Assembly and Maintenance of Centromeres in Filamentous Fungi

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，在核分裂期间将染色体运输到子核中。有缺陷的着丝粒导致染色体分离错误和非整倍体，被认为是癌症的原因之一。一个保守的着丝粒特异性组蛋白变体（CenH 3），重复的DNA和翻译后组蛋白修饰是普遍需要的着丝粒功能，但着丝粒组装和维护的机制尚未解决。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，两个丝状真菌，粗糙脉孢菌和禾谷镰刀菌，被用来作为强大的系统来测试DNA序列和异染色质的重要性，着丝粒功能。这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。人类着丝粒的大多数特征都在这些物种中发现，使它们成为优秀的参考生物。所有计划中的遗传学研究都是针对这些真菌的，但很难在哺乳动物中进行。这个项目借鉴了我们对脉孢菌的研究结果，这些结果表明目前的着丝粒维持模型是不够的。长期目标是确定着丝粒如何组装以及它们如何在丝状真菌中维持，丝状真菌是一组重要的-但在这方面仍然缺乏特征-人类，动物和植物病原体。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，并且在减数分裂期间CenH 3的掺入由CenH 3 mRNA介导的新机制控制。具体目标将通过以下方式测试这些假设：（1）表征着丝粒组分的关键特征（2）确定异染色质为什么对脉孢菌着丝粒的维持至关重要，以及（3）破译CenH 3调节的机制。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，并将进行绕过异染色质要求的突变体的新型抑制剂筛选。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）将补充遗传学和细胞学方法。已经积累了大量的支持性初步数据，解决潜在机制的大多数材料和方法都在手边，目前没有其他实验室正在研究丝状真菌的这个基本问题。拟议的实验不仅将提供急需的关键知识，最终用于指导新的抗真菌药物的开发，但也将导致更好地了解调控着丝粒组装和维护的表观遗传决定因素。 公共卫生相关性：在细胞分裂过程中，可能发生错误的染色体分离，这被认为是癌症和几种遗传性疾病的根本原因之一。人们对着丝粒是如何组装的还不是很清楚，但我们对染色体这些基本组成部分的了解大多来自于对简单模型系统的研究，如丝状真菌。我们的长期目标是阐明丝状真菌中着丝粒组装和遗传的机制，丝状真菌是目前特征不明显的人类病原体。该项目的一个转化目标是指导干扰染色体分离的药物的设计，这些药物可用于癌症研究和侵袭性真菌感染的治疗。

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. PUBLIC HEALTH RELEVANCE: During cell division, faulty chromosome segregation can occur, which has been implicated as one root cause of cancer and several inherited diseases. It is not well understood how centromeres assemble, but much of what we have learned about these essential components of chromosomes stems from studies with simple model systems, such as filamentous fungi. Our long-term goal is to shed light on mechanisms of centromere assembly and inheritance in filamentous fungi, a currently ill-characterized group of human pathogens. One translational goal of this project is to guide the design of drugs that interfere with chromosome segregation, which can be used for both cancer research and for the treatment of invasive fungal infections.