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于基于染色质的信号。拟议的研究集中在一个特别重要的例子，组蛋白H3赖氨酸27（H3 K27 me 3）的三甲基化。在果蝇、哺乳动物和植物中的研究表明，这种标记与发育过程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，H3 K27 me 3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了H3 K27 me 3区域，并鉴定了该生物体中潜在甲基化机制的元件，这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。H3 K27 me 3在其他更简单的系统（例如酵母）中没有发现。该项目的具体目标是：1。检测H3 K27 me 3的遗传力并确定从头K27甲基化的动力学。我们将测试在异位位点处诱导的H3 K27的甲基化在去除诱导构建体后是否得以维持。我们还将使用遗传和分子方法来确定从头H3 K27甲基化的动力学。2.鉴定H3 K27甲基化机制的组分。我们将描述K27甲基转移酶复合物的特征，并研究是什么读取H3 K27 me 3标记。将检测候选结合物（染色体结构域蛋白和SET-7复合物），并将使用蛋白质组学和遗传学（突变体狩猎）方法来鉴定该机制的非可疑元件。3.确定控制H3 K27 me 3的顺式作用序列。为了解决脉孢菌中H3 K27 me 3由与果蝇的PRE相当的序列指导的可能性，我们将：使用ChIP-Seq。绘制脉孢菌PRC 2组分的结合位点; B.测试天然H3 K27 me 3区域的缺失是否有H3 K27 me的丢失; c.测试候选对照区在异位位点指导H3 K27甲基化的能力。4.明确H3 K27甲基化在基因阻遏中的作用及机制。我们将探索可能控制K27 me 3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.为了测试SET-7复合体是否读取组蛋白标记。我们将使用我们收集的突变体来探索SET-7复合物对H3的N-末端的修饰在体内敏感的可能性，并且如果发现复合物在体外结合H3 K27 me，我们将通过测试其与修饰的肽的结合来跟进。 公共卫生相关性：组蛋白H3的赖氨酸27残基的三甲基化在发育期间基因的表观遗传抑制、X失活、基因组印记和癌症中基因的异常失活中起关键作用。了解这种表观遗传标记是如何调节的，将有助于更好地了解其在肿瘤发生中的作用，并最终发展治疗干预措施。粗糙脉孢菌是已知具有H3 K27 me 3标记的最简单的模式生物，并提供了一个系统，使我们对H3 K27 me 3标记的理解取得了很大进展。

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.