Oncogene (1998) 16, 1625 ± 1631 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 The EWS/ATF1 fusion protein contains a dispersed activation domain that functions directly Shu Pan, Koh Yee Ming, Theresa A Dunn, Kim KC Li and Kevin AW Lee Department of Biology, Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, P.R.C. Naturally occurring chromosomal fusion of the Ewings 1994). For all of the above malignancies, the EWS Sarcoma Oncogene (EWS) to distinct cellular transcrip- fusion proteins function as potent transcriptional tion factors, produces aberrant transcriptional activators activators (May et al., 1993b; Ohno et al., 1993; that function as dominant oncogenes. In Malignant Bailly et al., 1994; Brown et al., 1995; Lessnick et al., Melanoma of Soft Parts the N-terminal region of 1995; Fujimura et al., 1996) in a manner that is EWS is fused to C-terminal region of the cAMP- dependent on the EWS N-terminal region, hereafter inducible transcription factor ATF1. The EWS/ATF1 referred to as the EWS Activation Domain (EAD). It is fusion protein binds to ATF sites present in cAMP- envisioned that distinct tumors arise via de-regulation responsive promoters via the ATF1 bZIP domain and of dierent genes, depending on the fusion partner for activates transcription constitutively in a manner that is EWS. In cases where it has been examined, agents that dependent on an activation domain (EAD) present in antagonise EWS-fusion proteins also inhibit cellular EWS. To further de®ne the requirements for trans- proliferation (Ouchida et al., 1995; Kovar et al., 1996; activation we have performed mutational analysis of Yi et al., 1997; Tanaka et al., 1997), indicating that EWS/ATF1 in mammalian cells and report several new EWS fusions can play a role in both tumor formation ®ndings. First, trans-activation by EWS/ATF1 does not and maintenance. The molecular mechanism by which require dimerisation with other ATF family members EWS activates transcription is therefore of signi®cance, present in mammalian cells. Second, in contrast to the both for understanding tumorigenesis and for develop- earlier suggestion of an allosteric role, the EAD can act ment of potential therapeutic agents that target EWS. directly. Third, determinants of trans-activation are The mechanism(s) by which the EAD alters the dispersed throughout the EAD and cooperate synergis- activity of oncogenic fusion proteins is not well tically to produce potent trans-activation. We also report characterized at the molecular level. To date there is that the region of EWS containing the EAD can activate evidence that the EAD can increase transcriptional transcription in Yeast. This latter ®nding might enable a activity via both allosteric and direct mechanisms. genetic approach to understanding the mechanism of Studies of the native oncogenic fusion proteins EWS/ transcriptional activation by EWS and development of FLI1 (Ohno et al., 1993) and EWS/ATF1 (Fujimura et high-throughput screens for EWS inhibitors. al., 1996) have suggested an allosteric role, while fusion of EWS or the EAD (or the related protein TLS/FUS) Keywords: EWS/ATF1; oncogene; MMSP; transcrip- to GAL4 has indicated that the EAD contains an tional activation; yeast activation domain that functions directly (May et al., 1993b; Bailly et al., 1994; Sanchez-Garcia and Rabbitts, 1994; Lessnick et al., 1995; Kim et al., 1997). EWS/ATF1 is a much more potent activator Introduction than EWS/FLI1 when compared with their corre- sponding non-tumorigenic counterparts, ATF and FLI Chromosomal translocations involving fusion of the N- 1 respectively. EWS/ATF1 is *200-fold more active terminal region of the Ewings Sarcoma Oncogene than ATF1 (Brown et al., 1995) whereas EWS/FLI1 is (EWS) to a variety of cellular transcription factors, only 5 ± 10-fold more active than FLI1. The potency of produce dominant oncogenes that cause distinct trans-activation by EWS/ATF1 therefore oers advan- sarcomas (reviewed by Rabbitts, 1994; Ladanyi, tages for transcriptional studies of native oncogenic 1995). In Malignant Melanoma of Soft Parts fusion proteins containing EWS and possibly for (MMSP) a causative t(12;22) chromosomal transloca- examining the function of the normal EWS protein. tion gives rise to a fusion protein (EWS/ATF1, Figure Important functions of EWS/ATF1 are contributed 1) in which EWS is fused to the C-terminal region of by both fusion partners (Figure 1). ATF1 binds the cellular transcription factor ATF1 (Zucman et al., directly to cAMP-inducible promoters via the bZIP 1993a). In Ewings sarcoma, EWS is fused to the ETS domain (Hurst et al., 1991; Lee and Masson, 1993) and domain family members FLI1 (Delattre et al., 1992; activates transcription upon phosphorylation by PKA May et al., 1993a; Zucman et al., 1993b) or ERG1 (Ribeiro et al., 1994). The function of the normal EWS (Sorenson et al., 1994) and in Desmoplastic Small