Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity

In approximately 85% of Ewing sarcomas, chromosomal translocations give ris e to the chimeric gene EWS/FLI, encoding the N-terminus of the RNA binding protein EWS fused to the DNA-binding domain of the ETS protein FLI-1, EWS/F LI is a stronger transcriptional activator than wild-type FLI-1, although b oth proteins bind to the same DNA sequences in vitro. In addition, EWS/FLI, but not FLI-1, is a transforming oncogene in NIH3T3 fibroblasts. EWS/FLI i s thought to transform through its ability to deregulate the expression of target genes. We introduced several paint mutations into the ETS domain of EWS/FLI that abolished DNA-binding activity. Although two of these mutation s disrupted the transforming activity of EWS/FLI, one mutated protein conta ining a substitution of isoleucine 347 with glutamic acid (I347E) retained diminished transforming activity. In addition, EWS/FLI I347E did not activa te expression of the endogenous EWS/FLI target gene manic fringe (MFNG). Th ese studies demonstrate that a portion of the oncogenic activity of EWS/FLI is independent of nr DNA-binding activity.