Control and function of chromatin-mediated gene silencing in fungi

正常发育依赖于基因表达的调控，在许多生物体中，基因表达是通过改变染色质结构以沉默或激活基因的蛋白质复合物来实现的。染色质由DNA与组蛋白和其他蛋白质包装在一起组成。Polycomb抑制复合物（PRCs）如何沉默对发育重要的基因在任何生物中都没有完全理解。该项目旨在揭示特定基因是如何被PRCs靶向和灭活的，使用真菌作为方便的模型系统。社会影响将通过对基因调控、天然产物、生物燃料和发现推定的真菌致病因子的研究相结合来实现。这项工作的更广泛影响还将通过密集的长期研究项目为本科生的实验室教育提供帮助。其他推广工作包括为高中教师举办为期两天的基因组学研讨会，并向公众发表演讲。该项目探讨了丝状真菌禾谷镰刀菌中PRC 2和H3 K27甲基化导致的基于染色质的基因沉默机制。使用正向遗传学筛选，发现了两种影响H3 K27甲基化的新染色质蛋白（DIS 2和DIS 10）。这项工作的主要目标是揭示PRC 2靶向机制和PRC 2复合物的组成。当前项目的主要假设是：（1）镰刀菌DIS 2和DIS 10使用其推定的染色质结合基序与组蛋白或其他染色质蛋白上的特定区域相互作用，以将核心PRC 2靶向于将富集H3 K27三甲基化的染色质片段;（2）靶向基于便携式核酸信号。核心PRC 2亚基受DIS 2和DIS 10调节，并且自身调节转录因子和信号转导的主要调节因子的大网络。该项目的完成将有助于深入了解全球H3 K27甲基化网络。实现这一目标的方法有：（1）通过非定向选择和筛选、基因缺失和定点突变的正向和反向遗传学;（2）通过染色质免疫沉淀、全基因组表达分析和质谱的生物化学;（3）生物物理学研究以探测DIS 2或DIS 10与组蛋白或其它染色质蛋白之间的相互作用，和（4）通过检查与DNA相关的“沉默体”（含有PRC 2）的细胞学。结合这些技术，将允许PRC 2信号和参与基因沉默的新蛋白质的鉴定和表征。通过研究PRC 2靶向的机制，这项研究将提供关于真核生物中染色质介导的沉默的关键知识。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Normal development relies on regulated gene expression, which in many organisms is achieved by protein complexes that alter the structure of chromatin to silence or activate genes. Chromatin consists of DNA packaged together with histone and other proteins. How Polycomb Repressive Complexes (PRCs) silence genes that are important for development is not completely understood in any organism. This project seeks to uncover how specific genes are targeted and inactivated by PRCs, using fungi as convenient model systems. Societal impacts will be via a combination of research on gene regulation, natural products, biofuels and the discovery of putative fungal pathogenicity factors. The broader impact of the work will also provide hands on laboratory education of undergraduates via intensive long-term research projects. Other outreach efforts include two-day genomics workshops for high school teachers and presentations to the general public.This project addresses mechanisms of chromatin-based gene silencing by PRC2 and H3K27 methylation in the filamentous fungus, Fusarium graminearum. Using a forward genetics screen, two novel chromatin proteins that impact H3K27 methylation (DIS2 and DIS10) were discovered. The main objectives of this work are to uncover the mechanism for PRC2 targeting and the composition of PRC2 complexes. The main hypotheses for the current project are: (1) Fusarium DIS2 and DIS10 use their putative chromatin-binding motifs to interact with specific regions on histones or other chromatin proteins to target the core PRC2 to chromatin segments that will be enriched for H3K27 trimethylation; (2) targeting is based on portable, nucleic acid-based signals. Core PRC2 subunits are regulated by DIS2 and DIS10 and themselves regulate a large network of transcription factors and major regulators of signal transduction. Completion of this project will result in gaining insights into the global H3K27 methylation network. Methods used to achieve the aims are: (1) forward and reverse genetics by undirected selections and screens, gene deletions, and site-directed mutagenesis; (2) biochemistry by chromatin immunoprecipitation, genome-wide expression analyses and mass spectrometry; (3) biophysical studies to probe interactions between DIS2 or DIS10 and histone or other chromatin proteins, and (4) cytology by examination of "silencing bodies" (containing PRC2) in association with DNA. In combination, these techniques will allow identification and characterization of PRC2 signals and novel proteins involved in gene silencing. By studying mechanisms of PRC2 targeting, this research will provide key knowledge on chromatin-mediated silencing in eukaryotes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.