Control and Function of Chromatin-Mediated Gene Silencing in Fusarium

正常的发育依赖于受调控的基因表达，在许多生物体中，这是通过蛋白质复合体的平衡活动来实现的，这些复合体在正确的时间和正确的地点打开或关闭基因。一个尚未解决的主要问题是沉默蛋白如何定位和关闭它们的目标基因。这项研究的重点是发现沉默蛋白复合体是如何靶向基因并使其失活的，这种复合体被称为“多梳群”(PcG)复合体。作为一个研究系统，该项目将使用真菌禾谷镰刀菌，它作为有益的天然产品和生物燃料的来源具有重要的经济意义，作为植物病原体具有重要的农学意义。因此，研究结果可以转化为更好的方法来操纵真菌的有益特征，同时将其对作物的负面影响降至最低。该项目将通过为缺乏服务的学生提供研究培训机会，为高中科学教师提供实验室和专业发展研讨会，以及向公众提供科学演示来产生教育影响。该项目研究在一种新的模式生物--丝状真菌--禾谷镰刀菌中，通过PcG蛋白实现基于染色质的基因沉默。这项工作将利用镰刀菌使用一个多梳抑制复合体2(PRC2)将组蛋白H3赖氨酸27三甲基化(H3K27me3)传递到特定基因的事实。初步数据表明，基因识别是由PRC2亚基或相互作用的蛋白质介导的，随后修饰的基因以转录沉默的“PRC2小体”的形式隔离，与常染色质有明确的边界。该项目的主要目标是揭示PRC2靶向的机制，并确定PRC2复合体的组成。这些目标将通过多方面的方法实现，包括：通过非定向选择和筛选、基因删除和定点突变进行的正向和反向遗传学；通过染色质免疫沉淀、全基因组表达分析和质谱仪进行的生物化学；以及通过检查与DNA有关的PRC2小体的细胞学。这些技术结合在一起，将能够识别和表征参与基因沉默的PRC2信号和蛋白质。我们将对镰刀菌中PrC2介导的基因沉默的新成分进行鉴定，并绘制H3K27me3和PrC2在正常菌株和突变菌株中的分布图谱。这项研究将为真核生物中PrC2介导的沉默提供关键知识。

Normal development relies on regulated gene expression, which in many organisms is achieved by balanced activity of protein complexes that turn genes on or off at the right time and in the right place. A major unsolved question is how silencing proteins locate and turn off their target genes. The focus of this research is to discover how genes are targeted and inactivated by the silencing protein complex known as the "polycomb group" (PcG) complex. As a study system, the project will use the fungus, Fusarium graminearum, which has economic importance as a source of beneficial natural products and biofuels and agronomic importance as a plant pathogen. Thus, the results of the research could translate into better methods for manipulating the beneficial traits of the fungus, while minimizing its negative impact on crop plants. The project will have educational impacts by providing research training opportunities for underserved students, laboratory and professional development workshops for high school science teachers, and science presentations to the general public.This project addresses chromatin-based gene silencing by PcG proteins in a novel model organism, the filamentous fungus Fusarium graminearum. The work will take advantage of the fact that Fusarium uses one Polycomb Repressive Complex2 (PRC2) to deliver histone H3 lysine 27 trimethylation (H3K27me3) to specific genes. Preliminary data suggest that the gene recognition is mediated by PRC2 subunits or by interacting proteins and that the subsequently modified genes are sequestered as transcriptionally silent "PRC2 bodies" with clear boundaries to euchromatin. The main objectives of this project are to uncover the mechanism for PRC2 targeting and to define the composition of PRC2 complexes. The aims will be achieved by multi-faceted approaches, including: forward and reverse genetics by undirected selections and screens, gene deletions, and site-directed mutagenesis; biochemistry by chromatin immunoprecipitation, genome-wide expression analyses and mass spectrometry; and cytology by examination of PRC2 bodies in association with DNA. In combination, these techniques will allow identification and characterization of PRC2 signals and proteins involved in gene silencing. Novel components of PRC2-mediated gene silencing in Fusarium will be characterized, and distribution of H3K27me3 and PRC2 in normal and mutant strains will be mapped. The research will provide key knowledge on PRC2-mediated silencing in eukaryotes.