Preventing Aneuploidy in Aging Oocytes: Investigating the effects and mechanisms of cohesion enrichment in Drosophila melanogaster.

摘要 减数分裂I的总体目标是成功地将同源染色体分离成两个二倍体细胞。 不这样做会导致非整倍体，这是遗传疾病的主要原因，如21三体， 自然流产导致的流产。染色体不分离（NDJ）的发生率增加 随着卵母细胞年龄的增长呈指数增长，这种现象被称为母亲年龄效应。染色体相关 NDJ被假设是由卵母细胞不能维持交叉引起的， 同源染色体之间的交叉（CO）表现，直到排卵。姐妹染色单体 凝聚力（SCC）对于维持交叉至关重要;因此，SCC恶化被认为有助于年龄增长- 关于NDJ研究表明，SCC的整体减少会导致与年龄相关的NDJ，但目前 尚不清楚着丝粒周围SCC的缺失是否比缺失对年龄相关NDJ的影响更大。 SCC的染色体臂。由于臂SCC和着丝粒SCC在减数分裂中具有不同的作用， 了解每一个的作用可以提供对与年龄相关的NDJ的潜在机制的深入了解。在 在果蝇中，Nipped-B促进了臂内聚蛋白的加载，但控制着丝粒的过程 粘着蛋白负载仍然是未知的。最近，我发现了一个功能分离的等位基因，mcm 5A 7， 并证明了mcm 5A 7突变不影响MCM 5的复制作用;相反，这种突变 增加减数分裂NDJ推测通过减少装载的凝聚蛋白SMC 1在着丝粒。这 结果表明，MCM 5参与在减数分裂细胞的着丝粒上募集或建立SMC 1， MCM 5的作用以前没有描述过。如本建议书所述，我将测试 利用mcm 5A 7破坏果蝇年龄相关NDJ中着丝粒SCC与臂SCC的关系 着丝粒SCC和Nipped-B突变破坏臂SCC。然后我会用一个卵母细胞老化程序 以测量两种突变体中年龄相关NDJ的发生率。此外，通过蛋白质组学研究， 免疫荧光，我建议调查的机制，其中MCM 5的功能，以促进SMC 1在 减数分裂细胞中的着丝粒。总的来说，该领域缺乏关于以下方面重要性的知识： 年龄相关NDJ中的着丝粒SCC和臂SCC，以及MCM 5在建立着丝粒SCC中的作用 凝聚力，防止治疗的进展和如何防止年龄相关的基本理解 非整倍性拟议的研究将有助于填补这一根本空白，并推动 预防战略。

Abstract The overall goal for meiosis I is to successfully segregate homologous chromosomes into two diploid cells. Failure to do so results in aneuploidy, which is the leading cause of genetic disorders, such as Trisomy 21, and miscarriages due to spontaneous abortions. Incidences of chromosomal nondisjunction (NDJ) increase exponentially as an oocyte ages, a phenomenon termed the maternal-age effect. Age-related chromosomal NDJ is hypothesized to be caused by the inability of an oocyte to maintain a chiasma, the physical manifestation of a crossover (CO) between homologous chromosomes, until ovulation. Sister chromatid cohesion (SCC) is vital for maintenance of chiasmata; thus, SCC deterioration is thought to contribute to age- related NDJ. Studies have shown that an overall reduction of SCC causes age-related NDJ, but it is currently unknown whether the loss of SCC around the centromere has a greater impact on age-related NDJ than loss of SCC at the chromosome arms. Because arm SCC and centromere SCC have distinct roles in meiosis, understanding the role of each may provide insight into the mechanism underlying age-related NDJ. In Drosophila, loading of arm cohesion proteins is facilitated by Nipped-B, but the process governing centromere cohesin loading remains largely unknown. Recently, I characterized a separation-of-function allele, mcm5A7, and demonstrated that the mcm5A7 mutation does not affect the replication role of MCM5; instead, this mutation increases meiotic NDJ presumably by reducing loading of the cohesion protein SMC1 at the centromere. This result suggests that MCM5 is involved in recruiting or establishing SMC1 at the centromere in meiotic cells, a role for MCM5 that has not been previously described. As described in this proposal, I will test the importance of centromeric SCC versus arm SCC in age-related NDJ in Drosophila by utilizing mcm5A7 to disrupt centromeric SCC and a mutation in Nipped-B to disrupt arm SCC. I will then employ an oocyte aging protocol to measure the incidence of age-related NDJ in both mutants. Further, through proteomic studies and immunofluorescence, I propose to investigate the mechanism by which MCM5 functions to facilitate SMC1 at the centromere in meiotic cells. Overall, the lack of knowledge in the field regarding the importance of centromeric SCC and arm SCC in age-related NDJ, as well as the role of MCM5 in establishing centromeric cohesion, prevents progression of therapeutics and basic understanding in how to prevent age-related aneuploidy. The proposed research will help fill this fundamental gap and advance the development of prevention strategies.