Assembly and Maintenance of Centromeres in Filamentous Fungi

丝状真菌着丝粒的组装和维护

• 批准号: 8496084
• 负责人: Michael Freitag
• 金额: $ 25.98万
• 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/8496084
• 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）：中心粒构成了动物学的基础，即在核分裂期间将染色体传输到子核的纺锤微管的附着点。中心粒有缺陷会导致染色体分离和非整倍性，被视为癌症的一种原因。普遍需要一种保守的中心粒特异性组蛋白变体（CENH3），重复的DNA和翻译后组蛋白修饰，用于丝粒功能，但丝粒组装和维护的机制仍未解决。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离。在这里，两种丝状真菌，神经孢子骨和镰刀菌被用作强大的系统，用于测试DNA序列和异染色质对丝粒功能的重要性。两种真菌都缺乏串联重复，使室丝粒DNA与高通量测序分析相吻合。在这些物种中发现了人类中心粒的大多数特征，使其成为出色的参考生物。所有计划的遗传研究都对这些真菌很简单，但在哺乳动物中很难进行。该项目取决于我们与Neurospora的工作令人兴奋的结果，这表明当前的Centromere维护模型不足。长期目标是确定centromeres是如何组装的，以及它们如何在丝状真菌中维持，这是一个重要的人类，动物和植物病原体的重要 - 但在这方面仍然很差。这两个主要假设是，维持神经孢子的丝粒依赖着丝粒特异性核小体与异晶组蛋白修饰的相互作用，并且在减数分裂过程中，CENH3掺入由通过CENH3 mRNA介导的新机械学控制。具体目的将通过以下方式测试这些假设：（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神经孢子的丝粒以及（3）CENH3调节的解密机制至关重要。为了实现这些目标，将测试中心透明体DNA的体内成核染色质的倾向，并为突变体的新型抑制剂筛选绕过绕过异染色质需求的突变体。生物化学方法（染色质免疫沉淀，染色体构象捕获，丝粒蛋白的亲和力纯化）将补充遗传和细胞学方法。已经积累了大量支持的初步数据，大多数解决潜在机制的材料和方法都在手头，目前没有其他实验室正在处理丝状真菌的基本问题。提出的实验不仅最终将提供急需的关键知识，以指导新的抗真菌药物的开发，而且还将使人们更好地了解表观遗传决定因素，以调节丝粒组装和维护。