Circadian Clock: Differential Cryptochrome Functions

昼夜节律时钟：差异隐花色素功能

• 批准号: 7684820
• 负责人: STEVEN M. REPPERT
• 金额: $ 33.64万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-08 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7684820
• 关键词: Adult; Affinity Chromatography; Aloral; Animals; Behavior; Behavioral; Biochemical Genetics; Bombyx; Bombyx mori; Butterflies; Cell Line; Cells; Circadian Rhythms; Co-Immunoprecipitations; Complex; Crying; Deletion Mutation; Development; Drosophila genus; Evolution; Expressed Sequence Tags; Family; Feedback; Gene Mutation; Gene Silencing; Gene Targeting; Genes; Genetic; Goals; Homologous Gene; Human; In Vitro; Insecta; Jet Lag Syndrome; Knowledge; Lead; Libraries; Mammals; Mental disorders; Modeling; Molecular; Monitor; Moths; Nature; Output; Pathway interactions; Photoreceptors; Property; Proteins; Proteomics; Psychopathology; RNA Interference; Repression; Role; Signal Pathway; Signal Transduction; Sleep; Sleep Disorders; Testing; Time; Transcription Repressor/Corepressor; Zinc Fingers; circadian pacemaker; cryptochrome; egg; fly; hatching; improved; in vivo; innovation; loss of function mutation; novel; nuclease; public health relevance; shift work; tool

DESCRIPTION (provided by applicant): Paradigm shifting studies in the monarch butterfly (Danaus plexippus) have enhanced our view of the evolution and function of CRYPTOCHROME (CRY) proteins in animals. The butterfly's clock mechanism posits two CRY proteins as critical for circadian timing, giving rise to a NOVEL clockwork. Thus, in the same species, the two functionally distinct CRYs can be analyzed. In monarchs, a Drosophila-like CRY, designated CRY1, functions as a likely circadian photoreceptor, while a vertebrate-like CRY, designated CRY2, appears to function as the major transcriptional repressor of the clockwork transcriptional feedback loop. The analysis of distinct CRY function has been further aided by a monarch cell line (DpN1 cells), which expresses a light-driven clock, in which both monarch CRY1 and CRY2 function; no other light-sensitive insect cell line has been described. The Specific Aims of this proposal will expand our knowledge of the unique properties of the CRY proteins by defining distinct CRY mechanisms of action in lepidopterans (butterflies and moths) using molecular, biochemical, genetic, and behavioral approaches. Because of the parallel, complementary nature of circadian clock discoveries between flies and mammals, which can now be extended to lepidopterans, our overriding hypothesis is that further analysis of the two families of animal CRY proteins that are represented in insects will advance our fundamental understanding of animal clock mechanisms. The Specific Aims will 1) employ an RNAi screen in DpN1 cells to discover novel components of the insect CRY1 light-signaling pathway; 2) use a proteomics approach in DpN1 cells to identify novel interactors within the insect CRY2/CLK:CYC transcriptional complex, utilizing tandem affinity purification and co-immunoprecipitation to identify additional CRY2-, CLOCK- and CYCLE-interacting proteins; and 3) define in vivo the critical clockwork function of insect CRY2 by targeted gene inactivation in the commercial silkworm Bombyx mori using a zinc finger nuclease strategy. Our major goal is to define the molecular mechanisms of CRY function in the animal clockwork. The proposed studies are innovative and provide an integrated approach at defining differential insect CRY mechanisms of action. The results will also increase our fundamental understanding of clockwork function from an evolutionary perspective and demonstrate lepidopterans to be a useful model in which to study animal clock mechanisms. Understanding the molecular clock should increase our knowledge of how clock gene mutations contribute to psychopathology. Likewise, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and shift-work ailments, and of clock-related sleep and psychiatric disorders. PUBLIC HEALTH RELEVANCE Understanding the molecular clock, the focus of this proposal, should increase our knowledge of how clock gene mutations contribute to psychopathology and some sleep disorders. Likewise, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and shift-work ailments, and of clock-related sleep and psychiatric disorders.

描述（由申请人提供）：君主蝴蝶（Danaus plexippus）中的范式转移研究增强了我们对动物中隐性蛋白（CRY）蛋白的进化和功能的看法。蝴蝶的时钟机制假设两个哭泣的蛋白质对昼夜节律至关重要，从而产生了新颖的发条。因此，在同一物种中，可以分析两个在功能上不同的CRY。在Monarchs中，果蝇状的哭泣被指定为Cry1，起作用的昼夜节律感受器，而脊椎动物样的哭泣（指定为Cry2）似乎是发条转录反馈循环的主要转录阻遏物。君主细胞系（DPN1细胞）进一步辅助了对不同的CRY函数的分析，该君主细胞系（DPN1细胞）表达了一个轻度驱动的时钟，其中Monarch Cry1和Cry2函数均可进一步。尚未描述其他光敏的昆虫细胞系。该提案的具体目的将通过使用分子，生化，遗传和行为方法来定义鳞翅目（蝴蝶和飞蛾）的独特哭泣作用机制，扩大我们对哭泣蛋白独特特性的了解。由于蝇和哺乳动物之间的昼夜节律发现的平行，互补性，现在可以扩展到鳞翅目，我们的压倒性假设是，对昆虫中所代表的两个动物哭泣蛋白家族的进一步分析将推进我们对动物时钟机制的基本理解。具体目的是1）在DPN1细胞中采用RNAi筛选，发现昆虫Cry1光信号途径的新成分； 2）使用DPN1细胞中的蛋白质组学方法来识别昆虫Cry2/Clk中的新型相互作用：CYC转录复合物，利用串联亲和纯化和共免疫沉淀来识别其他CRY2-，时钟和周期性相互作用的蛋白质； 3）在体内使用锌指核酸酶策略在商业蚕bombyx mori中靶向基因失活的昆虫Cry2的关键发条函数。我们的主要目标是定义动物发条中哭泣功能的分子机制。拟议的研究具有创新性，并提供了一种综合方法来定义差异昆虫哭泣的作用机理。结果还将从进化的角度提高我们对发条功能的基本理解，并证明鳞翅目是研究动物时钟机制的有用模型。了解分子时钟应增加我们对时钟基因突变如何促进心理病理学的了解。同样，这种理解应导致对人类时钟进行药理学操纵的新策略，以改善喷气滞后和转移工作疾病的治疗，以及与时钟相关的睡眠和精神疾病的治疗。公共卫生的相关性理解分子时钟，这是该提案的重点，应该提高我们对时钟基因突变如何对精神病理学和某些睡眠障碍的贡献的了解。同样，这种理解应导致对人类时钟进行药理学操纵的新策略，以改善喷气滞后和转移工作疾病的治疗，以及与时钟相关的睡眠和精神疾病的治疗。