Control and function of heterochromatin in Neurospora crassa

我们的广泛目标是阐明真核基因组的结构及其工作原理。我们将重点关注 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 H3K9 的甲基化，以及组蛋白的甲基化 兼性异染色质中的 H3K27 (H3K27me)。我们已经证明丝状真菌脉孢菌 crassa 是一个非常有利于阐明两种形式的基本运作方式的遗传/分子系统 异染色质。该项目将涉及对相互关联的作用进行遗传和分子剖析 异染色质涉及的染色质特征，包括 DNA 信号、染色质修饰、组蛋白 营业额、核小体组织、核组织和其他因素。我们已经证明，一个保守的 蛋白质复合物 (PRC2) 负责兼性异染色质中的 H3K27me，并且该标记是 与高等生物一样具有压抑性。重要的是，H3K27me 对于脉孢菌的生存能力不是必需的，因此可以 用于在高等生物中困难或不可能的研究。我们研究的一个主要目标是 了解 H3K27me 介导的转录抑制机制。我们建立了正向遗传 计划识别 H3K27me 介导的沉默所需的基因，这已经确定了有趣的， 意外的染色质修饰剂。我们将扩大我们的选择范围，以识别更多的突变体，并将 描述已确定的因素，测试它们是否作用于 H3K27me 的上游或下游并确定 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。这将有助于深入了解 H3K27me 的镇压。此外，我们将重点关注色氨酸- 诱导型 H3K27me 标记位点 kyn-1，用于对 H3K27me 调节进行受控和深入剖析。我们 还将采取几种互补的方法来阐明是什么控制着这个基因组的位置 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） H3K27me 并揭示端粒重复能够诱导 H3K27me。我们将调查 TD H3K27me 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（例如置于核内） 周边）。使用基因敲除和我们的 LexA 系留系统将使我们能够测试必要性和 候选特征的充分性。我们还将通过实验剖析 TI 域，这可能会识别类似“PRE”的 参与 H3K27me 机制招募的元件和 DNA 结合因子。最后，我们将测试如何 更广泛的染色质环境控制 H3K27me，跟踪我们提出的组成性线索 异染色质、H3K36me、H3K56ac、核小体周转和转录均影响 H3K27me 分布。我们对脉孢菌异染色质的控制和功能的发现感到乐观 将阐明在高等真核生物中也起作用的基本过程。

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.