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

减数分裂是产生配子的特殊细胞分裂，配子具有亲本细胞染色体数量的一半。人类的减数分裂非常容易出错：高达30%的人类卵子染色体数目错误，导致流产和出生缺陷，如唐斯综合征。此外，产生错误卵子的风险随着女性年龄的增加而增加。然而，根本原因仍然不明。此外，我们对减数分裂期间指导染色体分离的基本生物学途径的知识极其有限。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，这个项目将采用裂变酵母，一个简单的单细胞真核生物，允许基本的分子机制进行详细解剖。所获得的知识将为未来的工作提供信息，旨在确定人类卵子形成缺陷的原因和衰老的影响。 该项目将集中在着丝粒周围的染色体区域的作用。该区域在减数分裂期间指导染色体分离中起着几种重要和专门的功能。近着丝粒影响减数分裂的重组，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，近着丝粒的一个关键和保守的特征是它高度富集在粘附素中，粘附素是在DNA复制后将新复制的染色体连接在一起的蛋白质复合物。在芽殖酵母中，一个专门的途径引导粘附素加载到着丝粒以富集着丝粒周围。然而，芽殖酵母着丝粒是不寻常的，因为它们缺乏“沉默”的异染色质，通常与许多其他真核生物（包括人类）的着丝粒有关。相反，裂殖酵母的近着丝粒是异染色质的，而且，这种近着丝粒异染色质已知是凝聚素富集所必需的。这导致了一种假设，即更复杂的近着丝粒通过两个独立的途径招募粘附素：运动舞蹈驱动和异染色质驱动的协会。本项目的目的是利用裂殖酵母来了解着丝粒周围区在减数分裂过程中染色体分离中的特殊功能是如何通过着丝粒运动和异染色质驱动的粘着蛋白之间的相互作用来实现的。芽殖酵母中的工作确定了一个保守的补丁上的Scc4亚基的粘着蛋白加载器的目标复合物的着丝粒。将突变裂殖酵母粘附素装载器上的等效保守区域，并评估其在近着丝粒处粘附素富集中的重要性。类似的方法将在动粒亚基中产生突变，粘着蛋白装载器停靠在动粒亚基上，这是通过对芽殖酵母的研究得知的。将对这些突变体进行功能测定，以确定减数分裂染色体分离中激动素驱动的和异染色质依赖的粘着蛋白缔合的确切功能。先进的活细胞成像方法将用于检查携带感兴趣的标记物并进行减数分裂的裂殖酵母细胞，以及分子生物学测定，如染色质免疫沉淀。最近在马斯顿实验室进行的一项功能基因组学筛选（未发表）调查了裂变酵母中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近着丝粒处起作用。将进行功能测定，以确定是否是这种情况，并确定它们与其他着丝粒周围调节剂的相互作用。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为从屏幕上功能性地排除最有趣的因素。

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.