MULTIPLE DOMAINS MEDIATE TRANSFORMATION BY THE EWINGS-SARCOMA EWS FLI-1 FUSION GENE/

The (11;22) chromosomal translocation found in Ewing's sarcoma and rel ated tumors fuses the amino terminus of the EWS protein to the DNA-bin ding domain of the FLI-1 transcription factor. In contrast to normal F LI-1, the EWS/FLI-1 fusion transforms NIH3T3 cells and this activity r equires both EWS and FLI-1 sequences. Reporter gene assays showed that the portion of EWS fused to FLI-1 encodes a strong transcriptional ac tivation domain. To determine whether this function is necessary for t ransformation by EWS/FLI-1, deletion analysis of EWS was performed. We found that the EWS domain could be functionally subdivided into two r egions: (i) an amino terminal domain (domain A) which transforms effic iently when fused to FLI-1 but has little transactivation activity in a model system and (ii) a distal region (domain B) which transactivate s efficiently but transforms less efficiently when fused to FLI-1. Rep lacement of the EWS domain with known heterologous transcriptional act ivation domains yielded chimeric FLI-1 fusions that in some instances could transform NM3T3 cells. Finally we demonstrate that EWS/FLI-1 and related FLI-1 chimeras are able to cooperate,vith another transcripti on factor to activate a model reporter gene. These results further dem onstrate that EWS/FLI-1 is an aberrant transcription factor and sugges t that the EWS domain mediates important protein-protein interactions with other factors resulting in the transcriptional modulation of targ et genes.