PRMT5-MEP50 Histone Arginine Methylation in Early Development

早期发育中的 PRMT5-MEP50 组蛋白精氨酸甲基化

基本信息

批准号：
8915230
负责人：
DAVID SHECHTER
金额：
$ 32.15万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8915230
关键词：
Affinity Androgens Animal Model Arginine Binding Biochemical Biochemistry Biological Biological Assay Biological Models Biological Process Cancerous Catalysis Chromatin Code Complex Crystallization Crystallography Defect Deposition Development Developmental Biology Diagnosis Electron Microscopy Embryo Embryonic Development Enzymes Epigenetic Process Family Foundations Funding Future Gene Expression Genes Genetic Transcription Genome Goals Harvest Health Histone Acetylation Histone Fold Histone H2A Histone H4 Histones In Vitro Injection of therapeutic agent Kinetics Knock-out Lead Life Malignant Neoplasms Mammalian Cell Maps Methylation Methyltransferase Modeling Molecular Molecular Chaperones Molecular Probes Molecular Profiling Mono-S Mus Oligonucleotides Pathology Pathway interactions Pattern Formation Peptides Phenotype Phosphatidyl-N-methylethanolamine N-methyltransferase Phosphorylation Positioning Attribute Post-Translational Protein Processing Process Protein-Arginine N-Methyltransferase Proteins Publications Rana Role Staging Stem Cell Development Structure Tail Testing United States National Institutes of Health Vertebrates WD Repeat Xenopus Xenopus laevis base cancer diagnosis cancer therapy clinically significant egg gastrulation gene repression genome-wide histone methylation histone modification human disease improved insight mutant overexpression pluripotency reconstruction tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): The goal of this project is to determine the biochemical mechanisms by which the essential histone-modifying PRMT5-MEP50 complex (protein arginine methyltransferase 5 - methylosome protein 50) guides early development. Arginine methylation of histones is carried out by a family of protein arginine methyltransferases (PRMTs) that have diverse roles. The primary focus of this proposal is PRMT5, the major Type II enzyme that catalyzes arginine mono- and symmetric-dimethylation (Rme1 and Rme2s). PRMT5-catalyzed histone H2A and H4 R3me2s is correlated with gene repression. PRMT5 is always found in complex with the WD-repeat protein MEP50 (also known as Wdr77 or androgen coreceptor p44). We recently solved the crystal structure of the Xenopus laevis PRMT5-MEP50 complex that provides insight into how MEP50 is required for PRMT5 histone methylation and showed that PRMT5-MEP50 is responsible for the abundant histone H2A and H4 R3 methylation in Xenopus eggs. Both PRMT5 and MEP50 are embryonic lethal when their expression is knocked out in mice or in frogs. We hypothesize that MEP50 targets egg and embryo chaperone-bound histones H2A and H4 for methylation by its partner PRMT5 to regulate early development. Our specific aims are: Aim 1) Test the hypothesis that MEP50 recognizes substrate for PRMT5. We will determine affinities for substrate by both MEP50 and PRMT5 using quantitative binding studies, perform co-crystallography with substrate peptides to identify domains of interaction, and assess the contribution of MEP50 substrate binding to PRMT5 catalysis using kinetic methyltransferase assays. Aim 2) Test the hypothesis that PRMT5-MEP50 methylation of the maternal store of pre-deposition histones is regulated by histone chaperones and post-translational modifications. For this aim we will determine how histone chaperones and maternal histone acetylation and phosphorylation modulate PRMT5-MEP50's activity towards H2A and H4. Aim 3) Test the hypothesis that PRMT5-MEP50 methylates histones H2A and H4 to regulate Xenopus early embryonic development. For this aim we will test the functional role of the maternal methylation and new zygotic methylation of histones by PRMT5-MEP50 in Xenopus laevis embryos. We will mimic the loss of maternally methylated histones through the overexpression of H2A and/or H4 R3Q mutants and probe the loss of zygotic PRMT5 and MEP50 by injection of antisense morpholino oligonucleotides. We will follow these treated embryos via phenotypic analysis, probe the coexistence of activation and repressive histone modifications with R3 methylation, and perform gene expression studies to probe the biological function of H2A and H4 R3 methylation. Our studies probe the biochemical mechanism of a major regulator of early embryonic development in a unique living vertebrate model organism. We have documented ability to effectively combine biochemical and biological studies that will allow us to dissect the function of this essential and highly conserve histone modifying complex. Our findings will inform future studies in both normal and cancerous mammalian cells.

描述（由申请人提供）：该项目的目的是确定生化机制，基本的组蛋白修饰PRMT5-MEP50复合物（蛋白质精氨酸甲基转移酶5-甲基化蛋白50）指导早期发育。组蛋白的精氨酸甲基化是由具有多种作用的蛋白质精氨酸甲基转移酶（PRMTS）进行的。该提案的主要重点是PRMT5，PRMT5是催化精氨酸单和对称二甲基化（RME1和RME2S）的主要II型酶。 PRMT5催化的组蛋白H2A和H4 R3ME2S与基因抑制相关。 PRMT5始终与WD重复蛋白MEP50（也称为WDR77或雄激素coecector p44）中发现。我们最近解决了爪蟾Laevis PRMT5-MEP50复合物的晶体结构，该复合物可深入了解PRMT5组蛋白甲基化所需的MEP50，并表明PRMT5-MEP50负责大量的组蛋白H2A和H4 R3甲基化的Xenopus卵中。当PRMT5和MEP50在小鼠或青蛙中敲出表达时，它们都是胚胎致死的。我们假设MEP50靶向卵和胚胎结合的组蛋白H2A和H4的伴侣PRMT5甲基化以调节早期发育。我们的具体目的是：目标1）测试MEP50识别PRMT5底物的假设。我们将使用定量结合研究来确定MEP50和PRMT5对底物的亲和力，与底物肽进行共结晶术以识别相互作用的域，并评估使用动力学甲基转移酶鉴定的MEP50底物与PRMT5催化的贡献。目的2）检验以下假设：孕产妇储存前沉积组蛋白的PRMT5-MEP50甲基化受组蛋白伴侣和翻译后修饰调节。为此，我们将确定组蛋白伴侣和母体组蛋白乙酰化和磷酸化如何调节PRMT5-MEP50对H2A和H4的活性。目标3）检验PRMT5-MEP50甲基化组蛋白H2A和H4的假设以调节早期胚胎发育。为此，我们将测试PRMT5-MEP50在Xenopus laevis胚胎中通过PRMT5-MEP50对组蛋白进行组蛋白的新二二甲基化的功能作用。我们将通过H2A和/或H4 R3Q突变体的过表达模仿母体甲基化组蛋白的丧失，并通过注射反义morpholino寡核苷酸，探测Zygotic PRMT5和MEP50的损失。我们将通过表型分析遵循这些处理过的胚胎，探测激活和抑制性组蛋白与R3甲基化的共存，并进行基因表达研究以探测H2A和H4 R3甲基化的生物学功能。我们的研究探究了独特的活脊椎动物模型生物体中早期胚胎发育的主要调节剂的生化机制。我们已经记录了有效结合生化和生物学研究的能力，这将使我们能够剖析这种基本且高度保护组蛋白修饰复合物的功能。我们的发现将为未来在正常和癌性哺乳动物细胞中的研究提供信息。