Control and Function of Histone H3 Lysine 27 Methylation in Neurospora

描述(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核心的例子上，组蛋白H3赖氨酸27(H3K27me3)的三甲基化。在果蝇、哺乳动物和植物上的研究表明，这一标记与发育过程中基因的长期抑制有关，也与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它的细节仍不清楚。最近在简单的真核生物粗糙脉孢子虫基因组中发现了H3K27me3区域，并对这种生物中潜在的甲基化机制的元件进行了鉴定，这为应用脉孢子虫分子和遗传学方法来探索这个组蛋白标记的功能和控制提供了机会。H3K27me3在其他较简单的系统(如酵母菌)中未发现。该项目的具体目标是：1.检测H3K27me3的遗传性，并确定从头K27甲基化的动力学。我们将测试在异位位点诱导的H3K27在诱导构建体移除后是否保持甲基化。我们还将使用遗传和分子方法来确定从头开始H3K27甲基化的动力学。2.鉴定H3K27甲基化机制的组成。我们将对K27甲基转移酶复合体进行鉴定，并研究H3K27me3标记的读数。将对候选结合蛋白(染色域蛋白和SET-7复合体)进行测试，并将使用蛋白质组学和遗传学(突变搜索)方法来识别机械的未被怀疑的元件。3.鉴定控制H3K27me3的顺式作用序列。为了解决脉孢子虫中H3K27me3由与果蝇PreS相似的序列控制的可能性，我们将：A.使用CHIP-Seq。绘制脉孢子菌PRC2组分的结合位点图；B.测试天然H3K27me3区域的缺失以寻找H3K27me的缺失；C.测试候选控制区在异位位点引导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甲基化是否导致转录起始或延伸受阻。5.测试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在体内探索SET-7复合体对H3 N末端修饰敏感的可能性，如果在体外发现该复合体与H3K27me结合，我们将通过测试其与修饰多肽的结合来进行后续研究。 公共卫生相关性：组蛋白H3的27个赖氨酸残基的三甲基化在发育过程中基因的表观遗传抑制、X失活、基因组印记以及癌症中基因的异常失活中起着关键作用。了解这种表观遗传标记是如何调控的，将有助于更好地了解它在肿瘤发生中的作用，并最终开发治疗干预措施。粗糙脉孢菌是已知的具有H3K27me3标志的最简单的模式生物，并提供了一个系统，使我们在理解H3K27me3标志方面取得重大进展。

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.