EWS fusion proteins (EFPs) caused by chromosomal translocations contain a transcriptional activation domain from EWS (EWS Activation Domain, EAD) and DNA binding domains of a variety of transcription factors. EFPs function as oncogenes and most likely, tumorigenesis arises via transcriptional de-regulation by EFPs. Studies on EAD action are therefore important for understanding trans-activation and oncogenesis....[ Read more ]

EWS fusion proteins (EFPs) caused by chromosomal translocations contain a transcriptional activation domain from EWS (EWS Activation Domain, EAD) and DNA binding domains of a variety of transcription factors. EFPs function as oncogenes and most likely, tumorigenesis arises via transcriptional de-regulation by EFPs. Studies on EAD action are therefore important for understanding trans-activation and oncogenesis.

The mechanism of trans-activation by the EAD is beginning to emerge. One subunit of human pol II, hsRPB7, interacts directly with the EAD, suggesting that hsRPB7 plays a role in trans-activation. hsRPB7 directly interacts with another subunit hsRPB4 to form a dissociable sub-complex of pol II. By exploiting yeast as a genetically manipulatable organism together with a yeast transcriptional assay for the EAD, I have probed the role of RPB4 and RPB7 in EAD action. Firstly, my results showed that trans-activation by the EAD is dependent on RPB4. Secondly, hsRPB4 could cooperate with hsRPB7 to support trans-activation, showing that RPB7 is also required. hsRPB4, however, could not cooperate with scRPB7 in trans-activation. These functional data correlate well with previously shown interactions between RPB4 and RPB7, strongly suggesting that RPB4 and RPB7 function as a complex in EAD-mediated trans-activation. Thirdly, RPB7 subunits from three other species could cooperate with scRPB4 to support trans-activation, and fusion of only 17 aa from the N-terminus of scRPB4 to hsRPB4 enabled hsRPB4 to functionally cooperate with scRPB7, indicating a strong structural and functional conservation of RPB4 and RPB7 subunits from humans to yeast. Finally, promoter-bound hsRPB7 activates transcription to a level comparable to the EAD, suggesting interaction of the EAD with RPB7 is sufficient to recruit the general transcription machinery to the promoter and activate transcription.

Overall, my results provide functional evidence for the role of RPB4 and RPB7 in EAD action. Besides, my results demonstrate that direct communication between a transcriptional activator and RNA polymerase is a possible transcription mechanism in a eukaryotic system and that RPB4/7 may be the direct targets of natural yeast and mammalian activators.