Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites
- 1Nationwide Children's Hospital
Abstract
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.
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BibTeX
@misc{chen2026fusion,
title = {{Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites}},
author = {R.-W. Chen and R. Zhou and E. J. Tokarsky and M. A. Boone and A. K. Byrum and M. G. Poirier and E. R. Theisen},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.16.752125},
url = {https://www.biorxiv.org/content/10.64898/2026.09.16.752125v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Fusion Oncoprotein EWSR1::FLI1 Invades Nucleosomes at Consensus ETS Motifs and GGAA Microsatellites
AU - R.-W. Chen
AU - R. Zhou
AU - E. J. Tokarsky
AU - M. A. Boone
AU - A. K. Byrum
AU - M. G. Poirier
AU - E. R. Theisen
PY - 2026
DA - 2026/09/16
PB - bioRxiv
DO - 10.64898/2026.09.16.752125
UR - https://www.biorxiv.org/content/10.64898/2026.09.16.752125v1
AB - 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.
ER -