Functional confirmation of EWS-FLI1 target genes

EWS-FLI1 represents a paradigm for cancer associated fusion genes that result in the expression of oncogenic chimeric transcription factors. In the preceding project P14299-B04 we cloned genomic DNA from Ewings sarcoma chromatin precipitating with EWS-FLI1 to characterize direct target genes within the authentic cellular context avoiding artificial model systems. We obtained a comprehensive list of 100 putative direct transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction only. One third of genes recovered showed a significant Ewings sarcoma specific expression pattern and were found to be altered upon RNA interference mediated knock-down of EWS-FLI1. So far, only one gene from the list, MK- STYX, has been further validated as a directly EWS-FLI1 regulated gene by in-vitro delineation of the EWS-FLI1 binding site, reporter gene assays, and demonstration of consistent expression in primary Ewings sarcoma. The current project aims at the investigation of the role of MK-STYX activation by EWS-FLI1 for Ewings sarcoma biology and at confirmation of further candidate EWS-FLI1 regulated genes from the list. Characterization of EWS-FLI1 binding sites within their individual specific genomic contexts shall lead to the definition of the architecture of EWS-FLI1 responsive promoters and to the identification of transcription factors that cooperate with EWS-FLI1 to mediate either transcriptional activation or repression of its target genes. By comparing the molecular signatures (as assessed by Affymetrix GeneChip analysis) of confirmed target genes with that of EWS-FLI1 after modulating the expression through either forced expression or RNAi mediated knock-down we will be able to define a hierarchy of genes downstream of the oncogenic transcription factor.

EWS-FLI1 represents a paradigm for cancer associated fusion genes that result in the expression of oncogenic chimeric transcription factors. In the preceding project P14299-B04 we cloned genomic DNA from Ewing`s sarcoma chromatin precipitating with EWS-FLI1 to characterize direct target genes within the authentic cellular context avoiding artificial model systems. We obtained a comprehensive list of 100 putative direct transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction only. One third of genes recovered showed a significant Ewing`s sarcoma specific expression pattern and were found to be altered upon RNA interference mediated knock-down of EWS-FLI1. So far, only one gene from the list, MK- STYX, has been further validated as a directly EWS-FLI1 regulated gene by in-vitro delineation of the EWS-FLI1 binding site, reporter gene assays, and demonstration of consistent expression in primary Ewing`s sarcoma. The current project aims at the investigation of the role of MK-STYX activation by EWS-FLI1 for Ewing`s sarcoma biology and at confirmation of further candidate EWS-FLI1 regulated genes from the list. Characterization of EWS-FLI1 binding sites within their individual specific genomic contexts shall lead to the definition of the architecture of EWS-FLI1 responsive promoters and to the identification of transcription factors that cooperate with EWS-FLI1 to mediate either transcriptional activation or repression of its target genes. By comparing the molecular signatures (as assessed by Affymetrix GeneChip analysis) of confirmed target genes with that of EWS-FLI1 after modulating the expression through either forced expression or RNAi mediated knock-down we will be able to define a hierarchy of genes downstream of the oncogenic transcription factor.