Assembly and Maintenance of Centromeres in Filamentous Fungi

丝状真菌着丝粒的组装和维护

• 批准号: 8328705
• 负责人: Michael Freitag
• 金额: $ 26.82万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8328705
• 关键词: 3' Untranslated Regions; Address; Affinity Chromatography; Alleles; Aneuploidy; Animals; Antifungal Agents; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biological Models; Boundary Elements; Bypass; Cancer Etiology; Cell Cycle; Cell Nucleus; Cell division; Centromere; ChIP-seq; Chromatin; Chromosome Segregation; Chromosomes; Complement; Complex; Cytosine; DNA; DNA Sequence; Data; Daughter; Defect; Deposition; Development; Disease; Drug Design; Epigenetic Process; Euchromatin; Fertility; Foundations; Fruit; Fusarium; Gene Expression Regulation; Genetic; Goals; Gray unit of radiation dose; Growth; Hand; Heterochromatin; Histone H3; Histones; Human; Human Characteristics; Inherited; Kinetochores; Knowledge; Lead; Learning; Light; Lysine; Maintenance; Mammals; Maps; Mediating; Meiosis; Messenger RNA; Methods; Methylation; Microtubules; Modeling; Modification; Molds; Molecular Conformation; Mutagenesis; Mycoses; Neurospora; Neurospora crassa; Nucleosomes; Organism; Plant Roots; Plants; Proteins; RNA; Regulation; Relative (related person); Reproduction spores; Research; Sequence Analysis; Structure; System; Tandem Repeat Sequences; Testing; Time; Tissues; Translating; Variant; Work; anticancer research; chromatin immunoprecipitation; fungus; heterochromatin-specific nonhistone chromosomal protein HP-1; histone methyltransferase; histone modification; in vivo; mutant; novel; nuclear division; pathogen; receptor; research study; stem; tool

DESCRIPTION (provided by applicant): Centromeres form the foundation of kinetochores, the attachment points for spindle microtubules that transport chromosomes into daughter nuclei during nuclear division. Defective centromeres result in faulty chromosome segregation and aneuploidy, implicated as one cause of cancer. A conserved centromere-specific histone variant (CenH3), repeated DNA and posttranslational histone modifications are universally required for centromere function, but mechanisms for centromere assembly and maintenance remain unresolved. The relative impact of DNA composition vs. epigenetic modifications is difficult to separate in most species. Here, two filamentous fungi, Neurospora crassa and Fusarium graminearum, are used as powerful systems to test the importance of DNA sequence and heterochromatin for centromere function. Both fungi lack tandem repeats, making the centromeric DNA amenable to high-throughput sequencing analyses. Most characteristics of human centromeres are found in these species, making them excellent reference organisms. All planned genetic studies are straightforward with these fungi but difficult to carry out in mammals. This project draws on exciting results from our work with Neurospora that suggest that current models for centromere maintenance are inadequate. Long-term goals are to determine how centromeres assemble and how they are maintained in filamentous fungi, an important - but in this respect still poorly characterized - group of human, animal and plant pathogens. The two major hypotheses are that maintenance of Neurospora centromeres relies on interactions of centromere-specific nucleosomes with heterochromatic histone modifications, and that incorporation of CenH3 during meiosis is controlled by a novel mechanism mediated via CenH3 mRNA. Specific aims will test these hypotheses by: (1) characterizing critical features of centromere components (2) determining why heterochromatin is essential for maintenance of Neurospora centromeres, and (3) deciphering mechanisms of CenH3 regulation. To accomplish these aims, centromeric DNA will be tested for the propensity to nucleate centromeric chromatin in vivo and a novel suppressor screen for mutants that bypass the requirement for heterochromatin will be carried out. Biochemical methods (chromatin immunoprecipitation, chromosome conformation capture, affinity purification of centromere proteins) will complement genetic and cytological approaches. Large amounts of supporting preliminary data have been accumulated, most materials and methods to address underlying mechanisms are at hand, and currently no other lab is working on this fundamental problem with filamentous fungi. The proposed experiments will not only provide much needed key knowledge eventually to be used to guide development of new antifungal drugs, but will also lead to a better understanding of epigenetic determinants for the regulation of centromere assembly and maintenance.

描述（由申请人提供）：着丝粒形成着丝粒的基础，着丝粒是纺锤体微管的附着点，在核分裂过程中将染色体运输到子核中。有缺陷的着丝粒会导致染色体分离错误和非整倍性，这可能是癌症的原因之一。保守的着丝粒特异性组蛋白变体 (CenH3)、重复的 DNA 和翻译后组蛋白修饰是着丝粒功能普遍需要的，但着丝粒组装和维持的机制仍未解决。在大多数物种中，DNA 组成与表观遗传修饰的相对影响很难分开。在这里，两种丝状真菌（粗糙脉孢菌和禾谷镰刀菌）被用作强大的系统来测试 DNA 序列和异染色质对着丝粒功能的重要性。两种真菌都缺乏串联重复序列，使得着丝粒 DNA 适合高通量测序分析。人类着丝粒的大部分特征都在这些物种中发现，使它们成为极好的参考生物。所有计划中的遗传学研究对于这些真菌来说都很简单，但在哺乳动物中却很难进行。该项目借鉴了我们与脉孢菌合作的令人兴奋的结果，这些结果表明当前着丝粒维护的模型是不够的。长期目标是确定丝状真菌中着丝粒的组装方式以及它们的维持方式，丝状真菌是人类、动物和植物病原体的重要组成部分，但在这方面仍知之甚少。两个主要假设是，脉孢菌着丝粒的维持依赖于着丝粒特异性核小体与异染色质组蛋白修饰的相互作用，以及减数分裂期间 CenH3 的掺入是由通过 CenH3 mRNA 介导的新机制控制的。具体目标将通过以下方式检验这些假设：（1）表征着丝粒成分的关键特征（2）确定为什么异染色质对于维持脉孢菌着丝粒至关重要，以及（3）破译CenH3调节机制。为了实现这些目标，将测试着丝粒DNA在体内使着丝粒染色质成核的倾向，并对绕过异染色质要求的突变体进行新型抑制筛选。生化方法（染色质免疫沉淀、染色体构象捕获、着丝粒蛋白的亲和纯化）将补充遗传和细胞学方法。已经积累了大量支持性的初步数据，解决潜在机制的大多数材料和方法都已掌握，目前没有其他实验室正在研究丝状真菌的这一基本问题。拟议的实验不仅将提供最终用于指导新抗真菌药物开发的急需的关键知识，而且还将有助于更好地理解着丝粒组装和维护调节的表观遗传决定因素。