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

• 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
• 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

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每日日出和日落的周期，并由相互作用分子网络组成的内源性生物钟控制。为了使生物钟能够控制生命的基本活动，生物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，尽管这些相互作用仍有待阐明。我们最近的研究已经清楚地建立了PER 2（时钟网络中的关键调节分子）和早幼粒细胞白血病蛋白（PML）之间的直接相互作用，PML最初被表征为负责急性早幼粒细胞白血病（APL）的肿瘤抑制蛋白。目前，PML被认为是一种大型核蛋白复合物的中心成分，PML核体（PML NB），其中PML被认为是各种功能网络中核蛋白的“招募者和组织者”。水平为核心 在生物钟机制中，核蛋白BMAL 1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PER 2直接相互作用，而且还促进了PER 2的核定位过程。最近，还发现BMAL 1与PML NB共定位。这些证据使我们假设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，我们建议调查的“招聘和组织者”功能的PML在生物钟机制，通过构建一个配置文件的亚细胞分布和相互作用的各种时钟调节器和PML。我们建议进行体外和体内实验，使用的工具，包括免疫荧光，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的组合。我们最近的研究表明，PML的乙酰化状态影响其与PER 2的相互作用。我们已经进行了小分子筛选，并确定了一种化合物，我们将其重新命名为PINEF，该化合物能够在没有PML的情况下促进PER 2进入核。我们还建议研究PML乙酰化的状态和PINEF在没有PML的情况下的作用如何改变亚细胞分布和各种时钟调节因子的相互作用。这些拟议的研究将导致一个更全面的时空分布和关键的时钟蛋白，包括BMAL 1，时钟，PER的，pers和PML的相互作用。这些发现将有助于更好地理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是否以及如何有助于不同功能网络之间的连接，包括昼夜节律钟调节网络。