NANOG STRUCTURE AND FUNCTION IN STEM CELL PLURIPOTENCY

项目摘要 干细胞多能性中的Nanog结构与功能 这项拟议的研究将确定表观遗传学Nanog相互作用的结构基础 调节细胞增殖和分化的重编程因子。自我更新需要Nanog 对于胚胎干细胞的研究和重新编程，将体细胞转化为多能干细胞。 Nanog与其他100种蛋白质相互作用，但本质上无序的区域使Nanog难以研究。 研究人员将利用他们在内在无序蛋白质方面的丰富经验来剖析Nanog 功能分为两个阶段：它们将决定磷酸化和自组装如何调节Nanog稳定性和 活动(目标1)，他们将决定Nanog和合作伙伴之间的互动如何产生多能性 (目标2)。这些实验将建立Nanog Hub功能的分子机制，并可能 为未来的药物发现工作生产先导化合物，以靶向Nanog依赖的细胞增殖和 多能性，这与利用干细胞或寻求消灭癌症的治疗干预有关 干细胞。 第一个目标将使用溶液核磁共振波谱和其他方法来揭示细胞内的磷酸化如何 Nanog害虫降解信号改变Ser-Pro二肽的局部骨架构象并影响Nanog 通过磷酸化依赖的脯氨酸异构酶Pin1在细胞中的水平。将使用重新编程的分析 测试扰乱Nanog/Pin1相互作用的效果。单分子荧光方法将用于 表征分离的Nanog C-末端结构域的结构和动力学，并确定N- 在体外和体内，末端结构域调控Nanog齐聚。这些实验将提供基础-- 在头脑中洞察到Nanog在组装状态下的可用性，认为这是其作为分子枢纽的功能的基础。 第二个目标将决定Nanog作为“通向多能性的门户”的机制。这个 调查人员将使用非天然氨基酸光交联法、化学交联法和质谱仪-- 基于蛋白质组学识别干细胞中的Nanog结合伙伴，以揭示两者之间的任何关联 多个结合配对，并映射支持结合的Nanog序列。他们将决定 这些相互作用通过干扰Nanog上的伴侣和/或其结合部位在体细胞中的功能重要性 细胞重编程分析。这些实验将证明关键合作伙伴对Nanog的重要性 并将揭示Nanog的哪些区域招募这些合作伙伴来推动多能性。双方都是合伙人 它们结合的Nanog区域可能是调节Nanog活性的有用靶点。他们会 确定Nanog如何影响转录因子Oct4和Sox2相互结合以及与 Nanog发起人。这些实验将揭示Nanog支架如何组装控制 干细胞的多能性。

Project Summary Nanog Structure and Function in Stem Cell Pluripotency This proposed research will determine the structural basis for interactions made by Nanog, an epigenetic reprogramming factor that regulates cellular proliferation and differentiation. Nanog is required for self-renewal of embryonic stem cells and for re-programming, the transformation of somatic cells to pluripotent stem cells. Nanog interacts with >100 other proteins, but intrinsically disordered regions make Nanog difficult to study. The investigators will use their extensive experience with intrinsically disordered proteins to dissect Nanog function in two stages: they will determine how phosphorylation and self-assembly regulate Nanog stability and activity (Aim 1), and they will determine how interactions between Nanog and partners give rise to pluripotency (Aim 2). These experiments will establish the molecular mechanisms of Nanog hub function, and perhaps produce lead compounds for future drug discovery efforts to target Nanog-dependent cell proliferation and pluripotency, which is relevant to therapeutic interventions that utilize stem cells or that seek to destroy cancer stem cells. The first Aim will use solution NMR spectroscopy and other methods to reveal how phosphorylation in the Nanog PEST degradation signal alters local backbone conformation at Ser-Pro dipeptides and affects Nanog levels in cells via the phosphorylation-dependent proline isomerase Pin1. Reprogramming assays will be used to test the effects of perturbing Nanog/Pin1 interaction. Single molecule fluorescence methods will be used to characterize the structure and dynamics of the isolated Nanog C-terminal domain and to determine how the N- terminal domain modulates Nanog oligomerization in vitro and in vivo. These experiments will provide funda- mental insight into Nanog availability in the assembled state thought to underlie its function as a molecular hub. The second Aim will determine the mechanisms by which Nanog acts as the ‘gateway to pluripotency’. The investigators will use unnatural amino acid photocrosslinking, chemical crosslinking and mass spectrometry- based proteomics to identify Nanog binding partners in stem cells, to uncover any correlations between multiple binding partners, and to map the Nanog sequences that support binding. They will determine the functional importance of these interactions by perturbing the partner and/or its binding site on Nanog in somatic cell reprogramming assays. These experiments will demonstrate the importance of key partners to Nanog function and will reveal which regions of Nanog recruit these partners to drive pluripotency. Both the partners and the regions of Nanog that they bind may be useful targets for modulating Nanog activity. They will determine how Nanog influences binding of the transcription factors Oct4 and Sox2 to each other and to the Nanog promoter. These experiments will reveal how Nanog scaffolds macromolecular assemblies that control stem cell pluripotency.