
The DBD-α4 helix of the EWSR1::FLI1 fusion protein is crucial for its cooperative binding at GGAA microsatellites, which underlies the formation of topologically associated domains (TADs), chromatin loops, enhancers, and productive transcription hubs that drive oncogenic gene expression in Ewing sarcoma.


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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, EWSR1::FLI1. EWSR1::FLI1 is the only genetic alteration in an otherwise unaltered genome of Ewing sarcoma tumors. The EWSR1 portion of the protein is an intrinsically disordered domain involved in transcriptional regulation by EWSR1::FLI1. 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 FLI1, DBD-α4 helix, is critical for the transcription function of EWSR1::FLI1. 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 EWSR1::FLI1 constructs with and without the DBD-α4 helix. Our studies revealed DBD-α4 helix is crucial for cooperative binding of EWSR1::FLI1 at GGAA microsatellites. This binding underlies many aspects of genome regulation by EWSR1::FLI1 such as formation of TADs, chromatin loops, enhancers and productive transcription hubs.

### Competing Interest Statement

The authors have declared no competing interest.

The DBD-α4 helix of EWSR1::FLI1 is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma

the-critical-role-of-the-dbd-α4-helix-in-ewsr1-fli1-mediated-genome-regulation-and-chromatin-restructuring-in-ewing-sarcoma


The Critical Role of the DBD-α4 Helix in EWSR1::FLI1-Mediated Genome Regulation and Chromatin Restructuring in Ewing Sarcoma
