Homotypic and heterotypic interactions of EWS, FLI1 and their oncogenic fusion protein.
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.
Spahn
Laura
L
Children's Cancer Research Institute, St Anna Kinderspital, A-1090 Vienna, Austria.
Siligan
Christine
C
Bachmaier
Radostina
R
Schmid
Johannes A
JA
Aryee
Dave N T
DN
Kovar
Heinrich
H
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
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