The DBD-α4 helix of EWS::FLI is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma.

Ewing sarcoma is the second most common bone cancer in children and young adults. In 85% of patients, a translocation between chromosomes 11 and 22 results in a potent fusion oncoprotein, EWS::FLI. EWS::FLI is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWS portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWS::FLI. The FLI portion of the fusion contains a DNA binding domain shown to bind core GGAA motifs and GGAA repeats. A small alpha-helix in the DNA binding domain of FLI, DBD-α4 helix, is critical for the transcription function of EWS::FLI. In this study, we aimed to understand the mechanism by which the DBD-α4 helix promotes transcription, and therefore oncogenic transformation. We utilized a multi-omics approach to assess chromatin organization, active chromatin marks, genome binding, and gene expression in cells expressing EWS::FLI constructs with and without DBD-α4 helix. Our studies revealed DBD-α4 helix is crucial for cooperative binding of EWS::FLI at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWS::FLI such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.