• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. May 1994; 14(5): 3230–3241.
PMCID: PMC358690

DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma.

Abstract

The 5' half of the EWS gene has recently been described to be fused to the 3' regions of genes encoding the DNA-binding domain of several transcriptional regulators, including ATF1, FLI-1, and ERG, in several human tumors. The most frequent occurrence of this situation results from the t(11;22)(q24;q12) chromosome translocation specific for Ewing sarcoma (ES) and related tumors which joins EWS sequences to the 3' half of FLI-1, which encodes a member of the Ets family of transcriptional regulators. We show here that this chimeric gene encodes an EWS-FLI-1 nuclear protein which binds DNA with the same sequence specificity as the wild-type parental FLI-1 protein. We further show that EWS-FLI-1 is an efficient sequence-specific transcriptional activator of model promoters containing FLI-1 (Ets)-binding sites, a property which is strictly dependent on the presence of its EWS domain. Comparison of the properties of the N-terminal activation domain of FLI-1 to those of the EWS domain of the fusion protein indicates that EWS-FLI-1 has altered transcriptional activation properties compared with FLI-1. These results suggest that EWS-FLI-1 contributes to the transformed phenotype of ES tumor cells by inducing the deregulated and/or unscheduled activation of genes normally responsive to FLI-1 or to other close members of the Ets family. ES and related tumors are characterized by an elevated level of c-myc expression. We show that EWS-FLI-1 is a transactivator of the c-myc promoter, suggesting that upregulation of c-myc expression is under control of EWS-FLI-1.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Allison LA, Wong JK, Fitzpatrick VD, Moyle M, Ingles CJ. The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function. Mol Cell Biol. 1988 Jan;8(1):321–329. [PMC free article] [PubMed]
  • Bartolomei MS, Halden NF, Cullen CR, Corden JL. Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II. Mol Cell Biol. 1988 Jan;8(1):330–339. [PMC free article] [PubMed]
  • Ben-David Y, Giddens EB, Letwin K, Bernstein A. Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1. Genes Dev. 1991 Jun;5(6):908–918. [PubMed]
  • Bentley DL, Groudine M. Sequence requirements for premature termination of transcription in the human c-myc gene. Cell. 1988 Apr 22;53(2):245–256. [PubMed]
  • Beug H, Hayman MJ, Graf T. Myeloblasts transformed by the avian acute leukemia virus E26 are hormone-dependent for growth and for the expression of a putative myb-containing protein, p135 E26. EMBO J. 1982;1(9):1069–1073. [PMC free article] [PubMed]
  • Bhat NK, Fisher RJ, Fujiwara S, Ascione R, Papas TS. Temporal and tissue-specific expression of mouse ets genes. Proc Natl Acad Sci U S A. 1987 May;84(10):3161–3165. [PMC free article] [PubMed]
  • Bosselut R, Duvall JF, Gégonne A, Bailly M, Hémar A, Brady J, Ghysdael J. The product of the c-ets-1 proto-oncogene and the related Ets2 protein act as transcriptional activators of the long terminal repeat of human T cell leukemia virus HTLV-1. EMBO J. 1990 Oct;9(10):3137–3144. [PMC free article] [PubMed]
  • Boulukos KE, Pognonec P, Rabault B, Begue A, Ghysdael J. Definition of an Ets1 protein domain required for nuclear localization in cells and DNA-binding activity in vitro. Mol Cell Biol. 1989 Dec;9(12):5718–5721. [PMC free article] [PubMed]
