Nucleolin recognition of MYC promoter G-quadruplex and its role in MYC regulation by MycG4-ligands

项目总结 MYC启动子G-四链的核仁识别及其在MycG4-配体调控MYC中的作用 G-四链(G4S)是一种非规范的DNA二级结构。C-Myc--中心驱动癌基因之一 在人类癌症中，在其近端的启动子区域有一个DNA G4基序，起到转录沉默的作用。 然而，对c-Myc启动子G4(MycG4)是如何调控的知之甚少。核仁素被鉴定为 主要的c-Myc G4结合蛋白，并显示出与MycG4的结合亲和力显著高于其已知的细胞 底物NRE RNA。核仁素在体内直接与MycG4启动子区域结合，并过表达 核仁会抑制c-Myc启动子的活性。我们假设核仁会识别c-Myc 启动子G4和MycG4靶向小分子通过相互作用调节c-Myc基因表达 核仁素-MycG4复合体。在这项建议中，我们将研究核仁素如何通过 确定核仁素-MycG4复合体的分子结构。我们还将研究小分子如何 与核仁素-MycG4复合体相互作用，以及相互作用如何影响c-Myc转录。C-Myc 启动子G4是一个有吸引力的抗癌药物靶点。我们的初步数据显示核仁素-MycG4复合体 显然参与了MycG4-配体对c-Myc的调节。这项拟议的研究是第一次 核仁素-MycG4复合体的结构和功能研究。对核仁素的结构层面的理解- MycG4复合体及其与小分子的相互作用将为MycG4的功能提供重要信息 和MycG4靶向药物开发。拟议的研究将使用高场核磁共振的组合 光谱学、X射线结晶学和其他生物物理、生化和细胞方法。我们有 组建了一支强大的协作团队。具体目的是：1)确定核仁的结构 与c-Myc启动子G-四链的复合体。核仁蛋白是一种多结构域蛋白质，含有四个串联的RNA- 结合结构域(RBD)。我们假设核仁会使用所有四种Rbd包裹MycG4并识别 G4的外环实现了高亲和力的结合。这个结构将是第一个展示不寻常的 DNA G4结构是由一种模块化的蛋白质识别的，可能是通过多价相互作用。2)调查 小分子如何与核仁-MycG4复合体相互作用及其对c-Myc基因调控的影响。我们的 初步数据显示，只有稳定核仁素-MycG4复合体的MycG4-配体才能降低c-Myc 级别。我们将使用体外和基于细胞的分析相结合的方法来鉴定能够稳定 核仁素-MycG4复合体，下调c-Myc。我们将确定c-Myc基因的特异性和 确定顶层化合物的主要细胞反应。对于形成稳定三元的MycG4-配体 对于核仁素-MycG4的络合物，我们还将确定核仁素-MycG4稳定的结构基础。

PROJECT SUMMARY Nucleolin recognition of MYC promoter G-quadruplex and its role in MYC regulation by MycG4-ligands G-quadruplexes (G4s) are non-canonical DNA secondary structures. c-Myc, one of the central driver oncogenes in human cancers, has a DNA G4 motif in its proximal promoter region that functions as a transcription silencer. However, little is known about how the c-Myc promoter G4 (MycG4) is regulated. Nucleolin is identified as the major c-Myc G4 binding protein and shows a remarkably higher binding affinity for MycG4 over its known cellular substrate NRE RNA. Nucleolin directly binds to the MycG4 promoter region in vivo and overexpression of nucleolin represses the activity of the c-Myc promoter. We hypothesize that nucleolin recognizes the c-Myc promoter G4 and that MycG4-targeted small molecules regulate c-Myc gene expression through interactions with the nucleolin-MycG4 complex. In this proposal, we will study how nucleolin recognizes MycG4 by determining the molecular structure of the nucleolin-MycG4 complex. We will also study how small molecules interact with the nucleolin-MycG4 complex and how the interactions affect c-Myc transcription. The c-Myc promoter G4 is an attractive anticancer drug target. Our preliminary data show that the nucleolin-MycG4 complex is clearly involved in the c-Myc regulation by MycG4-ligands. The proposed research represents the first structural and functional study of the nucleolin-MycG4 complex. A structural level understanding of the nucleolin- MycG4 complex and its interactions with small molecules will provide important information for MycG4 function and MycG4-targeted drug development. The proposed research will use a combination of high-field NMR spectroscopy, X-ray crystallography, and other biophysical, biochemical, and cellular approaches. We have assembled a strong collaboration team. The specific aims are: 1) To determine the structure of nucleolin in complex with the c-Myc promoter G-quadruplex. Nucleolin is a multi-domain protein containing four tandem RNA- binding domains (RBDs). We hypothesize that nucleolin uses all four RBDs to wrap around MycG4 and recognize the G4 external loops to achieve a high-affinity binding. This structure would be the first to show how the unusual DNA G4 structure is recognized by a modular protein, likely through multi-valent interactions. 2) To investigate how small molecules interact with the nucleolin-MycG4 complex and their effects on c-Myc gene regulation. Our preliminary data shows that only MycG4-ligands that stabilize the nucleolin-MycG4 complex can lower c-Myc levels. We will use a combination of in vitro and cell-based assays to identify compounds that can stabilize the nucleolin-MycG4 complex and downregulate c-Myc. We will determine the specificity for the c-Myc gene and determine major cellular response for the top compounds. For the MycG4-ligands that form the stable ternary complexes with nucleolin-MycG4, we will also determine the structural basis of the nucleolin-MycG4 stabilization.