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.