  • Boulukos KE, Pognonec P, Sariban E, Bailly M, Lagrou C, Ghysdael J. Rapid and transient expression of Ets2 in mature macrophages following stimulation with cMGF, LPS, and PKC activators. Genes Dev. 1990 Mar;4(3):401–409. [PubMed]
  • Brown TA, McKnight SL. Specificities of protein-protein and protein-DNA interaction of GABP alpha and two newly defined ets-related proteins. Genes Dev. 1992 Dec;6(12B):2502–2512. [PubMed]
  • Burtis KC, Thummel CS, Jones CW, Karim FD, Hogness DS. The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell. 1990 Apr 6;61(1):85–99. [PubMed]
  • Courey AJ, Tjian R. Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell. 1988 Dec 2;55(5):887–898. [PubMed]
  • Crozat A, Aman P, Mandahl N, Ron D. Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature. 1993 Jun 17;363(6430):640–644. [PubMed]
  • Dalton S, Treisman R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell. 1992 Feb 7;68(3):597–612. [PubMed]
  • Delattre O, Zucman J, Plougastel B, Desmaze C, Melot T, Peter M, Kovar H, Joubert I, de Jong P, Rouleau G, et al. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature. 1992 Sep 10;359(6391):162–165. [PubMed]
  • Dynlacht BD, Hoey T, Tjian R. Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation. Cell. 1991 Aug 9;66(3):563–576. [PubMed]
  • Fisher RJ, Mavrothalassitis G, Kondoh A, Papas TS. High-affinity DNA-protein interactions of the cellular ETS1 protein: the determination of the ETS binding motif. Oncogene. 1991 Dec;6(12):2249–2254. [PubMed]
  • Fujiwara S, Fisher RJ, Bhat NK, Diaz de la Espina SM, Papas TS. A short-lived nuclear phosphoprotein encoded by the human ets-2 proto-oncogene is stabilized by activation of protein kinase C. Mol Cell Biol. 1988 Nov;8(11):4700–4706. [PMC free article] [PubMed]
  • Gegonne A, Punyammalee B, Rabault B, Bosselut R, Seneca S, Crabeel M, Ghysdael J. Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein. New Biol. 1992 May;4(5):512–519. [PubMed]
  • Gégonne A, Bosselut R, Bailly RA, Ghysdael J. Synergistic activation of the HTLV1 LTR Ets-responsive region by transcription factors Ets1 and Sp1. EMBO J. 1993 Mar;12(3):1169–1178. [PMC free article] [PubMed]
  • Ghysdael J, Gegonne A, Pognonec P, Dernis D, Leprince D, Stehelin D. Identification and preferential expression in thymic and bursal lymphocytes of a c-ets oncogene-encoded Mr 54,000 cytoplasmic protein. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1714–1718. [PMC free article] [PubMed]
  • Ghysdael J, Yaniv M. Nuclear oncogenes. Curr Opin Cell Biol. 1991 Jun;3(3):484–491. [PubMed]
  • Gitlin SD, Bosselut R, Gégonne A, Ghysdael J, Brady JN. Sequence-specific interaction of the Ets1 protein with the long terminal repeat of the human T-lymphotropic virus type I. J Virol. 1991 Oct;65(10):5513–5523. [PMC free article] [PubMed]
  • Golay J, Introna M, Graf T. A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation. Cell. 1988 Dec 23;55(6):1147–1158. [PubMed]
  • Goldberg Y, Glineur C, Gesquière JC, Ricouart A, Sap J, Vennström B, Ghysdael J. Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein. EMBO J. 1988 Aug;7(8):2425–2433. [PMC free article] [PubMed]
  • Graham R, Gilman M. Distinct protein targets for signals acting at the c-fos serum response element. Science. 1991 Jan 11;251(4990):189–192. [PubMed]
  • Gunther CV, Nye JA, Bryner RS, Graves BJ. Sequence-specific DNA binding of the proto-oncoprotein ets-1 defines a transcriptional activator sequence within the long terminal repeat of the Moloney murine sarcoma virus. Genes Dev. 1990 Apr;4(4):667–679. [PubMed]
