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和它们是如何工作的。我们将重点关注 异染色质的作用。动植物和真菌的正常发育依赖于染色质特征 包括构成异染色质中DNA和组蛋白H3K9的甲基化，以及组蛋白的甲基化 H3K27(H3K27me)为兼性异染色质。我们已经证明了丝状真菌Neuropora CRASSA是一个非常有利的遗传/分子系统来阐明这两种形式的基本工作原理 异染色质。该项目将涉及基因和分子解剖相互关联的作用 异染色质中涉及的染色质特征，包括DNA信号、染色质修饰、组蛋白 周转、核小体组织、核组织等因素。我们已经证明了一个保守的 蛋白质复合体(PRC2)负责兼性异染色质中的H3K27me，该标记是 压抑性的，如在高等生物体中。重要的是，H3K27me对脉孢子菌的生存并不是必需的，允许 对于在高等生物体中很难或不可能进行的研究。我们研究的一个主要目标是 了解H3K27me介导的转录抑制的机制。我们建立了一个向前的基因 确定H3K27Me介导的沉默所需基因的方案，这已经确定了有趣的， 出乎意料的染色质修饰剂。我们都将扩大我们的选择范围，以确定更多的突变体，并将 描述已经确定的因素，测试它们是在H3K27Me的上游还是下游起作用，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。这将让我们深入了解H3K27Me的压制。此外，我们将重点研究色氨酸- 可诱导的H3K27me标记的基因座KYN-1，用于控制和深入剖析H3K27me的调控。我们 也将采取几种互补的方法来阐明是什么控制了这一基因的放置 表观遗传标记。我们最近的研究定义了端粒依赖(TD)和端粒非依赖(TI) H3K27me，并揭示端粒重复序列能够诱导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DNA)、端粒相关蛋白和核组织(例如，核的位置 外围)。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 候选要素的充分性。我们还将试验性地剖析TI域，它可能识别类似于Pre的 参与H3K27me机械招募的元件和DNA结合因子。最后，我们将测试 更广泛的染色质环境控制H3K27Me，遵循我们的线索，表明构成 异染色质、H3K36me、H3K56ac、核小体转换和转录都会影响H3K27me 分发。我们乐观地认为，我们在脉孢子瘤中异染色质的控制和功能方面的发现 将阐明也在高等真核生物中运行的基本过程。

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.