STAG2 loss amplifies EWS-FLI1-driven microsatellite enhancer activity promoting Ewing's sarcoma aggressiveness [RNA-Seq]

The lost sites are enriched for elements harboring 1–4 GGAA repeat motifs, with a simultaneous gain in EWS-FLI1 binding at multimeric enhancers containing ≥5 GGAA repeat sequences. Notably, reprogrammed EWS-FLI1 sites show concomitant changes in chromatin accessibility and H3K27ac abundance, which preferentially amplify EWS-FLI1 activity at multimeric enhancers and drive marked up‑regulation of canonical microsatellite‑regulated target genes in STAG2‑null cells.

By integrating Hi‑C–derived chromatin interaction maps with altered EWS-FLI1 occupancy, we derive distinct monomeric (1xGGAA) and multimeric (≥10xGGAA) EWS-FLI1 transcriptional signatures and show that STAG2 inactivation selectively augments the multimeric signature while attenuating monomeric activity. We further define a prognostic signature of GGAA-repeat enhancers that is significantly upregulated in patient tumors with aggressive clinical features and deleterious STAG2 alterations.

Together, these findings reveal that loss of STAG2–cohesin does not simply attenuate EWS-FLI1 function but reprograms its cistrome toward microsatellite multimeric GGAA neo‑enhancers, thereby amplifying a high‑risk EWS–FLI1 transcriptional state in Ewing sarcoma.