  • Hipskind RA, Rao VN, Mueller CG, Reddy ES, Nordheim A. Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF. Nature. 1991 Dec 19;354(6354):531–534. [PubMed]
  • Klemsz MJ, McKercher SR, Celada A, Van Beveren C, Maki RA. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncogene. Cell. 1990 Apr 6;61(1):113–124. [PubMed]
  • Lai ZC, Rubin GM. Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell. 1992 Aug 21;70(4):609–620. [PubMed]
  • Laimins LA, Gruss P, Pozzatti R, Khoury G. Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus. J Virol. 1984 Jan;49(1):183–189. [PMC free article] [PubMed]
  • LaMarco K, Thompson CC, Byers BP, Walton EM, McKnight SL. Identification of Ets- and notch-related subunits in GA binding protein. Science. 1991 Aug 16;253(5021):789–792. [PubMed]
  • Langer SJ, Bortner DM, Roussel MF, Sherr CJ, Ostrowski MC. Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression. Mol Cell Biol. 1992 Dec;12(12):5355–5362. [PMC free article] [PubMed]
  • Leprince D, Gegonne A, Coll J, de Taisne C, Schneeberger A, Lagrou C, Stehelin D. A putative second cell-derived oncogene of the avian leukaemia retrovirus E26. Nature. 1983 Nov 24;306(5941):395–397. [PubMed]
  • Lillie JW, Green MR. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. [PubMed]
  • Lim F, Kraut N, Framptom J, Graf T. DNA binding by c-Ets-1, but not v-Ets, is repressed by an intramolecular mechanism. EMBO J. 1992 Feb;11(2):643–652. [PMC free article] [PubMed]
  • Lipp M, Schilling R, Wiest S, Laux G, Bornkamm GW. Target sequences for cis-acting regulation within the dual promoter of the human c-myc gene. Mol Cell Biol. 1987 Apr;7(4):1393–1400. [PMC free article] [PubMed]
  • Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. [PubMed]
  • May WA, Gishizky ML, Lessnick SL, Lunsford LB, Lewis BC, Delattre O, Zucman J, Thomas G, Denny CT. Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5752–5756. [PMC free article] [PubMed]
  • McKeon C, Thiele CJ, Ross RA, Kwan M, Triche TJ, Miser JS, Israel MA. Indistinguishable patterns of protooncogene expression in two distinct but closely related tumors: Ewing's sarcoma and neuroepithelioma. Cancer Res. 1988 Aug 1;48(15):4307–4311. [PubMed]
  • Mermod N, O'Neill EA, Kelly TJ, Tjian R. The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell. 1989 Aug 25;58(4):741–753. [PubMed]
  • Metz T, Graf T. v-myb and v-ets transform chicken erythroid cells and cooperate both in trans and in cis to induce distinct differentiation phenotypes. Genes Dev. 1991 Mar;5(3):369–380. [PubMed]
  • Metz T, Graf T. Fusion of the nuclear oncoproteins v-Myb and v-Ets is required for the leukemogenicity of E26 virus. Cell. 1991 Jul 12;66(1):95–105. [PubMed]
  • Moreau-Gachelin F, Ray D, Mattei MG, Tambourin P, Tavitian A. The putative oncogene Spi-1: murine chromosomal localization and transcriptional activation in murine acute erythroleukemias. Oncogene. 1989 Dec;4(12):1449–1456. [PubMed]
  • Moreau-Gachelin F, Tavitian A, Tambourin P. Spi-1 is a putative oncogene in virally induced murine erythroleukaemias. Nature. 1988 Jan 21;331(6153):277–280. [PubMed]
  • Nonet M, Sweetser D, Young RA. Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II. Cell. 1987 Sep 11;50(6):909–915. [PubMed]
  • Nunn MF, Hunter T. The ets sequence is required for induction of erythroblastosis in chickens by avian retrovirus E26. J Virol. 1989 Jan;63(1):398–402. [PMC free article] [PubMed]
  • Nunn MF, Seeburg PH, Moscovici C, Duesberg PH. Tripartite structure of the avian erythroblastosis virus E26 transforming gene. Nature. 1983 Nov 24;306(5941):391–395. [PubMed]
