Control and function of heterochromatin in Neurospora crassa

粗糙脉孢菌异染色质的控制和功能

• 批准号: 9763633
• 负责人: Eric U. SELKER
• 金额: $ 51.31万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9763633
• 关键词: Affect; Animal Model; Animals; Chromatin; Chromatin Remodeling Factor; Chromosome Segregation; Chromosomes; DNA; DNA Binding; DNA Methylation; Development; Dissection; Elements; Environment; Epigenetic Process; Eukaryota; G-Quartets; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genome; Genomics; Heterochromatin; Histone H3; Histones; Lead; Lysine; Malignant Neoplasms; Mediating; Modification; Molds; Molecular; Molecular Genetics; Neurospora; Neurospora crassa; Normal Cell; Nuclear; Nucleosomes; Organism; Play; Positioning Attribute; Process; Proteins; Regulation; Repression; Role; Scheme; Signal Transduction; Stream; Structure; System; Testing; Therapeutic Intervention; Tryptophan; Work; X Inactivation; chromatin modification; fungus; gene repression; histone methylation; insight; knockout gene; mutant; plant fungi; protein complex; quadruplex DNA; recruit; scale up; telomere; therapeutic development; tumorigenesis

Our broad aim is to elucidate how eukaryotic genomes are structured and how they work. We will focus on the role of heterochromatin. Normal development of animals, plants and fungi relies on chromatin features including methylation of DNA and histone H3K9 in constitutive heterochromatin, and methylation of histone H3K27 (H3K27me) in facultative heterochromatin. We have shown that the filamentous fungus Neurospora crassa is an extraordinarily favorable genetic/molecular system to elucidate the basic workings of both forms of heterochromatin. The project will involve genetic and molecular dissection of the interconnected roles of chromatin features implicated in heterochromatin including DNA signals, chromatin modifications, histone turnover, nucleosome organization, nuclear organization, and other factors. We have shown that a conserved protein complex (PRC2) is responsible for H3K27me in facultative heterochromatin and that this mark is repressive as in higher organisms. Importantly, H3K27me is not essential for viability of Neurospora, allowing for studies that would be difficult or impossible in higher organisms. A major objective of our study is to understand the mechanism of H3K27me-mediated transcriptional repression. We built a forward genetic scheme to identify genes required for H3K27me-mediated silencing and this has already identified interesting, unanticipated chromatin modifiers. We will both scale up our selection to identify more mutants and will characterize the factors already identified, testing if they act up- or down-stream of H3K27me and determining how they affect gene expression, nucleosome positioning, epigenetic modifications, and other features of chromatin. This will give insight into repression by H3K27me. In addition, we will focus on a tryptophan- inducible H3K27me-marked locus, kyn-1, for a controlled and in depth dissection of H3K27me regulation. We will also take several complementary approaches to elucidate what controls the genomic placement of this epigenetic mark. Our recent studies defined telomere-dependent (TD) and telomere-independent (TI) H3K27me and revealed that telomere repeats are capable of inducing H3K27me. We will investigate the underlying mechanism of TD H3K27me, for example by testing the possible roles of structural features (e.g. G- quadruplex DNA), telomere-associated proteins, and nuclear organization (e.g. placement at nuclear periphery). Use of gene knockouts and our LexA tethering system will allow us to test both necessity and sufficiency of candidate features. We will also experimentally dissect TI domains, which may identify “PRE”-like elements and DNA binding factors involved in recruitment of H3K27me machinery. Finally, we will test how the broader chromatin environment controls H3K27me, following up our leads that suggest constitutive heterochromatin, H3K36me, H3K56ac, nucleosome turnover, and transcription all influence H3K27me distribution. We are optimistic that our findings on the control and function of heterochromatin in Neurospora will elucidate fundamental processes that also operate in higher eukaryotes.

我们的主要目标是阐明真核生物基因组的结构和工作方式。重点抓好 异染色质的作用动物、植物和真菌的正常发育依赖于染色质特征 包括组成型异染色质中DNA和组蛋白H3 K9的甲基化，以及组蛋白H3 K9的甲基化， H3 K27（H3 K27 me）在兼性异染色质中。我们已经证明丝状真菌脉孢菌 crassa是一个非常有利的遗传/分子系统，以阐明这两种形式的基本运作， 异染色质该项目将涉及遗传和分子解剖的相互关联的作用， 与异染色质有关的染色质特征，包括DNA信号、染色质修饰、组蛋白 周转、核小体组织、核组织和其他因素。我们已经证明，一个保守的 蛋白复合物（PRC 2）负责兼性异染色质中的H3 K27 me，并且该标记是 像高等生物一样压抑。重要的是，H3 K27 me对于脉孢菌的生存力不是必需的，这使得 在高等生物体中进行研究是困难或不可能的。我们研究的一个主要目的是 了解H3 K27 me介导的转录抑制机制。我们建立了一个正向遗传学 方案来鉴定H3 K27 me介导的沉默所需的基因，并且这已经鉴定了有趣的， 非预期的染色质修饰剂。我们都将扩大我们的选择，以确定更多的突变体， 表征已经确定的因素，测试它们是否在H3 K27 me的上游或下游起作用，并确定 它们如何影响基因表达、核小体定位、表观遗传修饰和其他特征， 染色质这将使我们深入了解H3 K27 me的抑制作用。此外，我们将重点关注色氨酸- 诱导型H3 K27 me标记基因座kyn-1，用于H3 K27 me调节的受控和深入剖析。我们 我也将采取几种互补的方法来阐明是什么控制着这种基因的基因组位置， 表观遗传标记我们最近的研究定义了端粒依赖性（TD）和端粒非依赖性（TI） H3 K27 me，并揭示端粒重复能够诱导H3 K27 me。我们将调查 TD H3 K27 me的潜在机制，例如通过测试结构特征（例如G- 四链体DNA）、端粒相关蛋白和核组织（例如，在核中的放置 外围）。使用基因敲除和我们的莱克萨拴系系统将使我们能够测试必要性和 候选特征的充分性。我们还将实验性地解剖TI域，其可以识别“PRE”样 元件和DNA结合因子参与募集H3 K27 me机制。最后，我们将测试 更广泛的染色质环境控制H3 K27 me，跟进我们的线索，表明组成性 异染色质、H3 K36 me、H3 K56 ac、核小体转换和转录都影响H3 K27 me 分布我们对我们在脉孢菌异染色质的控制和功能方面的发现感到乐观 将阐明在高等真核生物中也起作用的基本过程。