Transcriptomic analysis to functionally map the intrinsically disordered domain of EWS/FLI [Experiment 2]

The purpose of this study was to define unique classes of EWS/FLI target genes and the distinct set of structural features present in the EWS domain required for target gene regulation at different EWS/FLI response elements. This study reports the first genome-wide transcriptomic description of a partially-functional EWS/FLI mutant . We report new structure-function dependencies across different classes of response element and tie these dependencies to the ability of EWS/FLI to transform cells. Overall design: For knockdown of wildtype EWS/FLI and rescue with differing cDNAs in A673 Ewing sarcoma cell lines, shRNAs were retrovirally delivered on day 1. Selection in 2 µg/mL puromycin (Sigma P8833) occurred on days 2-5. Cells were infected with retrovirus containing the cDNA in the pMSCV-hygro backbone on day 5. Double selection in puromycin and 400 µg/mL hygromycin B (Thermo Fisher 10687010) began on day 6 and continued until day 16. Cells were subsequently harvested for RNA, protein, and seeded for soft agar assays.