EWS, but Not EWS-FLI-1, Is Associated with Both TFIID and RNA Polymerase II: Interactions between Two Members of the TET Family, EWS and hTAF_II68, and Subunits of TFIID and RNA Polymerase II Complexes

Abstract

The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAF II 68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF II s). In vitro binding studies revealed that both EWS and hTAF II 68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAF II 68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAF II 68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAF II 68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS-FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS-FLI-1 may play different roles in Pol II transcription.Structural alteration or aberrant expression of transcription factors is often a critical event in tumorigenic transformation (13,19, 22). Karyotypic analysis has revealed a tumor-specific t(11;22)(q24;q12) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor, suggesting that the product of this rearrangement is involved in the formation of these malignancies (34). This chromosomal translocation fuses the EWS gene on chromosome 22 to the FLI-1 gene on chromosome 11 (8). EWS is a protein with unknown function containing an RNA-binding motif and an activation domain(s) (18,24,25). In the EWS-FLI-1 fusion protein, the RNA-binding motif containing the C-terminal half of EWS is replaced by the DNA-binding domain (DBD) of the FLI-1 protein. FLI-1 is a member of the ETS family of transcription factors which activate specific target genes by binding to their cognate DNA sequences through their DNA-binding regions, usually located at their carboxyl termini (2, 37). The replacement of the native transcription activation domain(s) of FLI-1 by the N-terminal region of EWS converts the nontransforming activator, FLI-1, into a transforming protein with new transcriptional activation potential. In the EWS-FLI-1 fusion protein, both the N-terminal domain of EWS and the DBD of FLI-1 are necessary for the transforming activity (20). Recently, the EWS gene was also shown to be involved in tumorigenesis by chromosomal translocation with other genes encoding either other members of the ETS family (Erg, ETV1, E1A-F, and FEV) or other transcription factors, including ATF-1, WT1, and the nuclear orphan receptor TEC1 (...

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

“…In experiments using a carboxy terminus-speci®c antibody to precipitate germline EWS we were unable to see selective coprecipitation of hsRPB7. In accordance with our ®ndings, Tora and coworkers demonstrated recently that only trucated EWS proteins lacking the Cterminus, as is the case in chimeric EWS-FLI1 molecules, were able to associate with hsRPB7 (Bertolotti et al, 1998). Presumably, fusion to Fli1 causes as structural change of the amino terminal EWS domain making it accessible for interaction with hsRPB7.…”

“…Speci®cally hTAF II 68 and TLS have recently been shown to localize within the TFIID complex and evidence obtained by Tora and coworkers favours the idea that EWS can also be found as part of this general transcription factor (Bertolotti et al, 1996). However, in contrast to germline EWS, EWS-Fli1 does not appear to be stably associated with high molecular weight TFIID and RNA polymerase II complexes (Bertolotti et al, 1998). In concordance with Bertolotti's ®ndings, hsRPB7 was the only component of these transcription factors cloned in our two hybrid screen using the EWS amino terminus as a bait.…”

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.…”

Adenovirus-mediated p53 gene therapy in pediatric soft-tissue sarcoma cell lines: sensitization to cisplatin and doxorubicin

“…24,25 Both translocations involve the EWS transcription factor gene. 26,27 The gene fusion product of t (11;22) is EWS-FLI1, a gene with potent transforming capability in NIH3T3 cells. 28 p53 alterations in ES are not common (approximately 10%) but are associated with poor outcomes.…”