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The COOH-Terminal Domain of FLI-1 Is Necessary for Full Tumorigenesis and Transcriptional Modulation by EWS/FLI-1^{1 ,,2}

Department of Experimental Pathology and Laboratory Medicine [A. A., M. A. T.], Molecular Biology Institute [S. M. W., M. A. T., C. T. D.], Jonsson Comprehensive Cancer Center [M. A. T., C. T. D.], and Department of Pediatrics, Gwynne Hazen Cherry Memorial Labs [C. T. D.], University of California at Los Angeles, Los Angeles, California 90095

More than 85% of Ewing’s family tumors carry a specific chromosomal translocation that fuses the NH₂ terminus of the EWS gene to the COOH terminus of the FLI1 transcription factor. It has been shown previously that both the transactivation domain encoded by EWS and the DNA binding domain of FLI1 were necessary for transforming cells to anchorage independence. We now report that a COOH-terminal domain in addition to the FLI1 DNA binding domain is necessary to promote cellular transformation. NIH 3T3 cells expressing a COOH-terminal deletion mutant (EWS/FLI1 C) have a greatly reduced capability to form colonies in soft agar and tumors in severe combined immunodeficient mice. The rate of tumor formation for NIH 3T3 that express EWS/FLI1 C is 50 days, whereas EWS/FLI1 forms tumors within 22 days. In addition, cells expressing the EWS/FLI1 C mutant failed to completely demonstrate the round-cell histology that is seen in both Ewing’s tumor cell lines and NIH 3T3 cells expressing full-length EWS/FLI1. Northern and microarray analyses were performed to assess the effect of loss of the FLI1 COOH terminus on transcriptional modulation of EWS/FLI1 target genes. We found that although EWS/FLI1 C up-regulates smaller numbers of genes (21 genes) compared with EWS/FLI1 (34 genes), 41% of the EWS/FLI1 targets were also up-regulated by EWS/FLI1 C. On the other hand, EWS/FLI1 C is unable to down-regulate genes (3 genes) as efficiently as EWS/FLI1 (39 genes) with only one target gene repressed by both fusion constructs. Our study indicates that the EWS/FLI1 transcription factor has strong transcriptional activating as well as repressing properties and suggests that transcriptional activation and repression of target genes may occur through biochemically different mechanisms.

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