Role of Ror proteins in the mammalian circadian clock

描述(申请人提供)：昼夜节律存在于几乎所有的生物体中，以预测环境的每日变化并在时间上协调生物过程。虽然在确定哺乳动物生物钟的组成和控制生理的机制方面已经取得了很大进展，但知识库绝不是完整的。我们建议通过生成和整合不同类型的大规模数据，然后在体内验证这些数据，进一步阐明肝脏时钟的结构及其与新陈代谢的关系。为了分离和研究细胞自主时钟，我们已经确定了一个独特的细胞系，Met小鼠肝细胞-第3天(MMH-D3)，它保留了许多代谢功能，并表现出强大的昼夜节律。因此，我们将能够询问时钟和代谢途径，并研究它们的相互作用。我们已经获得了MMH-D3时间进程的转录和代谢数据(48小时内每2小时一次)，以确定循环实体。此外，我们将进行染色质免疫沉淀，然后对REV-ERB1进行测序(CHIP-SEQ)，REV-ERB1是时钟的一部分，也是脂肪代谢的调节因子，以探索时钟如何控制新陈代谢的机制之一。为了识别新的蛋白质，作为肝脏时钟的系统线索，我们将对超过6,000个分泌蛋白质的宝贵文库进行高通量筛选。据我们所知，这是为分析生物钟而收集的最广泛和最多样化的数据集之一。为了生成最全面的时钟模型，这些数据集不仅将被单独评估，而且将使用基于矢量自回归的方法被整合以重建生物网络。这样的网络能够预测不同层内和不同层之间的调节和相互作用，以及它们与代谢途径的关联。由网络模型产生的假说将在体内使用腺病毒技术进行验证，这也将使我们能够区分控制肝脏时钟的细胞自主信号和系统信号。生物钟显然对正常的生理功能有影响，因此，更好地了解生物钟工作将提高我们对人类疾病潜在机制的理解；这将完善诊断和治疗策略。

DESCRIPTION (provided by applicant): Circadian rhythms exist in almost all organisms to anticipate the daily changes in the environment and to temporally coordinate biological processes. Although much progress has been made to identify the components of the mammalian clock and the mechanisms by which it controls physiology, the knowledge base is by no means complete. We propose to further elucidate the architecture of the liver clock and its relationship with metabolism by generating and integrating divergent types of large-scale data, followed by validation of these data in vivo. In order to isolate and study the cell-autonomous clock, we have identified a unique cell line, Met Murine Hepatocyte-Day 3 (MMH-D3) that retains many metabolic functions and exhibits robust circadian rhythms. Thus we will be able to interrogate both the clock and metabolic pathways and study their interactions. We have already obtained transcriptomic and metabolomic data for MMH-D3 time-courses (every 2-hours for 48-hours) to identify cycling entities. Furthermore, we will perform chromatin immunoprecipitation followed by sequencing (ChIP-seq) for REV-ERB1, a nuclear hormone receptor that is part of the clock and a regulator of lipid metabolism, to explore one of the mechanisms of how the clock controls metabolism. To identify novel proteins that act as systemic cues for the liver clock, we will perform high- throughput screening of an invaluable library of over 6,000 secreted proteins. To our knowledge, this is one of the broadest and diverse data sets collected for the analysis of the circadian clock. To generate the most comprehensive model of the clock, these data sets will not only be evaluated individually, but will be integrated to reconstruct biological networks using methods based on vector auto-regression. Such networks have the ability to predict regulation and interaction within and between the different layers, as well as their associations with metabolic pathways. The hypotheses generated by the network model will be verified using adenovirus techniques in vivo, which will also enable us to differentiate between cell-autonomous and systemic signals governing the liver clock. The clock clearly has an influence on proper physiological function, thus a better understanding of circadian clockwork will improve our understanding of the mechanisms that underlie human disease; this will refine diagnostic and therapeutic strategies.