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

核仁素对 MYC 启动子 G-四链体的识别及其在 MycG4-配体调节 MYC 中的作用

基本信息

批准号：
10373013
负责人：
DANZHOU YANG
金额：
$ 34.97万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10373013
关键词：
Affect Affinity Antineoplastic Agents Binding Binding Proteins Binding Sites Biochemical Bioinformatics Biological Assay Biophysics C-terminal Cells ChIP-seq Chemicals Chromatin Collaborations Complex Computer Models DNA Data Disease Drug Targeting EMSA Elements Enzyme-Linked Immunosorbent Assay Fluorescence Fluorescence Resonance Energy Transfer G-Quartets Gene Expression Gene Expression Regulation Genetic Transcription Genomics Global Change Hela Cells Human In Vitro Libraries Ligands MYC Family Protein Malignant Neoplasms Methods Molecular Molecular Structure Mutation N-terminal NMR Spectroscopy Nuclear Extract Oncogenes Play Promoter Regions Proteins RNA RNA Recognition Motif Regulation Research Roentgen Rays Role Specificity Structure Tertiary Protein Structure X-Ray Crystallography base c-myc Genes cancer cell chromatin immunoprecipitation design drug development experience experimental study in vivo novel nucleolin overexpression promoter response screening small molecule targeted treatment transcriptome sequencing

项目摘要

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.

项目概要核仁素对 MYC 启动子 G-四链体的识别及其在 MycG4-配体调节 MYC 中的作用 G-四链体 (G4s) 是非规范的 DNA 二级结构。 c-Myc，核心驱动癌基因之一在人类癌症中，其近端启动子区域有一个 DNA G4 基序，起到转录沉默子的作用。然而，人们对 c-Myc 启动子 G4 (MycG4) 的调控机制知之甚少。核仁素被确定为主要的 c-Myc G4 结合蛋白，并且对 MycG4 的结合亲和力比其已知的细胞要高得多底物 NRE RNA。核仁素在体内直接结合 MycG4 启动子区域并过度表达核仁素抑制 c-Myc 启动子的活性。我们假设核仁素识别 c-Myc 启动子 G4 和 MycG4 靶向小分子通过相互作用调节 c-Myc 基因表达与核仁素-MycG4 复合物。在本提案中，我们将研究核仁素如何通过以下方式识别 MycG4：确定核仁素-MycG4 复合物的分子结构。我们还将研究小分子如何与核仁素-MycG4 复合物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。我们的初步数据表明核仁素-MycG4 复合物显然参与了 MycG4-配体对 c-Myc 的调节。拟议的研究代表了第一个核仁素-MycG4 复合物的结构和功能研究。对核仁素结构水平的理解 MycG4复合物及其与小分子的相互作用将为MycG4功能提供重要信息和 MycG4 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 射线晶体学以及其他生物物理、生物化学和细胞方法。我们有组建了一支强大的协作团队。具体目标是： 1) 确定核仁素的结构与 c-Myc 启动子 G-四链体的复合物。核仁蛋白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 来包裹 MycG4 并识别 G4外部环实现高亲和力结合。这个结构将是第一个展示不寻常的结构 DNA G4 结构被模块化蛋白质识别，可能是通过多价相互作用。 2）调查小分子如何与核仁素-MycG4 复合物相互作用及其对 c-Myc 基因调控的影响。我们的初步数据表明，只有稳定核仁素-MycG4复合物的MycG4配体才能降低c-Myc 水平。我们将结合使用体外和基于细胞的测定来鉴定可以稳定核仁素-MycG4 复合物并下调 c-Myc。我们将确定 c-Myc 基因的特异性和确定顶级化合物的主要细胞反应。对于形成稳定三元体的 MycG4 配体与核仁素-MycG4 形成复合物，我们还将确定核仁素-MycG4 稳定化的结构基础。