Molecular Mechanisms of Altered DNA Binding and Chromatin Regulation by FET::ETS Oncogenes

Many tumors are driven by chromosomal translocation-derived fusion transcription factors (TFs), which combine an intrinsically disordered low complexity domain (LCD) with a DNA-binding domain (DBD) from a TF. A major class of oncogenic fusion proteins involves a member of the FET family (FUS, EWSR1, or TAF15) fused to a TF DBD. These fusion oncoproteins exhibit neomorphic functions, including altered sequence-specific DNA binding and biomolecular condensate formation, allowing them to act as master regulators that reshape the genomic and epigenomic landscape essential for tumorigenesis. However, the molecular mechanisms governing their activity remain poorly understood. Our research seeks to define the biochemical and biophysical principles that drive fusion oncoprotein function in cancer. A major breakthrough in our work is the successful purification of full-length EWSR1::FLI1, the oncogenic driver of an aggressive pediatric tumor called Ewing sarcoma. This fusion protein, which contains the LCD from EWSR1 and the DBD from the ETS transcription factor FLI1, exemplifies how FET fusion oncoproteins acquire new regulatory functions through the interplay of their LCD and DBD. Our data indicate that fusion to the EWSR1 LCD fundamentally alters the DNA and nucleosome interactions of the FLI1 DBD, while features of the FLI1 domain in turn influence the ability of the EWSR1 LCD to nucleate transcriptional hubs at key regulatory sites. These findings support our hypothesis that fusion oncoproteins require an as-yet-undiscovered intramolecular crosstalk between their LCD and DBD. Our ability to produce high-quality EWSR1::FLI1 protein enables us to integrate biochemical, biophysical, single-molecule, and functional genomics approaches to dissect this mechanism. We will systematically investigate how interactions between FET LCDs and ETS DBDs confer neomorphic properties across multiple levels of chromatin regulation: DNA binding (Aim 1), nucleosome invasion (Aim 2), and 3D chromatin organization in transcriptional hubs (Aim 3). These mechanistic insights will not only advance our understanding of EWSR1::FLI1 but will also illuminate broader principles governing FET fusion oncoproteins as a class, laying the groundwork for novel therapeutic strategies targeting fusion-driven cancers. Project Number: 1R37CA299623-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Emily Theisen | Institution: RESEARCH INST NATIONWIDE CHILDREN'S HOSP, COLUMBUS, OH | Award Amount: $639,295 | Activity Code: R37 | Study Section: Cancer Genetics Study Section[CG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11298537