NANOG STRUCTURE AND FUNCTION IN STEM CELL PLURIPOTENCY

项目摘要 Nanog在干细胞多能性中的结构和功能 这项拟议中的研究将确定Nanog相互作用的结构基础，Nanog是一种表观遗传学， 调节细胞增殖和分化的重编程因子。Nanog是自我更新所必需的 胚胎干细胞和重新编程，体细胞转化为多能干细胞。 Nanog与超过100种其他蛋白质相互作用，但内在的无序区域使Nanog难以研究。 研究人员将利用他们对内在无序蛋白质的丰富经验来解剖Nanog 分两个阶段发挥作用：它们将决定磷酸化和自组装如何调节Nanog的稳定性， 活性（目标1），他们将确定Nanog和伴侣之间的相互作用如何产生多能性 (Aim 2）。这些实验将建立Nanog枢纽功能的分子机制， 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， 多能性，这与利用干细胞或寻求摧毁癌症的治疗干预有关 干细胞 第一个目标将使用溶液NMR光谱和其他方法来揭示磷酸化是如何在 Nanog PEST降解信号改变Ser-Pro二肽的局部骨架构象并影响Nanog 通过磷酸化依赖性脯氨酸异构酶Pin1在细胞中的水平。将使用重编程试验 以测试干扰Nanog/Pin1相互作用的影响。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-末端结构域是如何被分离的。 末端结构域在体外和体内调节Nanog寡聚化。这些实验将提供资金- 对组装状态下Nanog可用性的精神洞察，被认为是其作为分子枢纽功能的基础。 第二个目标将确定Nanog作为“通往多能性”的机制。的 研究人员将使用非天然氨基酸光交联，化学交联和质谱法- 基于蛋白质组学来识别干细胞中的Nanog结合伴侣，以揭示干细胞之间的任何相关性。 多个结合配偶体，并绘制支持结合的Nanog序列。他们将决定 这些相互作用的功能重要性，通过扰乱伴侣和/或其结合位点的Nanog体细胞中 细胞重编程测定。这些实验将证明关键合作伙伴对Nanog的重要性 功能，并将揭示Nanog的哪些区域招募这些合作伙伴来驱动多能性。两个合伙人 它们结合的Nanog区域可能是调节Nanog活性的有用靶点。他们将 确定Nanog如何影响转录因子Oct4和Sox 2相互结合以及与转录因子的结合。 Nanog启动子。这些实验将揭示Nanog支架如何控制大分子组装， 干细胞多能性。

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.