protein is not well characterised but it contains an Round Cell Tumor (DSRCT) EWS is fused to the RNA-binding domain at its C-terminus (Burd and Wilms tumor oncogene WT1 (Ladanyi and Gerald, Dreyfuss, 1994) suggesting that it plays a role in some aspect of RNA metabolism. However, recent ®ndings have revealed a high degree of homology between EWS and the human TBP-associated factor (hTAF Correspondence: KAW Lee ll68, Received 26 August 1997; revised 24 October 1997; accepted 24 Bertolotti et al., 1996) suggesting that EWS may October 1997 function directly as a transcription factor. In contrast Transcriptional activation by the EWS/ATF1 oncogene SPanet al 1626 to ATF1, EWS/ATF1 functions as a potent constitu- ATF1 does not activate promoters that do not contain tive activator of several PKA-inducible promoters ATF binding sites (Brown et al., 1995). (Brown et al., 1995; Fujimura et al., 1996) and Similar to the trans-activation domains present in activation is strictly dependent on the EAD (Brown many transcriptional activators, the EAD is rich in et al., 1995; Fujimura et al., 1996). With respect to proline, glutamine and serine/threonine residues and promoter speci®city, it has been shown that (1) EWS/ has thirty one copies of a consensus repeat SYGQQS ATF1 can activate a broad range of ATF-dependent (Delattre et al., 1992). The important structural promoters; (2) activation is dependent on the ATF features of the EAD and its mechanism of action binding site in the promoter; (3) EWS/ATF1 does not have not been thoroughly investigated, although it has activate all ATF-dependent promoters and (4) EWS/ been suggested that, for EWS/ATF1, the EAD plays a Figure 1 Functional regions of EWS/ATF1 and summary of EWS/ATF1 mutants. EWS/ATF1 contains the N-terminal region of EWS (residues 1 ± 325) and the C-terminal region of ATF1 (residues 66 ± 271). The EWS region present has a repetitive primary structure, with several prevalent amino acids (serine and threonine (*25%), proline (*10%), glutamine (*15%) and tyrosine (*10%)) dispersed evenly throughout. In addition, there are 31 copies of a repeat sequence, with the consensus SYGQQS, dispersed evenly throughout. EWS/ATF1 contains the C-terminal 75% ATF1 (residues 66 ± 271) but lacks the PKA phospho-acceptor site and does not function as a PKA-inducible activator. The bZIP domain (aa 214 ± 271) is necessary and sucient for dimerisation and DNA-binding and consists of the basic region (b) that directly contacts DNA and the leucine zipper (ZIP) that allows dimerisation. Q2 represents a glutamine-rich activation domain that has constitutive transcriptional activity (Brindle et al., 1993). For EWS/ATF1 mutants, the residues present are aligned with the intact protein at the top of the Figure and deletions are indicated with a dashed line. N-terminal deletions are named according to the number of EWS residues deleted and C-terminal deletions according to the number of EWS residues remaining. The EWS is represented by the striped box and the N-terminal 86 residues in D87C, D87CD and E(1 ± 87) by a shaded box. The black box in 4R and 8R represents one copy of the SYGQQS repeat sequence. Trans-activation assays in JEG3 cells were as described in Materials and methods. Reporter activity is CAT speci®c activity as determined by trans- activation of D(771)SomCat in the linear range for trans-activation and corrected for protein expression levels as determined by Western blot analysis of epitope-tagged proteins in transfected cells. For quantitation as %WT, the background activity (bkg) corresponds to the activity of D325 Transcriptional activation by the EWS/ATF1 oncogene SPanet all 1627 regulatory as opposed to a direct role in trans- Previous mutational analysis of EWS/ATF1 (Brown activation (Fujimura et al., 1996). Here we provide et al., 1995; Fujimura et al., 1996) demonstrated that evidence that the EAD can act directly but that the deletion of the N-terminal 78 residues of the EAD determinants of trans-activation are dispersed. Many strongly reduced activity (Figure 1) suggesting that this regions of the EAD have low activity by themselves region might be sucient for EAD function. We but cooperate synergistically to produce ecient trans- therefore tested this region fused to ATF1 residues activation. In addition, trans-activation by EWS/ATF1 66 ± 271 (D87C contains the N-terminal 86 residues of does not require dimerisation with endogenous EWS) for trans-activation and found that D87C gives partners of ATF1. We also show that the EAD can 80-fold higher activity than D325, while the N-terminal activate transcription in yeast. This latter ®nding might 86 residues of EWS by itself (E(1 ± 87)) has no activity enable a genetic approach to study the mechanism of (Figure 2). Thus the N-terminal 86 residues of the transcriptional activation by EWS and allow a high EAD fused to ATF1 has signi®cant activity.