Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites

Ewing sarcoma is an aggressive bone malignancy occurring in children, adolescents, and young adults. Most cases are caused by expression of the fusion oncoprotein EWSR1::FLI1, which contains the low complexity domain (LCD) of EWSR1 and the DNA-binding domain (DBD) of FLI1. Previous genomic studies indicate EWSR1::FLI1 accesses GGAA microsatellites in chromatin to function as a potent transcriptional regulator. Due to the technical challenges of purifying full-length EWSR1::FLI1, mechanistic studies biochemically characterizing its pioneer activities have been lacking. Here, we purified both full-length EWSR1::FLI1 and truncated DBD constructs to conduct biochemical and fluorescence-based experiments investigating interactions with different motifs in free DNA and nucleosomes. Both truncated and full-length EWSR1::FLI1 show efficient target binding in nucleosomes, and that the fourth alpha-helix in the FLI1 DBD enhances nucleosome-binding efficiency. Surprisingly, we also observe differences in both free DNA binding affinity and sequence preference between truncated and full-length proteins, though these changes are not apparent in nucleosome-binding assays. These findings reveal that EWSR1::FLI1 possesses a key pioneer factor property, efficiently targeting its binding site within nucleosomes, and that full-length EWSR1::FLI1 binding shifts to preferentially target GGAA repeats, even on motifs that bind a single EWSR1::FLI1 protein.