Mammalian circadian rhythms: from genes to mechanisms

项目摘要/摘要 生物钟在调节基本的细胞和生理过程中起着重要的作用。这个 哺乳动物的时钟由下丘脑协调的细胞自主振荡器组成 视交叉上核(SCN)执行组织和系统功能。十多个核心组件 已经确定了振荡器的类型；然而，关于监管方面的重大知识差距仍然存在 时钟系统中的机制/组件和组织特有的功能。我之前的研究提供了 对哺乳动物昼夜节律的重要见解。例如，我生成了PER2：：LUC报告小鼠 事实证明，它是钟表领域普遍使用的一种强大的试剂。我们最近报道了第二起- 一代报告鼠系PER2：：LucSV，并证明了PER2的一种新的miRNA调节 PER2在其自身转录中的积累和积极作用。与当前的Mira最相关 应用，我一直有兴趣将小鼠正向遗传筛选和机制研究相结合，以 探测基本时钟功能。此前，我们报道了通过老鼠筛选和 两种拮抗E3连接酶FBXL3和FBXL21在昼夜节律调节中的机制剖析。更多 最近，我们发现了一个GSK-3beta-FBXL21调节级联反应，控制着细胞的节律性降解 肌节蛋白TCAP与骨骼肌功能。在这些先前研究的基础上，目前的提案 目的是确定FBXL21的组织特异性昼夜节律机制，并识别新的时钟成分 来自流线型的老鼠筛选。我们将研究FBXL21的新靶点和功能 肌肉，包括骨骼肌和心肌，侧重于蛋白平衡和肌源性分化。 利用老鼠前向基因筛查方面的专业知识，我最近进行了一次基因筛查 使用高效育种/表型方案的显性表型。全外显子组测序及其变异 分析准确地发现了一个新的昼夜节律突变系，其昼夜轮转周期和年龄都延长了。 依赖性神经退行性变。我们将确定时钟的这种新的遗传成分，并表征 潜在的监管机制。总体而言，这些研究有望发现重要的机制和 哺乳动物昼夜节律的功能。我已经建立了综合研究能力，结合 小鼠遗传学和表型、生化/分子/细胞研究、成像方法和组学 平台，辅以广泛的合作者网络的专业知识。这些加在一起形成了一个极好的 为拟议的研究奠定了基础。最终目标是了解生物计时如何控制身体。 通过优化我们的生物钟，我们可以做些什么来保护我们的健康。

PROJECT SUMMARY / ABSTRACT Circadian clocks play fundamental roles in regulating essential cellular and physiological processes. The mammalian clock is comprised of cell-autonomous oscillators orchestrated by the hypothalamic suprachiasmatic nuclei (SCN) to perform tissue and systemic functions. More than a dozen core components of the oscillator have been identified; however, significant knowledge gaps remain regarding regulatory mechanisms/components and tissue-specific functions in the clock system. My previous research has provided important insights into mammalian circadian rhythms. For example, I generated Per2::Luc reporter mice which proved to be a powerful reagent ubiquitously employed in the clock field. We recently reported a second- generation reporter mouse line, Per2::LucSV, and demonstrated a novel miRNA regulation of PER2 accumulation and a positive role of PER2 in its own transcription. Most relevant to the current MIRA application, I have been interested in combining mouse forward genetic screening and mechanistic studies to probe fundamental clock functions. Previously we reported the identification by mouse screening and mechanistic dissection of two antagonistic E3 ligase, FBXL3 and FBXL21 in circadian period regulation. More recently, we uncovered a GSK-3beta-FBXL21 regulatory cascade controlling rhythmic degradation of the sarcomere protein TCAP and skeletal muscle function. Building on these prior studies, the current proposal aims to determine tissue-specific circadian mechanisms of FBXL21 and to identify novel clock components from a streamlined mouse screening. We will examine new targets and functions of FBXL21 in striated muscles including skeletal and cardiac muscles, focusing on proteostasis and myogenic differentiation. Leveraging expertise in mouse forward genetic screening, I recently performed a genetic screening for dominant phenotypes using an efficient breeding/phenotyping scheme. Whole-exome sequencing and variant analysis pinpointed a novel circadian mutant line with a lengthened circadian wheel-running period and age- dependent neurodegeneration. We will identify this new genetic component of the clock and characterize the underlying regulatory mechanisms. Overall, these studies promise to discover important mechanisms and functions of circadian rhythms in mammals. I have established an integrative research capability combining mouse genetics and phenotyping, biochemical/molecular/cellular studies, imaging methodologies, and omics platforms, complemented by expertise from a broad network of collaborators. These together form an excellent foundation for the proposed research. The ultimate goal is to understand how biological timing governs bodily function and what we can do to safeguard our health by optimizing our natural clock.