Molecular Basis of Histone Methylation by PRMT5

PRMT5 组蛋白甲基化的分子基础

基本信息

批准号：
10607316
负责人：
Sanim Rahman
金额：
$ 4.77万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10607316
关键词：
ARG2 gene Acute Myelocytic Leukemia Affinity Arginine Binding Binding Proteins Biochemical Biological Assay Biological Process Biophysics Cell Nucleus Chromatin Chromatin Modeling Colon Carcinoma Complex Cryoelectron Microscopy Cytoplasm Cytosol Data Deposition Development Drug Design Enzyme Kinetics Enzymes Epithelium Eukaryota Gene Expression Genes Genetic Transcription Histone H2A Histone H2B Histone H3 Histone H4 Histones Human In Vitro Knowledge Lysine Malignant Neoplasms Malignant neoplasm of lung Malignant neoplasm of ovary Malignant neoplasm of prostate Malignant neoplasm of urinary bladder Mesenchymal Metastasis Suppressor Genes Methylation Methyltransferase Modification Molecular Molecular Chaperones Nucleosomes Post-Translational Protein Processing Protein-Arginine N-Methyltransferase Proteins Research Research Personnel Role Small Interfering RNA Specificity Structure Substrate Specificity Techniques Testing Training Transcriptional Regulation Ubiquitination Work Writing career design dimer gene repression histone methylation histone methyltransferase histone modification in vivo inhibitor insight knock-down malignant stomach neoplasm overexpression protein function reconstitution recruit screening

项目摘要

Project Summary/Abstract The dynamic writing and erasing of histone post-translational modifications on nucleosomes regulate eukaryotic gene expression by tuning chromatin organization and recruiting chromatin-binding proteins. The methylation of arginines can activate or repress transcription depending on the histone residue and its methylation state. Protein arginine methyltransferase 5 (PRMT5), along with its obligate binding partner Methylosome protein 50 (MEP50), is the primary complex for the symmetric dimethylation of arginine across all eukaryotes. In addition, PRMT5- MEP50 can either activate or repress the transcription of several genes, depending on which residue the enzyme modifies. PRMT5-MEP50 catalyzes methylation on four histone residues, namely histone H2A-Arg3 (H2AR3), H3-Arg2 (H3R2), H3-Arg8 (H3R8), and H4-Arg3 (H4R3). Due to PRMT5-MEP50’s diverse roles in transcription regulation, PRMT5 is overexpressed in several cancers as it regulates the transcription of several metastasis suppressor genes and epithelial-mesenchymal transition activating genes. Despite PRMT5-MEP50’s importance in gene expression, very little is known of how PRMT5-MEP50 methylates histone and/or nucleosome substrates. However, recent work has revealed that PRMT5’s specificity is regulated by (1) recognition of cytosolic H2A-H2B dimers to methylate H2AR3 and (2) being able to preferentially methylate histone H4 in the presence of substrate adaptor Coordinator of PRMT5 (COPR5). Despite these findings, molecular determinants towards this specificity are still unknown. Using a combination of biochemical and structural approaches, I will investigate the mechanism of histone specificity and activity by the PRMT5-MEP50 complex. In Aim 1, I will determine contributions towards PRMT5-MEP50’s recognition of H2A-H2B dimers by quantifying the activity and binding of PRMT5-MEP50 on various histone H2A-containing substrates. To provide molecular detail of this recognition, I will determine the structure of PRMT5-MEP50 bound to H2A-H2B dimers using cryo- electron microscopy (cryo-EM). While screening substrates of H2A methylation, I discovered that PRMT5- MEP50 activity is stimulated by ubiquitination of histone H2BK120 (H2BK120-Ub). I will probe in vivo relevance of this crosstalk by siRNA knockdowns. I will then reveal the mechanism of this activation by quantifying activity and binding of PRMT5-MEP50 in the presence of H2BK120-Ub and resolving the EM structure of PRMT5- MEP50 bound to H2A-H2BK120-Ub dimers. In Aim 2, I will elucidate the function of COPR5 and the PRMT5- MEP50-COPR5 complex. My preliminary data revealed that COPR5 does not bind to nucleosomes and cannot recruit PRMT5-MEP50 to the nucleosome, conflicting previous speculations of COPR5’s function. Therefore, I will identify COPR5’s preferred histone-containing substrate and quantify COPR5’s binding and contribution to the enzymatic activity of PRMT5-MEP50. Finally, I will solve the structure of PRMT5-MEP50-COPR5 bound to its histone substrate by cryo-EM. Together, this proposal will construct a molecular framework of PRMT5- MEP50’s substrate specificity to aid in structure-based drug design, by revealing substrate-specific interactions.

