title: SRE elements are binding sites for the fusion protein EWS-FLI-1.
abstract: EWS-FLI-1 is a chimeric protein produced in most Ewing's sarcomas. It results from the fusion of the N-terminal-encoding region of the EWS gene to the C-terminal DNA-binding domain (the ETS domain) encoded by the FLI-1 ets family gene. Both EWS-FLI-1 and FLI-1 proteins function as transcription factors that bind specifically to ets sequences (the ets boxes) present in promoter elements. EWS- FLI-1 is a powerful transforming protein, whereas FLI-1 is not. In a search for potential DNA binding sites for these two proteins, we have tested their ability to recognize the serum responsive element (SRE) in the c-fos promoter. This cis element contains an ets box which can be occupied by members of the ETS protein family which do not bind DNA autonomously but form a ternary complex with a second protein, p67SRF (serum responsive factor). We demonstrate here that EWS-FLI-1, but not FLI-1, is able to form a ternary complex on the c-fos SRE. Using a GST pull-down assay, we show that both FLI-1 and EWS-FLI-1 interact in vitro with SRF in the absence of DNA. In electromobility shift assays, EWS-FLI-1 binding to the SRE is detectable in the absence of SRF whereas the binding of FLI-1 is not, suggesting that the interaction with DNA is the step which limits ternary complex formation by FLI-1. Deletion of the N-terminal portion of FLI-1 resulted in a protein which behaved as EWS-FLI-1, suggesting the existence of an N- terminal inhibitory domain in the normal protein. Taken together, our data indicate that there are intrinsic differences in the binding of EWS-FLI-1 and FLI-1 proteins to distinct ets sequences.
authors.affiliation: Laboratoire de Biologie des Tumeurs Humaines, CNRS URA 1156, Villejuif, France.
authors.forename: L
authors.initials: L
authors.lastname: Magnaghi-Jaulin
authors.forename: H
authors.initials: H
authors.lastname: Masutani
authors.forename: P
authors.initials: P
authors.lastname: Robin
authors.forename: M
authors.initials: M
authors.lastname: Lipinski
authors.forename: A
authors.initials: A
authors.lastname: Harel-Bellan
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016762:Genes, fos; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins / Q000235:genetics; D050783:Proto-Oncogene Proteins c-ets; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D015534:Trans-Activators; D014157:Transcription Factors