Importance of kinetochore-driven cohesion loading at a heterochromatic pericentromere for accurate chromosome segregation during meiosis

着丝粒周围异染色质着丝点驱动的内聚力对于减数分裂过程中染色体精确分离的重要性

• 批准号: 1941193
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1941193
• 关键词: Importance; kinetochore; driven; cohesion; loading

Meiosis is the specialized cell division that generates gametes, which have half the number of chromosomes of the parental cell. Human meiosis is extremely error-prone: up to 30% of all human eggs have the wrong number of chromosomes, causing miscarriages and birth defects such as Downs syndrome. Moreover, the risk of producing a faulty egg increases with the age of the female. However, the underlying causes remain unknown. Furthermore, our knowledge of the basic biological pathways that direct chromosome segregation during meiosis is extremely limited. This project aims to uncover fundamental mechanisms of chromosome segregation during meiosis. Since meiosis is highly conserved, this project will employ fission yeast, a simple unicellular eukaryote that allows basic molecular mechanisms to be dissected in detail. The knowledge gained will inform future work aimed at identifying the causes of defective egg formation in humans and the influence of ageing. The project will focus on the role of the pericentromere, the chromosomal region surrounding the centromere. This region plays several important and specialized functions in directing chromosome segregation during meiosis. Pericentromeres influence meiotic recombination, regulate the linkages between chromosomes and direct the orientation of chromosome attachment to microtubules. A key and conserved feature of the pericentromere, that underlies all of these functions, is that it is highly enriched in cohesin, the protein complex that links the newly duplicated chromosomes together after DNA replication. In budding yeast, a dedicated pathway directs cohesin loading to the centromere to enrich the pericentromere. However, budding yeast centromeres are unusual since they lack the "silent" heterochromatin, typically associated with centromeres of many other eukaryotes, including humans. In contrast, fission yeast pericentromeres are heterochromatic, moreover, this pericentromeric heterochromatin is known to be required for cohesin enrichment. This leads to the hypothesis that more complex pericentromeres recruit cohesin through two independent pathways: kinetochore-driven and heterochromatin-driven association. The goal of this project is use fission yeast to understand how the interplay between kinetochore-driven and heterochromatin-driven cohesin association contributes to the specialized function of the pericentromere in chromosome segregation during meiosis. Work in budding yeast identified a conserved patch on the Scc4 subunit of the cohesin loader that targets the complex to centromeres. The equivalent conserved region on the fission yeast cohesin loader will be mutated and its importance in cohesin enrichment at the pericentromere will be assessed. Similar approaches will generate mutations in the kinetochore subunits onto which the cohesin loader docks, informed by studies on budding yeast. These mutants will be subjected to functional assays to determine the exact function of kinetochore-driven and heterochromatin-dependent cohesin association in meiotic chromosome segregation. Advanced live cell imaging methods will be used to examine fission yeast cells carrying markers of interest and undergoing meiosis will be used, alongside molecular biological assays, such as chromatin immunoprecipitation. A recent functional genomics screen carried out in the Marston lab (unpublished) surveyed essentially all non-essential genes of fission yeast for roles in meiotic chromosome segregation. Several of the genes newly found to have roles in meiosis are hypothesized to work at the pericentromere. Functional assays will be carried out to determine if this is the case and to identify their interactions with other pericentromere regulators. This will include live cell microscopy, proteomic approaches and bioinformatics analysis. Specific follow up experiments will be designed to functionally characterise the most interesting factors from the screen.

减数分裂是产生配子的特殊细胞分裂，配子的染色体数是亲本细胞的一半。人类减数分裂极易出错：高达30%的人类卵子染色体数目错误，导致流产和唐斯综合征等出生缺陷。此外，随着女性年龄的增长，产生有缺陷卵子的风险也会增加。然而，根本原因仍不清楚。此外，我们对在减数分裂过程中直接进行染色体分离的基本生物学途径的了解极其有限。该项目旨在揭示减数分裂过程中染色体分离的基本机制。由于减数分裂是高度保守的，这个项目将使用分裂酵母，这是一种简单的单细胞真核生物，可以详细剖析基本的分子机制。所获得的知识将为未来旨在确定人类缺陷卵子形成原因和衰老影响的工作提供依据。该项目将侧重于着丝粒周围的染色体区域--着丝粒周围的染色体区域的作用。在减数分裂过程中，这个区域在指导染色体分离方面发挥着几个重要而特殊的功能。着丝粒影响减数分裂重组，调节染色体之间的连接，并指导染色体附着在微管上的方向。作为所有这些功能的基础，包着丝粒的一个关键和保守的特征是它高度富含粘附素，这是一种蛋白质复合体，在DNA复制后将新复制的染色体连接在一起。在萌芽酵母中，一条专门的途径将粘附素装载到着丝粒以丰富着丝粒。然而，发芽酵母着丝粒是不寻常的，因为它们缺乏“沉默的”异染色质，这通常与包括人类在内的许多其他真核生物的着丝粒有关。相比之下，分裂酵母的着丝粒是异染色质，而且，这种着丝粒周围的异染色质被认为是凝集素浓缩所必需的。这导致了一种假设，即更复杂的近着丝粒通过两条独立的途径招募粘附素：动粒驱动和异染色质驱动的关联。这个项目的目标是利用分裂酵母来了解动粒驱动和异染色质驱动的粘连蛋白协会之间的相互作用如何有助于减数分裂过程中染色体分离中的着丝粒周围的特殊功能。对发芽酵母的研究发现，在粘附素加载器的Scc4亚单位上有一个保守的补丁，该补丁针对着丝粒的复合体。裂解酵母粘附素加载器上的等量保守区将发生突变，并将评估其在近着丝点粘附素浓缩中的重要性。根据对发芽酵母的研究，类似的方法将在粘附素加载器停靠的动粒亚基中产生突变。这些突变体将接受功能分析，以确定动粒驱动和异染色质依赖的粘附素关联在减数分裂染色体分离中的确切功能。先进的活细胞成像方法将用于检测携带感兴趣标记并正在进行减数分裂的分裂酵母细胞，并将与染色质免疫沉淀等分子生物学分析一起使用。最近在马斯顿实验室进行的一项功能基因组学筛查(未发表)基本上调查了分裂酵母的所有非必需基因在减数分裂染色体分离中的作用。新发现的几个在减数分裂中起作用的基因被假设在着丝粒周围起作用。将进行功能分析，以确定是否是这种情况，并确定它们与其他着丝粒调控因子的相互作用。这将包括活细胞显微镜、蛋白质组学方法和生物信息学分析。具体的后续实验将被设计成从功能上表征屏幕上最有趣的因素。