The Drosophila FET ortholog Cabeza is an essential cofactor for ETV4-mediated activation of GGAA microsatellite neoenhancers.

The conversion of transcriptionally silent GGAA microsatellites (GGAAµSats) into functional enhancers by FET::ETS oncogenic fusions is a hallmark of Ewing sarcoma. However, emerging evidence implicates non-fused, full-length oncogenic ETS transcription factors in activating these repeats in other malignancies. Evaluating the in vivo transcriptional requirements of various human ETS factors in Drosophila, we found that human ETV4 associates with and robustly activates GGAAµSats in a tissue-specific manner. This activation is strongly inhibited by the human ETS repressor ETV6. Taking advantage of low genetic redundancy in Drosophila, we identified Cabeza (Caz), the single fly FET ortholog, as a necessary cofactor for ETV4-mediated transcription at GGAAµSats. Conversely, EWS::FLI1-mediated transcriptional activation of GGAAµSats is entirely independent of endogenous Caz, highlighting the distinct mechanics of covalent tethering versus non-covalent physical complexes. Collectively, our findings provide in vivo evidence that non-fused ETS factors cooperate with endogenous FET proteins to drive transcription from silent GGAA repeats, mechanistically validating this regulatory transformation known to operate as an oncogenic mechanism beyond Ewing sarcoma.