Abstract B019: Full-length EWSR1::FLI1 reveals low-complexity domain regulation of DNA and nucleosome binding

Abstract Ewing sarcoma is the second most common childhood bone cancer. In approximately 85% of cases, the tumor is caused by expression of the fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 is generated from a chromosome translocation event between chromosomes 11 and 22. This combines the low complexity domain (LCD) of EWSR1 with the DNA-binding domain (DBD) of FLI1. Previous genomic studies have shown that EWSR1::FLI1 is a potent genome regulator at GGAA microsatellites. However, due to the technical difficulty in purifying the full-length EWSR1::FLI1, there has been a lack of biochemical studies characterizing the properties of the fusion protein. In this study, we successfully purified the full-length EWSR1::FLI1 and conducted a series of in vitro biochemical and fluorescence-based experiments to investigate its interaction with DNA and nucleosomes. We found that truncated EWSR1::FLI1 shows pioneering activities, and an alpha-helix in the FLI1 DBD further facilitates its binding to nucleosomes. Surprisingly, we observed that the DNA-binding sequence preference differs between full-length and truncated proteins. This suggests that the EWSR1 LCD might also be involved in regulating sequence-specific DNA binding. Our findings provide new insights into how EWSR1::FLI1 binds to DNA and nucleosomes as a pioneer factor to alter genome regulation in Ewing sarcoma. Citation Format: Ruo-Wen Chen, John E. Tokarsky, Andrea K. Byrum, Megann A. Boone, Michael G. Poirier, Emily R. Theisen. Full-length EWSR1::FLI1 reveals low-complexity domain regulation of DNA and nucleosome binding [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B019.