Phase separation of the oncogenic fusion protein EWS::FLI1 is modulated by its DNA-binding domain

Ewing sarcoma (EwS) is an aggressive cancer of bone and soft tissue that predominantly affects children and young adults. A chromosomal translocation joins the low-complexity domain (LCD) of the RNA-binding protein EWS (EWS LCD ) with the DNA-binding domain of Friend leukemia integration 1 (FLI1 DBD ), creating EWS::FLI1, a potent fusion oncoprotein essential for EwS development and responsible for over 85% of EwS tumors. EWS::FLI1 forms biomolecular condensates in vivo and promotes tumorigenesis through mediation of aberrant transcriptional changes and by interfering with the normal functions of nucleic acid-binding proteins like EWS through a dominant-negative mechanism. In particular, the expression of EWS::FLI1 in EwS directly interferes with the biological functions of EWS leading to alternate splicing events and defects in DNA-damage repair pathways. Though the EWS LCD is capable of phase separation, here we report a direct interaction between FLI1 DBD and EWS LCD that enhances condensate formation and alters the physical properties of the condensate. This effect was conserved for three related E-twenty-six transformation-specific (ETS) DNA-binding domains (DBDs) while DNA binding blocked the interaction with EWS LCD and inhibited EWS::FLI1 condensate formation. NMR spectroscopy and mutagenesis studies confirmed that ETS DBDs transiently interact with EWS LCD via the ETS DBDs “wings.” Together these results revealed that ETS DBDs, particularly FLI1 DBD , enhance EWS LCD condensate formation and rigidity, supporting a model in which electrostatic and structural interactions drive condensate dynamics with implications for EWS::FLI1-mediated transcriptional regulation in EwS.