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

项目摘要 核仁素识别MYC启动子G-quadruplex及其在MycG 4配体调控MYC中的作用 G-四链体（G4）是一种非典型的DNA二级结构。c-Myc，一种中心驱动癌基因 在人类癌症中，在其近端启动子区具有DNA G4基序，其作为转录沉默子发挥作用。 然而，关于c-Myc启动子G4（MycG 4）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，并且显示出比其已知的细胞内结合蛋白对MycG 4的显著更高的结合亲和力。 底物NRE RNA。核仁素在体内直接与MycG 4启动子区结合， 核仁素抑制c-Myc启动子的活性。我们假设核仁素识别c-Myc 启动子G4和MycG 4靶向小分子通过相互作用调节c-Myc基因表达 与核仁素-MycG 4复合物。在本研究中，我们将研究核仁素如何识别MycG 4， 确定核仁素-MycG 4复合物的分子结构。我们还将研究小分子 与核仁素-MycG 4复合物相互作用以及相互作用如何影响c-Myc转录。的c-Myc 启动子G4是一个有吸引力的抗癌药物靶点。我们的初步数据表明，核仁素-MycG 4复合物 明确参与MycG 4配体对c-Myc的调控。这项研究代表了第一个 核仁素-MycG 4复合物的结构和功能研究。对核仁素的结构水平的理解- MycG 4复合物及其与小分子的相互作用将为研究MycG 4的功能提供重要信息 和MycG 4靶向药物开发。拟议的研究将结合使用高场核磁共振 光谱学、X射线晶体学和其他生物物理学、生物化学和细胞学方法。我们有 组建了强大的合作团队。具体的目的是：1）确定核仁素的结构， 与c-Myc启动子G-四链体的复合物。核仁素是一种多结构域蛋白，含有四个串联的RNA- 结合域（RBD）。我们假设核仁素使用所有四个RBD包裹MycG 4并识别 G4外部环以实现高亲和力结合。这个结构将是第一个展示不寻常的 DNA G4结构被模块化蛋白质识别，可能通过多价相互作用。2)探讨 小分子如何与核仁素-MycG 4复合物相互作用及其对c-Myc基因调控的影响。我们 初步数据显示，只有稳定核仁素-MycG 4复合物的MycG 4配体才能降低c-Myc 程度.我们将使用体外和基于细胞的测定相结合的方法来鉴定可以稳定细胞的化合物。 核仁素-MycG 4复合物并下调c-Myc。我们将确定c-Myc基因的特异性， 确定主要的细胞反应的顶部化合物。对于形成稳定的三元结构的MycG 4-配体， 与核仁素-MycG 4的复合物，我们还将确定核仁素-MycG 4稳定化的结构基础。

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.