  • Nye JA, Petersen JM, Gunther CV, Jonsen MD, Graves BJ. Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif. Genes Dev. 1992 Jun;6(6):975–990. [PubMed]
  • Pognonec P, Boulukos KE, Gesquière JC, Stéhelin D, Ghysdael J. Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins. EMBO J. 1988 Apr;7(4):977–983. [PMC free article] [PubMed]
  • Pognonec P, Boulukos KE, Ghysdael J. The c-ets-1 protein is chromatin associated and binds to DNA in vitro. Oncogene. 1989 Jun;4(6):691–697. [PubMed]
  • Pongubala JM, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML. PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity. Mol Cell Biol. 1992 Jan;12(1):368–378. [PMC free article] [PubMed]
  • Pugh BF, Tjian R. Transcription from a TATA-less promoter requires a multisubunit TFIID complex. Genes Dev. 1991 Nov;5(11):1935–1945. [PubMed]
  • Rao VN, Reddy ES. A divergent ets-related protein, elk-1, recognizes similar c-ets-1 proto-oncogene target sequences and acts as a transcriptional activator. Oncogene. 1992 Jan;7(1):65–70. [PubMed]
  • Reddy ES, Rao VN. erg, an ets-related gene, codes for sequence-specific transcriptional activators. Oncogene. 1991 Dec;6(12):2285–2289. [PubMed]
  • Roussel MF, Davis JN, Cleveland JL, Ghysdael J, Hiebert SW. Dual control of myc expression through a single DNA binding site targeted by ets family proteins and E2F-1. Oncogene. 1994 Feb;9(2):405–415. [PubMed]
  • Roussel MF, Cleveland JL, Shurtleff SA, Sherr CJ. Myc rescue of a mutant CSF-1 receptor impaired in mitogenic signalling. Nature. 1991 Sep 26;353(6342):361–363. [PubMed]
  • Roussel MF, Shurtleff SA, Downing JR, Sherr CJ. A point mutation at tyrosine-809 in the human colony-stimulating factor 1 receptor impairs mitogenesis without abrogating tyrosine kinase activity, association with phosphatidylinositol 3-kinase, or induction of c-fos and junB genes. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6738–6742. [PMC free article] [PubMed]
  • Schneikert J, Lutz Y, Wasylyk B. Two independent activation domains in c-Ets-1 and c-Ets-2 located in non-conserved sequences of the ets gene family. Oncogene. 1992 Feb;7(2):249–256. [PubMed]
  • Seth A, Papas TS. The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated. Oncogene. 1990 Dec;5(12):1761–1767. [PubMed]
  • Seth A, Watson DK, Blair DG, Papas TS. c-ets-2 protooncogene has mitogenic and oncogenic activity. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7833–7837. [PMC free article] [PubMed]
  • Tei H, Nihonmatsu I, Yokokura T, Ueda R, Sano Y, Okuda T, Sato K, Hirata K, Fujita SC, Yamamoto D. pokkuri, a Drosophila gene encoding an E-26-specific (Ets) domain protein, prevents overproduction of the R7 photoreceptor. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6856–6860. [PMC free article] [PubMed]
  • Thompson CB, Wang CY, Ho IC, Bohjanen PR, Petryniak B, June CH, Miesfeldt S, Zhang L, Nabel GJ, Karpinski B, et al. cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1. Mol Cell Biol. 1992 Mar;12(3):1043–1053. [PMC free article] [PubMed]
  • Thompson CC, Brown TA, McKnight SL. Convergence of Ets- and notch-related structural motifs in a heteromeric DNA binding complex. Science. 1991 Aug 16;253(5021):762–768. [PubMed]
  • Turc-Carel C, Philip I, Berger MP, Philip T, Lenoir GM. Chromosome study of Ewing's sarcoma (ES) cell lines. Consistency of a reciprocal translocation t(11;22)(q24;q12). Cancer Genet Cytogenet. 1984 May;12(1):1–19. [PubMed]
