EWS/FLI is a translocation-encoded oncogenic transcription factor specific to Ewing sarcoma. The amino- terminal EWS portion harbors transcriptional activation and repression functions, and has potentially important biophysical properties, including the ability to polymerize and/or to undergo phase-separation. The carboxyl- terminal FLI portion contains an ETS-type DNA binding domain. In addition to standard ETS response elements, EWS/FLI binds repetitive GGAA-microsatellite sequences both in vitro and in vivo, and these microsatellite sequences confer EWS/FLI responsiveness to reporter and target genes. Thousands of GGAA- microsatellites are dispersed throughout the human genome, and are highly enriched in the promoters of EWS/FLI-upregulated target genes required for Ewing sarcoma development. GGAA-microsatellites exhibit significant variations (indels, transitions/transversions, length variations, etc.) across different loci in the human genome and across individuals in the population. These variations have significant functional consequences for gene expression and tumor development, and ?optimal? GGAA-microsatellite variants exist that drive the highest-levels of requisite gene expression. The foundational principle of this application is that GGAA- microsatellites are critical EWS/FLI regulatory elements required for Ewing sarcoma development. The isolated FLI DNA binding domain binds short stretches of GGAA-repeats. We have now identified regions of the EWS-domain absolutely required for EWS/FLI binding to longer ?optimal? GGAA-microsatellites in vitro and in vivo, and these also modulate transcriptional activation from ?promoter-like? microsatellites. We also recently distinguished between ?promoter-like? GGAA-microsatellites distal ?enhancer-like? GGAA- microsatellites. For example, certain artificial EWS/FLI alleles activate gene expression from ?promoter-like? microsatellites, but not from ?enhancer-like? microsatellites, and we also observed distinct functions in vivo. Finally, we recently identified ?faux-microsatellite? sequences that are EWS/FLI-bound and associated with gene repression rather than activation. Based on published and preliminary data, we hypothesize a new model: that oncogenic gene regulation in Ewing sarcoma depends on a complex interplay between the biophysical/biochemical properties of EWS/FLI and the location and specific DNA sequences of GGAA- microsatellite response elements. We propose experiments designed to test this hypothesis, and in doing so, to decipher the molecular mechanisms that EWS/FLI uses to cause Ewing sarcoma.