View Program - 63rd Annual Drosophila Research Conference

Premature loss of sister chromatid cohesion during the meiotic arrest in human oocytes is a major contributor to the maternal age effect, a phenomenon in which the probability of an aneuploid pregnancy increases as a woman ages. Our laboratory has previously uncovered evidence for a cohesion rejuvenation program in Drosophila oocytes that operates during prophase, after cohesive linkages are originally established during S phase. To better understand the behavior of rejuvenation-specific cohesin, we designed an “allele-switch” transgene that expresses endogenous levels of the cohesin subunit Smc1 tagged with mCherry which is flanked by FRT sites. Induction of Flippase using the prophase specific matα-Gal4 driver causes excision of the mCherry tag, resulting in expression of Smc1 tagged with superfolder GFP (sGFP). This tool allows us to specifically visualize chromatin-associated cohesin that is synthesized after the canonical cohesion establishment that occurs during oocyte DNA replication. Upon switch induction with the matα driver, newly synthesized Smc1-sGFP begins to load onto the meiotic chromosomes as early as oogenesis stage 2. Meiotic prophase lasts ~6 days in Drosophila oocytes. Surprisingly, almost the entire cohesin population appears to turn over in < 2 days (by oogenesis stage 5). Turnover at centromeres is slower than at arms. Matα-induced knockdown (KD) of the cohesin subunit Smc3 abolishes chromosomal loading of newly synthesized Smc1-sGFP. This failure to load rejuvenation-specific cohesin during prophase leads to premature loss of arm cohesion in metaphase I arrested oocytes, as evidenced by fluorescence in situ hybridization that uses Oligo-PAINT probes to visualize X-chromosome arm cohesion. Together these results indicate that rejuvenation in Drosophila oocytes is required to maintain levels of cohesion that are sufficient for accurate chromosome segregation. Future experiments will utilize the allele-switch transgene to investigate cohesin loading dynamics when cohesion regulators or rejuvenation specific proteins are knocked down during prophase. These experiments will allow us to better understand the mechanism(s) underlying cohesion rejuvenation during meiotic prophase in Drosophila oocytes.

Cohesin dynamics during meiotic prophase in Drosophila oocytes