An RNAi screen to identify proteins required for cohesion rejuvenation during meiotic prophase in Drosophila oocytes
Accurate chromosome segregation during meiosis requires maintenance of sister chromatid cohesion, initially established during premeiotic S phase. In human oocytes, DNA replication and cohesion establishment occur decades before chromosome segregation and deterioration of meiotic cohesion is one factor that leads to increased segregation errors as women age. Our previous work led us to propose that a cohesion rejuvenation program operates to establish new cohesive linkages during meiotic prophase in Drosophila oocytes and depends on the cohesin loader Nipped-B and the cohesion establishment factor Eco. In support of this model, we recently demonstrated that chromosome-associated cohesin turns over extensively during meiotic prophase and failure to load cohesin onto chromosomes after premeiotic S phase results in arm cohesion defects in Drosophila oocytes. To identify proteins required for prophase cohesion rejuvenation but not S phase establishment, we conducted a Gal4-UAS inducible RNAi screen that utilized two distinct germline drivers. Using this strategy, we identified 29 gene products for which hairpin expression during meiotic prophase, but not premeiotic S phase, significantly increased segregation errors. Prophase knockdown of Brahma or Pumilio, two positives with functional links to the cohesin loader, caused a significant elevation in the missegregation of recombinant homologs, a phenotype consistent with premature loss of arm cohesion. Moreover, fluorescence in situ hybridization confirmed that Brahma, Pumilio and Nipped-B are required during meiotic prophase for maintenance of arm cohesion. Our data support the model that Brahma and Pumilio regulate Nipped-B dependent cohesin loading during rejuvenation. Future analyses will better define the mechanism(s) that govern meiotic cohesion rejuvenation and whether additional prophase-specific positives function in this process.