The knockout mice for EWSR1 displayed impaired differentiation of pre-B lymphocytes, defects in meiosis of sperm, and reduction of mitochondria through degradation of PGC1a and of mitochondria function in brown fat and skeletal muscles of EWSR1-deficient mice ( Li et al., 2007 Park et al., 2015). Multiple phenotypes were also reported using animal models with genetically ablated EWSR1. Other reports demonstrated that EWSR1 promotes homologous recombination by suppressing R-loop formation ( Gorthi et al., 2018). The EWSR1 regulates splicing through interaction with splicing factor SF1 as well as small nuclear ribonucleoprotein-specific protein U1C that is required for the early spliceosome formation ( Zhang et al., 1998 Knoop and Baker, 2000). For example, EWSR1 interacts with the subunits of TFIID and RNA PolII, and regulates transcription of Oct4 and Brn3a ( Zhang et al., 1998 Gascoyne et al., 2004 Lee et al., 2005). The EWSR1 has various activities in multiple biological phenomena. Subsequent studies showed that EWSR1 is fused to various types of transcription factors in multiple sarcomas (e.g., EWSR1-WT1 desmoplastic small round cell tumor, EWSR1-ATF1 clear cell sarcoma, EWSR1-CHOP myxoid liposarcoma, EWSR1-NR4A3 extraskeletal myxoid chondrosarcoma) ( Lee et al., 1997 Panagopoulos et al., 2002 Matsui et al., 2006 Filion et al., 2009). The Ewing sarcoma region 1 gene ( EWSR1) was originally identified in the pediatric bone cancer, Ewing sarcoma, as a part of an aberrant fusion gene with FLI1 ( Delattre et al., 1992). Together, we demonstrate that EWSR1 prevents the induction of lagging chromosomes, and of aneuploidy through the interaction with Aurora B. The EWSR1-mCherry rescued the high incidence of aneuploidy of EWSR1 knockdown cells, whereas EWSR1-mCherry:R565A failed to rescue the phenotype. Since our previous study demonstrated that EWSR1 interacts with the key mitotic kinase, Aurora B, we generated replacement lines of EWSR1-mCherry and EWSR1:R565A-mCherry (a mutant that has low affinity for Aurora B) in the ( AID-EWSR1/AID-EWSR1) DLD-1 cells. Significantly, the EWSR1 knockdown (AUX+) cells induced higher incidence of aneuploidy compared to the control (AUX-) cells. Despite these defects, the EWSR1 knockdown cells did not undergo mitotic arrest, suggesting that the cell lacks the error correction mechanism. 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. During anaphase, the EWSR1 knockdown (AUX+) cells displayed higher incidence of lagging chromosomes compared to the control (AUX-) cells. When both EWSR1 genes of DLD-1 cell were tagged with mini-AID at its 5′-end using a CRISPR/Cas9 system, treatment of the ( AID-EWSR1/AID-EWSR1) DLD-1 cells with a plant-based Auxin (AUX) led to the significant levels of degradation of AID-EWSR1 proteins. 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.
Our previous study demonstrated that the loss of ewsr1a (homologue of human EWSR1) in zebrafish leads to the high incidence of mitotic dysfunction, of aneuploidy, and of tumorigenesis in the tp53 mutant background. Due to formation of the EWSR1/FLI1 fusion gene in the tumor genome, the cell loses one wild type EWSR1 allele. Molecular Biosciences, University of Kansas, Lawrence, KS, United StatesĮWSR1 (Ewing sarcoma breakpoint region 1) was originally identified as a part of an aberrant EWSR1/FLI1 fusion gene in Ewing sarcoma, the second most common pediatric bone cancer.
0 kommentar(er)