  • Urness LD, Thummel CS. Molecular interactions within the ecdysone regulatory hierarchy: DNA binding properties of the Drosophila ecdysone-inducible E74A protein. Cell. 1990 Oct 5;63(1):47–61. [PubMed]
  • Usheva A, Maldonado E, Goldring A, Lu H, Houbavi C, Reinberg D, Aloni Y. Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein. Cell. 1992 May 29;69(5):871–881. [PubMed]
  • Wang CY, Petryniak B, Ho IC, Thompson CB, Leiden JM. Evolutionarily conserved Ets family members display distinct DNA binding specificities. J Exp Med. 1992 May 1;175(5):1391–1399. [PMC free article] [PubMed]
  • Wasylyk B, Hahn SL, Giovane A. The Ets family of transcription factors. Eur J Biochem. 1993 Jan 15;211(1-2):7–18. [PubMed]
  • Wasylyk B, Wasylyk C, Flores P, Begue A, Leprince D, Stehelin D. The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation. Nature. 1990 Jul 12;346(6280):191–193. [PubMed]
  • Wasylyk C, Flores P, Gutman A, Wasylyk B. PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes. EMBO J. 1989 Nov;8(11):3371–3378. [PMC free article] [PubMed]
  • Wasylyk C, Kerckaert JP, Wasylyk B. A novel modulator domain of Ets transcription factors. Genes Dev. 1992 Jun;6(6):965–974. [PubMed]
  • Watanabe H, Wada T, Handa H. Transcription factor E4TF1 contains two subunits with different functions. EMBO J. 1990 Mar;9(3):841–847. [PMC free article] [PubMed]
  • Webster NJ, Green S, Jin JR, Chambon P. The hormone-binding domains of the estrogen and glucocorticoid receptors contain an inducible transcription activation function. Cell. 1988 Jul 15;54(2):199–207. [PubMed]
  • Whang-Peng J, Triche TJ, Knutsen T, Miser J, Douglass EC, Israel MA. Chromosome translocation in peripheral neuroepithelioma. N Engl J Med. 1984 Aug 30;311(9):584–585. [PubMed]
  • Whang-Peng J, Triche TJ, Knutsen T, Miser J, Kao-Shan S, Tsai S, Israel MA. Cytogenetic characterization of selected small round cell tumors of childhood. Cancer Genet Cytogenet. 1986 Apr 1;21(3):185–208. [PubMed]
  • Woods DB, Ghysdael J, Owen MJ. Identification of nucleotide preferences in DNA sequences recognised specifically by c-Ets-1 protein. Nucleic Acids Res. 1992 Feb 25;20(4):699–704. [PMC free article] [PubMed]
  • Xin JH, Cowie A, Lachance P, Hassell JA. Molecular cloning and characterization of PEA3, a new member of the Ets oncogene family that is differentially expressed in mouse embryonic cells. Genes Dev. 1992 Mar;6(3):481–496. [PubMed]
  • Yamaguchi Y, Satake M, Ito Y. Two overlapping sequence motifs within the polyomavirus enhancer are independently the targets of stimulation by both the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the Ha-ras oncogene. J Virol. 1989 Mar;63(3):1040–1048. [PMC free article] [PubMed]
  • Zucman J, Delattre O, Desmaze C, Epstein AL, Stenman G, Speleman F, Fletchers CD, Aurias A, Thomas G. EWS and ATF-1 gene fusion induced by t(12;22) translocation in malignant melanoma of soft parts. Nat Genet. 1993 Aug;4(4):341–345. [PubMed]
  • Zucman J, Delattre O, Desmaze C, Plougastel B, Joubert I, Melot T, Peter M, De Jong P, Rouleau G, Aurias A, et al. Cloning and characterization of the Ewing's sarcoma and peripheral neuroepithelioma t(11;22) translocation breakpoints. Genes Chromosomes Cancer. 1992 Nov;5(4):271–277. [PubMed]
  • Zucman J, Melot T, Desmaze C, Ghysdael J, Plougastel B, Peter M, Zucker JM, Triche TJ, Sheer D, Turc-Carel C, et al. Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J. 1993 Dec;12(12):4481–4487. [PMC free article] [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...