{"doi":"10.1101/2020.10.29.355859","title":"The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma","abstract":"Abstract Background Ewing 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. Methods We 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. Results We 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. Conclusions The 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2020,"id":125543,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9558,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":488652,"name":"Cenny Taslim","orcid":"0000-0003-3302-9099","position":1,"is_corresponding":false},{"id":571713,"name":"Jesse C. Crow","orcid":"0000-0001-7369-0653","position":2,"is_corresponding":false},{"id":572134,"name":"Julia Selich‐Anderson","orcid":null,"position":3,"is_corresponding":false},{"id":488650,"name":"Iftekhar A. Showpnil","orcid":"0000-0002-3168-8114","position":4,"is_corresponding":false},{"id":571714,"name":"Benjamin D. Sunkel","orcid":"0000-0003-1376-9567","position":5,"is_corresponding":false},{"id":558985,"name":"Meng Wang","orcid":"0000-0002-3457-5908","position":6,"is_corresponding":false},{"id":469267,"name":"Benjamin Z. Stanton","orcid":"0000-0002-2613-2955","position":7,"is_corresponding":false},{"id":488654,"name":"Emily R. Theisen","orcid":"0000-0003-2923-1198","position":8,"is_corresponding":false},{"id":465978,"name":"Stephen L. Lessnick","orcid":"0000-0001-9645-4434","position":9,"is_corresponding":false},{"id":571712,"name":"Megann A. Boone","orcid":"0000-0002-7806-8027","position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-18T23:15:15.482227Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}