The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

Boone, M. A.; Taslim, C.; Crow, J. C.; Selich-Anderson, J.; Showpnil, I. A.; Sunkel, B. D.; Wang, M.; Stanton, B. Z.; Theisen, E. R.; Lessnick, S. L.

doi:10.1101/2020.10.29.355859

bioRxiv · 10.1101/2020.10.29.355859

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

Boone, M. A.·Taslim, C.·Crow, J. C.·Selich-Anderson, J.·Showpnil, I. A.·Sunkel, B. D.·Wang, M.·Stanton, B. Z.·Theisen, E. R.·Lessnick, S. L.

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Abstract

BackgroundEwing sarcoma is an aggressive bone cancer in children and young adults that contains a pathognomonic chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the low-complexity amino-terminal portion of EWS to the carboxyl-terminus of FLI. The FLI portion contains an ETS DNA-binding domain and adjacent amino- and carboxyl-regions. Early studies using non-Ewing sarcoma cellular models provided conflicting information on the role of these adjacent regions in the oncogenic function of EWS/FLI. We therefore sought to define the specific contributions of each FLI region to EWS/FLI activity in an appropriate Ewing model, and in doing so, to better understand Ewing sarcoma development mediated by the fusion protein. MethodsWe used a "knock-down/rescue" system to replace endogenous EWS/FLI expression with mutant forms of the protein in Ewing sarcoma cells and tested these for oncogenic transformation using soft-agar colony forming assays. These data were complemented by DNA-binding assays using fluorescence anisotropy, genomic localization assays using CUT&RUN, transcriptional regulation studies using luciferase reporter assays and RNA-sequencing, as well as chromatin accessibility assays using ATAC-sequencing. ResultsWe found that the DNA-binding domain and short flanking regions of FLI were required for oncogenic transformation, gene expression, genomic localization and chromatin accessibility when fused to the amino-terminal EWS-portion from EWS/FLI, but that the remaining regions of FLI were dispensable for these functions. Removal of a carboxyl-terminal alpha-helix from the short flanking regions of the DNA-binding domain of FLI created a hypomorphic EWS/FLI that retained normal DNA binding, genomic localization, and chromatin accessibility, but had significantly restricted transcriptional activity and a near total loss of oncogenic transformational capacity. ConclusionsThe DNA-binding domain and carboxyl-terminal short flanking region of FLI are the only portions of FLI required for EWS/FLI-mediated oncogenic transformation in a Ewing sarcoma cellular context. In addition to the well-defined DNA-binding function of FLI, this additional alpha-helix immediately downstream of the DNA-binding domain contributes a previously-undescribed function in gene regulation and oncogenic transformation. Understanding the function of this critical region could provide new therapeutic opportunities to target EWS/FLI in Ewing sarcoma.

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Boone, M. A., Taslim, C., Crow, J. C., Selich-Anderson, J., Showpnil, I. A., Sunkel, B. D., Wang, M., Stanton, B. Z., Theisen, E. R., Lessnick, S. L.. 2020-10-30. The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma. https://doi.org/10.1101/2020.10.29.355859

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