Ancient and animal-specific regulatory modes of EWS::FLI1 revealed by a minimal yeast model.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::friend leukemia integration 1 (FLI1) fusion protein. To assess whether EWS::FLI1's neomorphic functions depend on evolutionarily recent cofactors, we expressed EWS::FLI1 in Saccharomyces cerevisiae, a minimal system in which the BAF complex is strongly diverged and ETS transcription factors (ETS-TFs), polycomb group (PcG) proteins, and CBP/p300 are absent. We used co-IP/mass spectrometry to map the yeast interactome, ChIP-seq to identify gDNA binding sequences, RNA-seq for gene expression, and engineered reporters to test conversion of GGAA tandem repeats (GGAAμSat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was limited and distinct from its human counterpart, sharing core machinery (RNA polymerase II, FACT) but lacking BAF/SWI-SNF and spliceosome complexes, and showing enrichment for SAGA. EWS::FLI1 binds to hundreds of yeast genomic sites with preference for putative ETS-TF consensus sequences and CA dinucleotide repeats, and redirects RNA polymerase II to EWS::FLI1-bound loci. Yet, EWS::FLI1-expressing cells presented only minimal transcriptional dysregulation, in contrast to the extensive changes observed in human and Drosophila cells. Finally, EWS::FLI1 successfully converted silent GGAAμSat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, suggesting that EWS::FLI1 can mobilize functionally related, nonhomologous pathways to establish neoenhancers at GGAAμSat sites. Altogether, our results indicate that EWS::FLI1's core ability to drive GGAAμSat-dependent gene expression is a conserved, ancient property, while GGAAμSat-independent extensive transcriptome reprogramming depends on animal-specific cofactors and pathways.