Chromosomal localization of Ewing sarcoma EWSR1/FLI1 protein promotes the induction of aneuploidy

Abstract

Ewing sarcoma is a pediatric bone cancer that expresses the chimeric protein EWSR1/FLI1. We previously demonstrated that EWSR1/FLI1 impairs the localization of Aurora B kinase to the midzone (the midline structure located between segregating chromosomes) during anaphase. While localization of Aurora B is essential for faithful cell division, it is unknown whether interference with midzone organization by EWSR1/FLI1 induces aneuploidy. To address this, we generated stable Tet-on inducible cell lines with EWSR1/FLI1, using CRISPR/Cas9 technology to integrate the transgene at the safe-harbor AAVS1 locus in DLD-1 cells. Induced cells expressing EWSR1/FLI1 displayed an increased incidence of aberrant localization of Aurora B, and greater levels of aneuploidy, compared with noninduced cells. Furthermore, the expression of EWSR1/FLI1-T79A, containing a threonine (Thr) to alanine (Ala) substitution at amino acid 79, failed to induce these phenotypes, indicating that Thr 79 is critical for EWSR1/FLI1 interference with mitosis. In contrast, the phosphomimetic mutant EWSR1/FLI1-T79D (Thr to aspartic acid (Asp)) retained the high activity as wild-type EWSR1/FLI1. Together, these findings suggest that phosphorylation of EWSR1/FLI1 at Thr 79 promotes the colocalization of EWSR1/FLI1 and Aurora B on the chromosomes during prophase and metaphase and, in addition, impairs the localization of Aurora B during anaphase, leading to induction of aneuploidy. This is the first demonstration of the mechanism for EWSR1/FLI1-dependent induction of aneuploidy associated with mitotic dysfunction and the identification of the phosphorylation of the Thr 79 of EWSR1/FLI1 as a critical residue required for this induction.

Modification of Phosphorylation Sites in the Yeast Lysine Methyltransferase Set5 Exerts Influences on the Mitogen-Activated Protein Kinase Hog1 under Prolonged Acetic Acid Stress

“…These results also suggest dynamic regulation of the central protein kinase Hog1 under different environmental conditions. The phosphomimetic and alanine substitution mutations are widely used to study protein phosphorylation ( 37 , 38 ). However, no studies have been performed to test whether such modifications affect protein folding, stability, or expression levels.…”

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

“…The PCR products of hEWSR1 , hEWSR1:R565A and mCherry were inserted to SalI and MluI sites of pMK243 that had been modified at its multicloning sites in the previous study (Tet-OSTIR1-PURO) ( Natsume et al, 2016 ; Hassebroek et al, 2020 ; Park et al, 2021 ).…”

“…The cells grown in a T25 flasks at ∼70%–80% confluency were synchronized to mitosis using the thymidine/nocodazole protocol ( Kotýnková et al, 2016 ; Park et al, 2021 ). The mitotic cells were collected in a 15 mL tube, washed for 3 times with McCoy’s 5A media (without FBS), and it were resuspended in 1 mL of PBS.…”

EWSR1 prevents the induction of aneuploidy by regulating the localization of Aurora B at inner centromere

“…The PCR products of hEWSR1, hEWSR1:R565A and mCherry were inserted to SalI and MluI sites of pMK243 that was modified at its multicloning sites in the previous study (Tet-OSTIR1-PURO) (Natsume et al, 2016, Hassebroek et al, 2020, Park et al, 2021)…”

“…The cells grown in a T25 flasks at ∼70-80% confluency were synchronized to mitosis by following the Thymidine/Nocodazole protocol ((Kotýnková et al, 2016, Park et al, 2021). Mitotic cells were collected in a 15 ml tube and the cells were washed for 3 times with McCoy’s 5A media (without FBS), and it were resuspended in 1 mL of PBS.…”