Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

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 twohybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that speci®cally interacts with the amino terminus of EWS. This association was con®rmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 speci®cally 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 in¯uence promoter selectivity.

The RNA Pol II sub-complex hsRpb4/7 is required for viability of multiple human cell lines

“…cerevisiae (sc) Rpb4/7 form a dissociable complex with Pol II (Edwards et al, 1991;Khazak et al, 1998) and scRpb4/7 activates promoter-specific transcription in vitro (Edwards et al, 1991;Jensen et al, 1998). The Rpb4/7 sub-complex also interacts with general transcription factors TFIIB and TFIIF (Bushnell and Kornberg, 2003;Chung et al, 2003), transcriptional activator proteins (Bertolotti et al, 1998;Petermann et al, 1998;Shen et al, 1999;Todorova, 2009) and the Pol II CTD phosphatase Fcp1 (Kimura et al, 2002;Kamenski et al, 2004). Intriguingly non-transcriptional functions of Rpb4/7 have also emerged (Choder, 2004;Sampath and Sadhale, 2005).…”

“…The von Hippel-Lindau (VHL) tumor suppressor protein (Na et al, 2003) directly interacts with and destabilises hsRpb7 via the ubiquitin-proteasome pathway. hsRpb7 also interacts physically (Bertolotti et al, 1998;Petermann et al, 1998;Todorova, 2009) and functionally (Zhou and Lee, 2001) with Ewing's family oncoproteins (Romeo and Dei Tos, 2010;Kovar, 2011) and may play a role in the Ewing's family of tumors. It is therefore of significance to understand the role of hsRpb4/7 in mammalian cell proliferation and survival.…”

A repetitive element containing a critical tyrosine residue is required for transcriptional activation by the EWS/ATF1 oncogene

“…For DHRs 1 ± 4, amino acid substitutions in each position relative to consensus are summarized (box marked DHRs (Z33)) other regions of the EAD (Pan et al, 1998). Second, hsRPB7 is known to bind to residues 1 ± 82 of the EAD (Petermann et al, 1998) and hsRPB7 is required for EAD-mediated trans-activation (Zhou and Lee, 2001). …”

“…Second, multiple dispersed elements are required for full EAD activity (Pan et al, 1998;Lessnick et al, 1995;Kim et al, 1998). Third, binding of the EAD to the RNA polymerase II subunit hsRPB7 (Petermann et al, 1998;Bertolotti et al, 1998) and the correlation between hsRPB7 binding and trans-activation (Li and Lee, 2000), suggested an important role for hsRPB7. Such a role has recently been demonstrated directly using a yeast assay for EAD-mediated trans-activation (Zhou and Lee, 2001).…”

RNA splicing mediated by YB‐1 is inhibited by TLS/CHOP in human myxoid liposarcoma cells

“…The mechanism by which these fusion proteins induce cellular transformation has been thought to be aberrant transcriptional activation. Supporting this hypothesis is the fact that the N-terminal domains of TLS, EWS and TAFI168 are known to associate with components of the RNA Pol I1 complex [7,8,28,38]. Furthermore, the N-terminus of TLS is rich in glutamine, tyrosinc, and serine residues that are commonly found in transcriptional activators [8,29].…”