Assembly and Maintenance of Centromeres in Filamentous Fungi

描述(申请人提供)：着丝粒构成着丝点的基础，动点是纺锤体微管的连接点，在核分裂期间将染色体输送到子核中。着丝粒缺陷会导致错误的染色体分离和非整倍体，这可能是癌症的原因之一。着丝粒的功能普遍需要一种保守的着丝粒特异性组蛋白变异体(CenH3)、重复的DNA和翻译后的组蛋白修饰，但着丝粒的组装和维持机制仍未解决。在大多数物种中，DNA组成和表观遗传修饰的相对影响很难分开。在这里，两种丝状真菌，粗糙脉孢霉和禾谷镰刀菌，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。这两种真菌都缺乏串联重复序列，这使得着丝粒DNA适合高通量测序分析。人类着丝粒的大部分特征都存在于这些物种中，这使它们成为极好的参考生物。对于这些真菌，所有计划中的遗传研究都是直截了当的，但很难在哺乳动物身上进行。这个项目从我们对脉孢子虫的研究中得出了令人兴奋的结果，表明目前的着丝粒维持模型是不充分的。长期目标是确定着丝状真菌中着丝粒是如何组装和维持的，丝状真菌是一组重要的但在这方面仍然缺乏特征的人类、动物和植物病原体。两个主要的假设是，着丝粒的维持依赖于着丝粒特异的核小体与异染色组蛋白修饰的相互作用，以及CenH3在减数分裂中的掺入是由一种新的机制控制的，该机制由CenH3mRNA介导。特定的目标将通过：(1)表征着丝粒成分的关键特征(2)确定为什么异染色质对维持脉孢子虫着丝粒是必不可少的，以及(3)破译CenH3调节的机制来检验这些假说。为了实现这些目标，着丝粒DNA将在体内测试着丝粒染色质成核的倾向，并将对绕过异染色质要求的突变体进行一种新的抑制筛选。生物化学方法(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。已经积累了大量的支持初步数据，大多数材料和方法都在手头，以解决潜在的机制，目前还没有其他实验室正在研究这个丝状真菌的基本问题。拟议的实验不仅将提供急需的关键知识，最终将用于指导新的抗真菌药物的开发，而且还将导致更好地理解调控着丝粒组装和维持的表观遗传决定因素。

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.