ABSTRACT
As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast two-hybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that specifically interacts with the amino terminus of EWS. This association was confirmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 specifically increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may influence promoter selectivity.
Subject(s)
Gelsolin , Oncogene Proteins, Fusion/metabolism , RNA Polymerase II/metabolism , Receptors, Cytoplasmic and Nuclear , Transcription Factors/metabolism , 3T3 Cells , Animals , Artificial Gene Fusion , Binding Sites , Cloning, Molecular , Gene Expression , Humans , Mice , Microfilament Proteins , Nucleic Acid Hybridization , Oncogene Proteins, Fusion/genetics , Proteins/genetics , Proto-Oncogene Protein c-fli-1 , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , RNA-Binding Protein EWS , Saccharomyces cerevisiae , Sarcoma, Ewing , Trans-Activators , Transcription Factors/genetics , Transcriptional Activation , Tumor Cells, CulturedABSTRACT
The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is assayed in parallel for combinations with different DNA-binding domain hybrids. Further characterization of inserts does not require plasmid isolation and intermediate hosts.