项目概要/摘要核小体上组蛋白翻译后修饰的动态写入和擦除调节真核生物通过调整染色质组织和招募染色质结合蛋白来调节基因表达。精氨酸可以根据组蛋白残基及其甲基化状态激活或抑制转录。精氨酸甲基转移酶 5 (PRMT5) 及其专性结合伴侣甲基糖体蛋白 50 (MEP50)，是所有真核生物中精氨酸对称二甲基化的主要复合物。 MEP50 可以激活或抑制多个基因的转录，具体取决于酶的残基 PRMT5-MEP50 催化四个组蛋白残基的甲基化，即组蛋白 H2A-Arg3 (H2AR3)， H3-Arg2 (H3R2)、H3-Arg8 (H3R8) 和 H4-Arg3 (H4R3) 由于 PRMT5-MEP50 在转录中的不同作用。 PRMT5 在多种癌症中过度表达，因为它调节多种转移的转录尽管 PRMT5-MEP50 很重要，但抑制基因和上皮间质转化激活基因。在基因表达中，人们对 PRMT5-MEP50 如何甲基化组蛋白和/或核小体知之甚少然而，最近的工作表明 PRMT5 的特异性受 (1) 识别的调节。胞质 H2A-H2B 二聚体甲基化 H2AR3，并且 (2) 能够优先甲基化组蛋白 H4 尽管存在这些发现，但 PRMT5 的底物接头协调子 (COPR5) 的存在仍是分子决定因素。这种特异性仍然未知，我将结合使用生化和结构方法。研究 PRMT5-MEP50 复合物的组蛋白特异性和活性机制。在目标 1 中，我将通过量化确定 PRMT5-MEP50 识别 H2A-H2B 二聚体的贡献 PRMT5-MEP50 对各种含组蛋白 H2A 的底物的活性和结合提供分子。为了了解这一识别的细节，我将使用冷冻技术确定与 H2A-H2B 二聚体结合的 PRMT5-MEP50 的结构在筛选 H2A 甲基化底物时，我发现 PRMT5-。 MEP50 活性由组蛋白 H2BK120 (H2BK120-Ub) 泛素化刺激，我将探讨体内相关性。然后我将通过量化活性来揭示这种激活的机制。以及在 H2BK120-Ub 存在下 PRMT5-MEP50 的结合并解析 PRMT5-的 EM 结构 MEP50 与 H2A-H2BK120-Ub 二聚体结合在目标 2 中，我将阐明 COPR5 和 PRMT5- 的功能。 MEP50-COPR5 复合物。我的初步数据显示 COPR5 不结合核小体，也不能结合。将 PRMT5-MEP50 招募到核小体，这与之前对 COPR5 功能的推测相矛盾。将确定 COPR5 首选的含组蛋白底物，并量化 COPR5 的结合和贡献最后，我将解析PRMT5-MEP50-COPR5的结构。通过冷冻电镜分析其组蛋白底物，该提案将共同构建 PRMT5- 的分子框架。 MEP50 的底物特异性通过揭示底物特异性相互作用来帮助基于结构的药物设计。