title: Homotypic and heterotypic interactions of EWS, FLI1 and their oncogenic fusion protein.
abstract: In Ewing's sarcoma family tumors, the ets transcription factor gene FLI1 is rearranged with one EWS allele resulting in coexpression of germline EWS and chimeric EWS-FLI1 proteins. Here, we investigated the potential of germline EWS, FLI1 and EWS-FLI1 to oligomerize. In two functional in vivo tests, fluorescence resonance energy transfer (FRET) and the mammalian two-hybrid (MTH) assay, self-association of EWS and EWS-FLI1, but not of FLI1 was detected. In addition, interaction of EWS-FLI1 with EWS and FLI1 was observed. GST pull-down assays and immunoprecipitation experiments largely confirmed these results. The EWS N-terminal domain present in both EWS and EWS-FLI1 was found to contribute to homotypic and heterotypic interactions of these proteins. However, in the context of germline EWS, the presence of the whole or part of the C-terminal RNA-binding domain greatly supported the self-association potential of the protein. Involvement of an RNA component in EWS oligomerization was confirmed by sensitivity of the corresponding GST pull-down assay to RNaseA treatment. In contrast, EWS-FLI1 was able to self-associate and also bind to FLI1 via its C-terminal domain, which comprises the FLI1 DNA-binding motif. Accordingly, the EWS-FLI1 interaction was not disrupted by RNaseA treatment. Despite its potential to oligomerize, EWS-FLI1 bound to a tandem ets-binding site of the TGFbeta type II receptor promoter as a monomer. Therefore, the functional consequences of homo- and hetero-oligomerization of EWS and EWS-FLI1 proteins remain to be elucidated.
authors.affiliation: Children's Cancer Research Institute, St Anna Kinderspital, A-1090 Vienna, Austria.
authors.forename: Laura
authors.initials: L
authors.lastname: Spahn
authors.forename: Christine
authors.initials: C
authors.lastname: Siligan
authors.forename: Radostina
authors.initials: R
authors.lastname: Bachmaier
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Schmid
authors.forename: Dave N T
authors.initials: DN
authors.lastname: Aryee
authors.forename: Heinrich
authors.initials: H
authors.lastname: Kovar
mesh_terms: D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D015514:Oncogene Proteins, Fusion / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D034802:RNA-Binding Protein EWS; D012512:Sarcoma, Ewing / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D004268:DNA-Binding Proteins; C106031:EWS-FLI fusion protein; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D034802:RNA-Binding Protein EWS; D015534:Trans-Activators; D014157:Transcription Factors