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性模式遵循日出和日落的每日循环，并由由交互分子网络组成的内源性生物时钟控制。为了使生物钟对生命的基本活性进行控制，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，睡眠清醒循环，生长和代谢调节的信号网络，尽管这些相互作用仍尚待阐明。我们最近的研究清楚地确定了PER2（时钟网络中的关键调节分子）与临时细胞性白血病蛋白（PML）之间的直接相互作用，该蛋白（PML）最初以肿瘤抑制蛋白为特征，负责肿瘤抑制蛋白，负责急性临床前临床细胞性白血病（APL）。目前，PML被称为大型核蛋白质复合物PML核体（PML NB）的核心成分，其中建议PML充当来自各种功能网络的核蛋白的“招募者和组织者”。核心 昼夜节律时钟机制，核蛋白BMAL1和时钟形成了驱动关键时钟调节器的转录表达式的异二聚体，包括PER和CRY基因以及许多时钟输出基因。我们发现PML不仅与核中的PER2直接相互作用，还促进了PER2核定位的过程。最近，还发现BMAL1与PML NB共定位。这些证据线使我们提出了这样的假设：PML在昼夜节律时钟机制的核调节网络中扮演“招聘者和组织者”的作用。在这里，我们建议通过构建各种时钟调节器和PML的亚细胞分布以及相互作用的曲线来研究PML在昼夜节律机制中的“招聘和组织者”功能。我们建议使用包括免疫荧光，倒数共免疫沉淀，重组表达构建体，基因工程性细胞系和动物模型在内的工具组合进行体外和体内实验。我们最近的研究表明，PML的乙酰化状态会影响其与PER2的相互作用。我们进行了一个小的分子筛选，并确定了一种我们将Pinef重命名的化合物，该化合物能够在没有PML的情况下促进PER2核进入。我们还建议研究PML乙酰化的状态以及在没有PML的情况下PINEF的作用如何改变细胞分布的特征以及各种时钟调节剂的相互作用。这些提出的研究将导致关键时钟蛋白的分布和相互作用（包括BMAL1，Clock，Pers，Pers，Crys和pml）的分布和相互作用的更全面时空特征。这些发现将有助于更好地理解哺乳动物昼夜节律的时钟机制。此外，正如许多参与许多不同细胞途径的核蛋白已定位在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之间的连接，包括昼夜节律时钟调节网络。