In-situ identification of target genes for the oncogenic EWS-FLI1 transcription factor in its authentic cellular milieu

Research project P 14299 EWS-FLI1 target genes Heinrich KOVAR 06.03.2000 Dysregulation of many transcription factors is associated with the development of human neoplasia. Transcription factors regulate cell growth, differentiation, and apoptosis by binding to specific DANN sequences within the promoter regions of growth-regulatory genes and modulating expression of these genes. The EWS-FLI1 aberrant transcription factor, specifically expressed in Ewing`s sarcoma family tumors (EFT) as a result of chromosomal rearrangement, belongs to a large protein family that regulate expression of overlapping or unique sets of genes. Here, we propose to isolate EWS-FLI1 bound sequences in the context of EFT chromatin. In addition, we suggest to follow changes in EFT gene expression profiles when EWS-FLI1 expression is inducibly i) enforced or ii) repressed. These two complementary approaches should allow cellular milieu. Also, by studying different EWS- FLI1 fusion types it should be possible to pinpoint target genes, whose expression may correlate to the clinically observed disparate outcome of disease, and which may therefore be of prognostic relevance.

In this project we isolated for the first time genes directly regulated by the tumor specific fusion protein EWS-FLI1 from Ewings sarcoma chromatin based on physical in-vivo interaction only. Ewings sarcoma family tumors (ESFT) constitute a group of highly aggressive tumors affecting bone and soft tissues in children and young adults. The tissue of origin is not known but limited neural differentiation has so far been considered as evidence for a neural descent of this desease. ESFT are genetically characterized by a specific chromosomal rearrangement that leads to the expression of a fusion gene, EWS-FLI1, whose product binds to DNA and compromises gene regulation. Consequently, the altered activity of EWS-FLI1 regulated genes is considered responsible for the malignant behavior of the tumor cells and considerable interest exists in the identification of these genes as potential targets for novel treatment strategies. In this project we therefore aimed at the isolation of directly EWS- FLI1 regulated genes from ESFT cells. We optimized a physical method to precipitate genomic DNA bound to EWS-FLI1 in-vivo and used this technique to clone genes in an unbiased approach. For functional validation, an RNA interference strategy was developed to modulate EWS-FLI1 in ESFT cells and follow associated changes in the expression of cloned genes. Ninety nine genes were identified of which one third showed altered EWS-FLI1 dependent expression in ESFT cell lines. These genes were found to be non-randomly distributed in the genome, many of them clustering on chromosomes 3 and 8. EWS-FLI1 binding sites were predominantly identified in the gene regions assumed to serve gene regulatory function. Importantly, a significant number of the identified genes are involved in neural development suggesting that the partial neural differentiation of ESFT cells is a consequence of the EWS-FLI1 gene fusion and not a feature of the ESFT progenitor cell as previously assumed. When gene expression profiles of ESFT were compared to those of other childhood tumors, most of the isolated genes displayed an ESFT specific pattern. However, analysis of several ESFT cell lines revealed that binding of EWS- FLI1 to its target genes is not sufficient for aberrant gene regulation and, for a subset of EWS-FLI1 bound genes, inter-individual variation has been observed in the transcriptional response. The consequences of EWS-FLI1 binding to one of the isolated genes assumed to play a role in intracellular signal transduction was studied in great detail and the architecture of a bona-fide EWS-FLI1 binding site was defined. Two further genes from our screen were shown to be involved in growth regulation of ESFT cells. This study represents a precedent for the successful isolation of genes associated with an oncogenic DNA binding protein from its authentic cellular context.