AUTHOR=Kim Haeyoung , Park Hyewon , Schulz Evan T. , Azuma Yoshiaki , Azuma Mizuki 

TITLE=EWSR1 prevents the induction of aneuploidy through direct regulation of Aurora B

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=11

YEAR=2023

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2023.987153

DOI=10.3389/fcell.2023.987153

ISSN=2296-634X

ABSTRACT=<p><italic>EWSR1 (Ewing sarcoma breakpoint region 1)</italic> was originally identified as a part of an aberrant <italic>EWSR1/FLI1</italic> fusion gene in Ewing sarcoma, the second most common pediatric bone cancer. Due to formation of the <italic>EWSR1/FLI1</italic> fusion gene in the tumor genome, the cell loses one wild type <italic>EWSR1</italic> allele. Our previous study demonstrated that the loss of <italic>ewsr1a</italic> (homologue of human <italic>EWSR1</italic>) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the <italic>tp53</italic> 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 <italic>EWSR1</italic> genes of DLD-1 cell were tagged with <italic>mini-AID</italic> at its 5′-end using a CRISPR/Cas9 system, treatment of the (<italic>AID-EWSR1/AID-EWSR1</italic>) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. During anaphase, the <italic>EWSR1</italic> 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 <italic>EWSR1-mCherry</italic> and <italic>EWSR1:R565A-mCherry</italic> (a mutant that has low affinity for Aurora B) in the (<italic>AID-EWSR1/AID-EWSR1</italic>) 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.</p>