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。 如果做不到这一点，就会导致非整倍体，这是遗传疾病的主要原因，如21三体，以及 自然流产导致的流产。染色体不分离(NDJ)发生率增加 随着卵母细胞的老化，这种现象被称为母体年龄效应。年龄相关染色体 NDJ被推测是由于卵母细胞无法维持交叉，物理上 同源染色体之间交换(CO)的表现，直到排卵。姐妹染色单体 凝聚力(SCC)对交叉的维持至关重要；因此，SCC的恶化被认为与年龄有关。 相关NDJ。研究表明，SCC的全面减少会导致与年龄相关的NDJ，但目前 未知着丝粒周围的SCC丢失是否比丢失对年龄相关性NDJ的影响更大 染色体臂上的鳞状细胞癌。由于臂鳞状细胞癌和着丝粒鳞状细胞癌在减数分裂中具有不同的作用， 了解两者的作用可能有助于深入了解与年龄相关的NDJ的机制。在……里面 果蝇，臂结合蛋白的装载是由NIPD-B促进的，但控制着丝粒的过程 粘附素的载量在很大程度上仍不清楚。最近，我鉴定了一个功能分离的等位基因mcm5A7， 并证明了mcm5a7突变不影响mcm5的复制作用；相反，该突变 增加减数分裂NDJ可能是通过减少着丝粒上凝聚蛋白SMC1的负载来实现的。这 结果表明，MCM5参与了减数分裂细胞着丝粒上SMC1的募集或建立，a 以前没有描述过的MCM5的角色。正如本提案中所描述的，我将测试 利用mcm5A7干扰果蝇年龄相关性NDJ着丝粒SCC与ARM SCC的比较 着丝粒SCC和NIPD-B突变破坏ARM SCC。然后我会采用卵母细胞老化方案 以测量两个突变体中年龄相关性NDJ的发生率。此外，通过蛋白质组学研究和 免疫荧光，我建议研究MCM5促进SMC1的作用机制 减数分裂细胞中的着丝粒。总体而言，该领域缺乏关于以下方面重要性的知识 着丝粒SCC和ARM SCC在年龄相关性NDJ中的作用以及MCM5在建立着丝粒中的作用 凝聚力，防止治疗进展和对如何预防年龄相关的基本理解 非整倍体。拟议的研究将有助于填补这一根本空白，并推动 预防策略。

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.