Control and Function of Histone H3 Lysine 27 Methylation in Neurospora

脉孢菌中组蛋白 H3 赖氨酸 27 甲基化的控制和功能

基本信息

批准号：
8295616
负责人：
Eric U. SELKER
金额：
$ 26.93万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8295616
关键词：
Address Animal Model Animals Arabidopsis Binding Binding Sites Biochemical Caenorhabditis elegans Catalytic Domain ChIP-on-chip ChIP-seq Characteristics Chromatin Cis-Acting Sequence Collection Complex DNA Methylation Development Drosophila genus Elements Epigenetic Process Eukaryota Fission Yeast Gene Expression Gene Silencing Genes Genetic Genome Genomic Imprinting Heritability Histone H3 Histones Homologous Gene Human In Vitro Kinetics Lead Lysine Maintenance Malignant Neoplasms Mammals Maps Methods Methylation Methyltransferase Modeling Modification Molecular Genetics Neurospora Neurospora crassa Organism PRC1 Protein Peptides Phase Plants Play Polycomb Process Proteins Proteomics Reading Reporting Repression Research Research Personnel Role Saccharomyces cerevisiae Signal Transduction Site Sports System Tertiary Protein Structure Testing Therapeutic Intervention Transcription Elongation Transcription Initiation Work X Inactivation Yeasts base chromatin protein fly follow-up gene repression in vivo insight member mutant plant fungi protein complex research study telomere tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Normal development of animals, plants and fungi rely heavily on chromatin-based signals. The proposed research focuses on a particularly central example, trimethylation of histone H3 Lysine 27 (H3K27me3). Work in flies, mammals and plants have implicated this mark in long-term repression of genes in development, as well as in X-inactivation, genomic imprinting and cancer. Details of how H3K27me3 functions and how it is controlled remain unknown, however. The recent discovery of regions of H3K27me3 in the genome of the simple eukaryote, Neurospora crassa, and identification of elements of the underlying methylation machinery in this organism provide an opportunity to apply the exceptional power of Neurospora molecular and genetic methods to explore the function and control of this histone mark. H3K27me3 has not been found in other simpler systems (e.g. yeasts). Specific aims of the project are: 1. To test for heritability of H3K27me3 and to determine the kinetics of de novo K27 methylation. We will test if methylation of H3K27 induced at an ectopic site is maintained after the inducing construct is removed. We will also use genetic and molecular methods to determine the kinetics of de novo H3K27 methylation. 2. To identify the components of the H3K27 methylation machinery. We will characterize the K27 methyltransferase complex and investigate what reads the H3K27me3 mark. Candidate binders (chromo domain proteins and the SET-7 complex) will be tested and proteomic and genetic (mutant hunt) methods will be used to identify unsuspected elements of the machinery. 3. To identify cis-acting sequences that control H3K27me3. To address the possibility that H3K27me3 in Neurospora is directed by sequences comparable to PREs of Drosophila, we will: a. use ChIP-Seq. to map binding sites of Neurospora PRC2 components; b. test deletions of native H3K27me3 regions for loss of H3K27me; c. test candidate control regions for the ability to direct H3K27 methylation at an ectopic site. 4. To define the role and mechanism of H3K27 methylation in gene repression. We will explore conditions that may control K27me3 genes and determine whether repression by K27 methylation results from a block in transcription initiation or elongation. 5. To test whether the SET-7 complex reads histone marks. We will explore the possibility that the SET-7 complex is sensitive to modifications in the N-terminus of H3 in vivo using our collection of mutants, and if the complex is found to bind H3K27me in vitro, we will follow up by testing it's binding to modified peptides. PUBLIC HEALTH RELEVANCE: Trimethylation of the lysine 27 residue of histone H3 plays a critical role in the epigenetic repression of genes during development, X-inactivation, genomic imprinting, and the aberrant inactivation of genes in cancer. Understanding how this epigenetic mark is regulated will lead to better insight of its role in tumorigenesis and ultimately to the development of therapeutic interventions. Neurospora crassa is the simplest model organism known to have the H3K27me3 mark and provides a system in which to make great strides in our understanding of the H3K27me3 mark.

描述（由申请人提供）：动物，植物和真菌的正常发育严重依赖于基于染色质的信号。拟议的研究重点是一个特别中心的例子，即组蛋白H3赖氨酸27（H3K27ME3）的三甲基化。果蝇，哺乳动物和植物的工作使这一标记在长期抑制发育中的基因以及X灭活，基因组印迹和癌症中。然而，H3K27ME3的功能及其如何控制的详细信息仍然未知。在简单真核生物，神经孢子骨的基因组中发现了H3K27me3区域的最新发现，并鉴定了这种生物体中潜在甲基化机制的元素，这提供了一个机会，可以运用神经孢子的分子和遗传方法的特殊力量，以探索这种历史蛋白的功能和控制功能和控制。 H3K27me3在其他更简单的系统（例如酵母）中尚未发现。该项目的具体目的是：1。测试H3K27me3的遗传力并确定从头K27甲基化的动力学。我们将测试在去除诱导构建体后，在异位部位诱导的H3K27的甲基化是否维持。我们还将使用遗传和分子方法来确定从头H3K27甲基化的动力学。 2。确定H3K27甲基化机制的组件。我们将表征K27甲基转移酶复合物，并研究读取H3K27me3标记的内容。将测试候选粘合剂（Chromo域蛋白和SET-7复合物），并将使用蛋白质组学和遗传（突变狩猎）方法来识别机械的未经引起的元素。 3。识别控制H3K27me3的顺式作用序列。为了解决Neurospora中H3K27me3的可能性，由与果蝇的序列相当的序列指向，我们将：使用chip-seq。绘制神经孢子prc2成分的结合位点； b。 H3K27ME损失的天然H3K27ME3区域的测试缺失； c。测试候选控制区，可在异位部位指导H3K27甲基化的能力。 4。定义H3K27甲基化在基因抑制中的作用和机制。我们将探索可能控制K27me3基因的条件，并确定K27甲基化抑制是否是由于转录起始或伸长率的块而导致的。 5。测试Set-7复合物是否读取组蛋白标记。我们将探讨使用我们的突变体集合在体内H3中的Set-7复合物对H3的N末端的修饰敏感的可能性，并且如果发现该复合物可以在体外结合H3K27ME，我们将通过测试其与改性肽的结合来跟进。公共卫生相关性：赖氨酸27组蛋白H3残基的三甲基化在发育，X灭活，基因组印记和癌症基因的异常失活过程中对基因的表观遗传抑制至关重要。了解这种表观遗传标记的调节将如何更好地了解其在肿瘤发生中的作用，并最终导致治疗干预措施的发展。 Neurospora crassa是已知具有H3K27me3标记的最简单模型生物，并提供了一个系统，可以在我们对H3K27me3标记的理解中取得长足进步。