An hsRPB4/7-dependent yeast assay for trans-activation by the EWS oncogene

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Abstract
Chromosomal fusions of the N-terminal region of the Ewings Sarcoma Oncogene (EWS-Activation-Domain, EAD) to the DNA-binding domains of a variety of cellular transcription factors, produce oncogenic proteins (EWS-fusion proteins (EFPs)) that cause a variety of malignancies. The EAD can act as a potent transcriptional activation domain and is required for the oncogenic activity of EFPs. Previous studies demonstrating a physical interaction between the EAD and the human RNA Polymerase II subunit hsRPB7 suggest a crucial role for RPB7 and its partner, RPB4, in EAD function. Homologues of hsRPB4/7 exist in S. cerevisiae, and here we describe an RPB4/7-dependent yeast assay for EAD-mediated trans-activation. Conditional yeast strains lacking RPB4 are defective for trans-activation by a Gal4/EAD fusion protein at the permissive temperature. Introduction of hsRPB4 alone is unable to rescue trans-activation, while a combination of hsRPB4 and hsRPB7 significantly rescues activity. These findings provide the first functional evidence for a direct role of the RPB4/7 complex in EAD-mediated trans-activation. In addition, the yeast assay provides a tractable system for further molecular analysis of EAD and RPB4/7 action.