Abstract B025: Multiple domains contribute to nucleosome invasion by EWSR1::FLI1

Abstract

Ewing sarcoma is a rare and aggressive pediatric cancer caused by a translocation-derived fusion oncoprotein that fuses the N-terminal low complexity domain (LCD) of a FET protein (FUS, EWSR1, or TAF15) with the C-terminal portion of an ETS transcription factor from the ERG or PEA3 subfamilies. These fusion oncoproteins retain the conserved ETS winged helix-loop-helix (wHLH) DNA binding domain and function as aberrant transcription factors to alter gene regulation and promote oncogenesis. The most common fusion in this disease is EWSR1::FLI1. Because EWSR1::FLI1 functions as a master regulator of cell fate, it is proposed to be a pioneer transcription factor (PF). PFs recognize and bind their motifs in closed chromatin, facilitate chromatin opening, and recruit ATP-dependent chromatin remodelers to establish lineage-specific gene expression programs. EWSR1::FLI1 has been shown to bind at both the FLI1 high affinity (HA) motif (5’-ACCGGAAGTG-3’) and repetitive GGAA microsatellites. Genomic profiling shows that EWSR1::FLI1-bound regions become more accessible with GGAA microsatellite binding frequently resulting in de novo enhancer formation. However, other studies have shown context-specific binding of EWSR1::FLI1 to GGAA repeats in closed chromatin and the importance of binding at HA ETS sites for tumor initiation. The ability of EWSR1::FLI1 to recognize and bind nucleosomal motifs and the molecular mechanisms by which it may do so remain poorly understood. Here we use purified recombinant truncated FLI1 wHLH and full-length EWSR1::FLI1 constructs in in vitro biochemical and fluorescence experiments to better understand how the DBD of EWSR1::FLI1 interacts with DNA and nucleosomes. While both the FLI1 wHLH and full-length EWSR1::FLI1 display efficient nucleosome invasion, we identify significant differences in sequence-specific binding to both DNA and nucleosomes between the full-length protein and truncated constructs, suggesting the EWSR1 LCD modulates both DNA sequence recognition and nucleosome engagement. Our findings reveal new insights into how EWSR1::FLI1 binds to DNA motifs in chromatin and suggest that the EWSR1 LCD may contribute to both altered sequence-specific DNA binding and nucleosome engagement. These studies establish an experimental framework for future investigations of the different factors that influence nucleosome engagement by EWSR1::FLI1 to cause Ewing sarcoma in cells.