Abstract 4968: Modes of co-factor recruitment by an oncogenic fusion protein

Abstract

Abstract Ewing's sarcoma (ESFT) is a highly aggressive pediatric cancer of the bone and characterized by the chimeric ETS transcription factor EWS-FLI1 as a result of a chromosomal translocation. By whole genome gene expression and ChIP-seq analyses we obtained evidence that EWS-FLI1 directly regulates E2F target genes and possibly cooperates with E2F transcription factors. Furthermore, several E2F factors were themselves inferred to be direct targets of EWS-FLI1. To unravel the hierarchical structure of this EWS-FLI1 transcriptional network module we performed luciferase reporter gene assays in combination with DNA motif mutation analyses. The promoter regions of nine candidates for direct target genes of EWS-FLI1 and E2F factors were chosen for in-depth analysis. Corroborating the transcriptomic data, promoter activity of all 9 genes showed reduced levels upon knockdown of EWS-FLI1. The strongest modulatory effect of EWS-FLI1 silencing was observed for the E2F3 promoter, and mutation of a single ETS binding site lead to a comparable reduction of luciferase activity as knockdown of EWS-FLI1. These results validate E2F3 as a directly EWS-FLI1 regulated gene in Ewing's sarcoma. For the other EWS-FLI1 target candidates mutation analysis lead to more complex results. In the RAD51 promoter simultaneous mutation of two ETS binding sites was necessary to obtain a significant reduction of luciferase activity. Furthermore, mutation of a single E2F binding site abolished the effect of EWS-FLI1 knockdown. In promoters of two other candidates, GEMIN4 and ATAD2, mutation of the ETS site resulted in only a moderate reduction of reporter activity, and additional destruction of an E2F binding site was required to achieve a significant decrease in promoter activity. Our results imply that EWS-FLI1 uses several modes of interaction with E2F factors to control common target genes. First, EWS-FLI1 directly activates E2F3 and other E2F factors which, in turn, co-operate with EWS-FLi1 on target gene promoters in a feed-forward loop. Second, results for the RAD51 promoter are compatible with a mechanism where a repressive E2F factor is exchanged for an activating E2F factor via binding of EWS-FLI1. Finally, EWS-FLI1 might have a more indirect role in the regulation of ATAD2 and GEMIN4 where most of the transcriptional effect seems to be exerted by E2F factors. Acknowledgement: This study was funded by the 6th framework program of the European Commission, (“E.E.T. Pipeline” contract LSHC-CT-2006-037260) and by the Austrian genome research program “GEN-AU Ch.I.L.D.“ (GZ 200.136/1 - VI/1/2005). R. Schwentner is a recipient of a DOC-fFORTE-fellowship of the Austrian Academy of Sciences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4968.