Control and Function of Histone H3 Lysine 27 Methylation in Neurospora

描述（由申请人提供）：动物、植物和真菌的正常发育严重依赖染色质信号。提出的研究重点是一个特别中心的例子，组蛋白H3赖氨酸27 （H3K27me3）的三甲基化。对苍蝇、哺乳动物和植物的研究表明，这种标记与发育过程中基因的长期抑制、x失活、基因组印记和癌症有关。然而，H3K27me3如何起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神经孢子虫的基因组中发现了H3K27me3区域，并鉴定了该生物体内潜在甲基化机制的元件，这为应用神经孢子虫分子和遗传学方法的特殊能力来探索这一组蛋白标记的功能和控制提供了机会。H3K27me3在其他更简单的系统（如酵母）中未被发现。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。我们将测试在移除诱导结构体后，在异位位点诱导的H3K27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甲基化机制的组成部分。我们将描述K27甲基转移酶复合物的特征，并研究读取H3K27me3标记的是什么。候选结合物（染色质结构域蛋白和SET-7复合物）将被测试，蛋白质组学和遗传学（突变体搜寻）方法将被用来识别该机制的未知元素。3. 鉴定控制H3K27me3的顺式作用序列。为了解决神经孢子虫中的H3K27me3是由与果蝇的PREs相似的序列指导的可能性，我们将：a.使用ChIP-Seq。绘制神经孢子虫PRC2组分的结合位点；b.检测原生H3K27me3区域的缺失以检测H3K27me的缺失；c.测试候选控制区在异位位点指导H3K27甲基化的能力。4. 明确H3K27甲基化在基因抑制中的作用及机制。我们将探索可能控制K27me3基因的条件，并确定K27甲基化的抑制是否来自转录起始或延伸的阻断。5. 检测SET-7复合体是否读取组蛋白标记。我们将利用我们收集的突变体来探索SET-7复合物在体内对H3 n端修饰敏感的可能性，如果在体外发现该复合物与H3K27me结合，我们将通过测试其与修饰肽的结合来跟进。

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.