EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B.

was originally identified as a part of an aberrant fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the fusion gene in the tumor genome, the cell loses one wild type allele. Our previous study demonstrated that the loss of (homologue of human ) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the mutant background. To dissect the molecular function of EWSR1, we successfully established a stable DLD-1 cell line that enables a conditional knockdown of EWSR1 using an Auxin Inducible Degron (AID) system. When both genes of DLD-1 cell were tagged with at its 5'-end using a CRISPR/Cas9 system, treatment of the () DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. This defect was proceeded by a lower incidence of the localization of Aurora B at inner centromeres, and by a higher incidence of the protein at Kinetochore proximal centromere compared to the control cells during pro/metaphase. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of and (a mutant that has low affinity for Aurora B) in the () DLD-1 cells. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B.