth circadian pacemaker feeding genetically modified cells in vivo innovation mouse model mutant novel therapeutics promoter protein function public health relevance research study response small molecule spatiotemporal tool transcription factor PML tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): The repetitive activity pattern of all life on earth follows the daily cycle of sunrise and sunset and is controlled by an endogenous biological clock composed of a network of interactive molecules. For the biological clock to exert its control over the fundamental activities of life, the molecules in the clock network must interact with molecules of other functional networks, such as signaling networks that control feeding, sleep-awake cycle, growth and metabolic regulation, though these interactions remain to be elucidated. Our recent investigations have clearly established direct interactions between PER2, a key regulatory molecule in the clock network, and promyelocytic leukemia protein (PML), which was initially characterized as a tumor suppressor protein responsible for acute promyelocytic leukemia (APL). Currently PML is known as the central component of a large nuclear protein complex, PML nuclear bodies (PML NBs), where PML is suggested to act as a "recruiter and organizer" for nuclear proteins from various functional networks. At the core of the circadian clock mechanism, nuclear proteins BMAL1 and CLOCK form the heterodimers that drive the transcriptional expressions of the key clock regulators, including Per and Cry genes, as well as many clock output genes. We found PML not only interacts directly with PER2 in the nucleus, it also facilitates the process of PER2 nuclear localization. Recently, BMAL1 was also found to colocalize with PML NBs. These lines of evidence, led us to the hypothesis that PML plays the role of "recruiter and organizer" for the nuclear regulatory network of the circadian clock mechanism. Here we propose to investigate the "recruiter and organizer" function of PML in the circadian clock mechanism by constructing a profile of subcellular distributions and interactions of various clock regulators and PML. We propose to carry out in vitro and in vivo experiments using a combination of tools including immunofluorescence, reciprocal coimmunoprecipitation, recombinant expression constructs, genetically engineered cell lines and animal models. Our recent studies have revealed that the acetylation status of PML affects its interaction with PER2. We have undertaken a small molecule screen and identified a compound, which we have renamed PINEF, that is capable of facilitating PER2 nuclear entry in the absence of PML. We also propose to study how the status of PML acetylation and the actions of PINEF in the absence of PML may alter the profile of subcellular distributions and interactions of various clock regulators. These proposed studies will result in a more comprehensive spatiotemporal profile of the distribution and interactions of key clock proteins, including BMAL1, CLOCK, PERs, CRYs, and PML. The findings will contribute to a better understanding of the mammalian circadian clock mechanism. In addition, as dozens of nuclear proteins involved in many different cellular pathways have been localized in PML NBs, this study will help to further understand if and how PML contributes to the connections among different functional networks, including the circadian clock regulatory network.

描述（由申请人提供）：地球上所有生命的重复活动模式遵循日出和日落的每日周期，并由由相互作用分子网络组成的内源生物钟控制。为了使生物钟对生命的基本活动发挥控制作用，时钟网络中的分子必须与其他功能网络的分子相互作用，例如控制进食、睡眠-觉醒周期、生长和代谢调节的信号网络，尽管这些网络相互作用仍有待阐明。我们最近的研究清楚地确定了生物钟网络中的关键调节分子 PER2 与早幼粒细胞白血病蛋白 (PML) 之间的直接相互作用，PML 最初被定性为导致急性早幼粒细胞白血病 (APL) 的肿瘤抑制蛋白。目前，PML 被认为是大型核蛋白复合物 PML 核体 (PML NB) 的核心组成部分，其中 PML 被认为充当来自各种功能网络的核蛋白的“招募者和组织者”。其核心是在生物钟机制中，核蛋白 BMAL1 和 CLOCK 形成异二聚体，驱动关键时钟调节因子的转录表达，包括 Per 和 Cry 基因，以及许多时钟输出基因。我们发现PML不仅与细胞核中的PER2直接相互作用，而且还促进了PER2核定位的过程。最近，还发现 BMAL1 与 PML NB 共定位。这些证据使我们得出这样的假设：PML 在生物钟机制的核调节网络中扮演着“招募者和组织者”的角色。在这里，我们建议通过构建各种时钟调节器和 PML 的亚细胞分布和相互作用的概况来研究 PML 在生物钟机制中的“招募者和组织者”功能。我们建议使用免疫荧光、相互免疫共沉淀、重组表达构建体、基因工程细胞系和动物模型等工具组合来进行体外和体内实验。我们最近的研究表明，PML 的乙酰化状态影响其与 PER2 的相互作用。我们进行了小分子筛选并鉴定了一种化合物，我们将其重新命名为 PINEF，它能够在没有 PML 的情况下促进 PER2 进入核。我们还建议研究 PML 乙酰化的状态和 PML 缺失时 PINEF 的作用如何改变亚细胞分布的概况和各种时钟调节器的相互作用。这些拟议的研究将得出关键时钟蛋白（包括 BMAL1、CLOCK、PER、CRY 和 PML）的分布和相互作用的更全面的时空概况。这些发现将有助于更好地了解哺乳动物的生物钟机制。此外，由于参与许多不同细胞通路的数十种核蛋白已定位于 PML NB 中，这项研究将有助于进一步了解 PML 是否以及如何促进不同功能网络（包括生物钟调节网络）之间的连接。