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

• 批准号: 8454417
• 负责人: Eric U. SELKER
• 金额: $ 25.96万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 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/8454417
• 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

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.

描述（由申请人提供）：动物、植物和真菌的正常发育在很大程度上依赖于基于染色质的信号。拟议的研究重点关注一个特别重要的例子，即组蛋白 H3 赖氨酸 27 (H3K27me3) 的三甲基化。对果蝇、哺乳动物和植物的研究表明，该标记与发育中基因的长期抑制以及 X 失活、基因组印记和癌症有关。然而，H3K27me3 如何发挥作用以及如何控制的细节仍然未知。最近在简单真核生物粗糙脉孢菌基因组中发现的 H3K27me3 区域，以及对该生物体中潜在甲基化机制元件的鉴定，为应用脉孢菌分子和遗传方法的特殊能力来探索该组蛋白标记的功能和控制提供了机会。在其他更简单的系统（例如酵母）中尚未发现 H3K27me3。该项目的具体目标是： 1. 测试H3K27me3的遗传力并确定K27从头甲基化的动力学。我们将测试在异位位点诱导的 H3K27 甲基化在诱导构建体被去除后是否得以维持。我们还将使用遗传和分子方法来确定 H3K27 从头甲基化的动力学。 2. 鉴定H3K27甲基化机制的组成部分。我们将表征 K27 甲基转移酶复合物并研究 H3K27me3 标记的读取内容。将测试候选结合物（色域蛋白和 SET-7 复合物），并使用蛋白质组学和遗传学（突变体狩猎）方法来识别机器中未被怀疑的元件。 3. 鉴定控制H3K27me3的顺式作用序列。为了解决脉孢菌中 H3K27me3 受与果蝇 PRE 类似的序列指导的可能性，我们将：使用 ChIP 测序。绘制脉孢菌 PRC2 成分的结合位点图； b.测试天然 H3K27me3 区域的删除是否丢失 H3K27me； c.测试候选控制区域在异位位点引导 H3K27 甲基化的能力。 4. 明确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 甲基化的抑制是否是转录起始或延伸受阻的结果。 5.测试SET-7复合物是否读取组蛋白标记。我们将利用我们收集的突变体探索体内 SET-7 复合物对 H3 N 末端修饰敏感的可能性，如果发现该复合物在体外结合 H3K27me，我们将通过测试它与修饰肽的结合来进行后续研究。