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Mol Cell Biol. 1995 August; 15(8): 4623–4630.
PMCID: PMC230703

Identification of target genes for the Ewing's sarcoma EWS/FLI fusion protein by representational difference analysis.


The EWS/FLI-1 fusion gene results from the 11;22 chromosomal translocation in Ewing's sarcoma. The product of the gene is one of a growing number of structurally altered transcription factors implicated in oncogenesis. We have employed a subtractive cloning strategy of representational difference analysis in conjunction with a model transformation system to identify genes transcribed in response to EWS/FLI. We have characterized eight transcripts that are dependent on EWS/FLI for expression and two transcripts that are repressed in response to EWS/FLI. Three of the former were identified by sequence analysis as stromelysin 1, a murine homolog of cytochrome P-450 F1 and cytokeratin 15. Stromelysin 1 is induced rapidly after expression of EWS/FLI, suggesting that the stromelysin 1 gene may be a direct target gene of EWS/FLI. These results demonstrate that expression of EWS/FLI leads to significant changes in the transcription of specific genes and that these effects are at least partially distinct from those caused by expression of germ line FLI-1. The representational difference analysis technique can potentially be applied to investigate transformation pathways activated by a broad array of genes in different tumor systems.

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Selected References

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  • Bailly RA, Bosselut R, Zucman J, Cormier F, Delattre O, Roussel M, Thomas G, Ghysdael J. DNA-binding and transcriptional activation properties of the EWS-FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma. Mol Cell Biol. 1994 May;14(5):3230–3241. [PMC free article] [PubMed]
  • Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, Moore PS. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science. 1994 Dec 16;266(5192):1865–1869. [PubMed]
  • Chen L, Hardwick JP. Identification of a new P450 subfamily, CYP4F1, expressed in rat hepatic tumors. Arch Biochem Biophys. 1993 Jan;300(1):18–23. [PubMed]
  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. [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]
  • Delattre O, Zucman J, Melot T, Garau XS, Zucker JM, Lenoir GM, Ambros PF, Sheer D, Turc-Carel C, Triche TJ, et al. The Ewing family of tumors--a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med. 1994 Aug 4;331(5):294–299. [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]
  • Dunn T, Praissman L, Hagag N, Viola MV. ERG gene is translocated in an Ewing's sarcoma cell line. Cancer Genet Cytogenet. 1994 Aug;76(1):19–22. [PubMed]
  • Fujii Y, Hongo T, Nakagawa Y, Nasuda K, Mizuno Y, Igarashi Y, Naito Y, Maeda M. Cell culture of small round cell tumor originating in the thoracopulmonary region. Evidence for derivation from a primitive pluripotent cell. Cancer. 1989 Jul 1;64(1):43–51. [PubMed]
  • Fujimura Y, Yamamoto H, Hamazato F, Nozaki M. One of two Ets-binding sites in the cytokeratin EndoA enhancer is essential for enhancer activity and binds to Ets-2 related proteins. Nucleic Acids Res. 1994 Feb 25;22(4):613–618. [PMC free article] [PubMed]
  • Giovannini M, Biegel JA, Serra M, Wang JY, Wei YH, Nycum L, Emanuel BS, Evans GA. EWS-erg and EWS-Fli1 fusion transcripts in Ewing's sarcoma and primitive neuroectodermal tumors with variant translocations. J Clin Invest. 1994 Aug;94(2):489–496. [PMC free article] [PubMed]
  • Hachitanda Y, Tsuneyoshi M, Enjoji M, Nakagawara A, Ikeda K. Congenital primitive neuroectodermal tumor with epithelial and glial differentiation. An ultrastructural and immunohistochemical study. Arch Pathol Lab Med. 1990 Jan;114(1):101–105. [PubMed]
  • Hamazato F, Fujimura Y, Tamai Y, Takemoto Y, Matsushiro A, Nozaki M. Sequence specific binding of Ets-1 to the mouse cytokeratin EndoA gene enhancer. Biochem Biophys Res Commun. 1993 Apr 30;192(2):430–438. [PubMed]
  • Hubank M, Schatz DG. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 1994 Dec 25;22(25):5640–5648. [PMC free article] [PubMed]
  • Ichikawa H, Shimizu K, Hayashi Y, Ohki M. An RNA-binding protein gene, TLS/FUS, is fused to ERG in human myeloid leukemia with t(16;21) chromosomal translocation. Cancer Res. 1994 Jun 1;54(11):2865–2868. [PubMed]
  • Jeon IS, Davis JN, Braun BS, Sublett JE, Roussel MF, Denny CT, Shapiro DN. A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1. Oncogene. 1995 Mar 16;10(6):1229–1234. [PubMed]
  • Khokha R, Waterhouse P, Yagel S, Lala PK, Overall CM, Norton G, Denhardt DT. Antisense RNA-induced reduction in murine TIMP levels confers oncogenicity on Swiss 3T3 cells. Science. 1989 Feb 17;243(4893):947–950. [PubMed]
  • Kikuta Y, Kusunose E, Endo K, Yamamoto S, Sogawa K, Fujii-Kuriyama Y, Kusunose M. A novel form of cytochrome P-450 family 4 in human polymorphonuclear leukocytes. cDNA cloning and expression of leukotriene B4 omega-hydroxylase. J Biol Chem. 1993 May 5;268(13):9376–9380. [PubMed]
  • Kikuta Y, Kusunose E, Kondo T, Yamamoto S, Kinoshita H, Kusunose M. Cloning and expression of a novel form of leukotriene B4 omega-hydroxylase from human liver. FEBS Lett. 1994 Jul 4;348(1):70–74. [PubMed]
  • Ladanyi M, Gerald W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell tumor. Cancer Res. 1994 Jun 1;54(11):2837–2840. [PubMed]
  • Lessnick SL, Braun BS, Denny CT, May WA. Multiple domains mediate transformation by the Ewing's sarcoma EWS/FLI-1 fusion gene. Oncogene. 1995 Feb 2;10(3):423–431. [PubMed]
  • Lewin B. Oncogenic conversion by regulatory changes in transcription factors. Cell. 1991 Jan 25;64(2):303–312. [PubMed]
  • Liang P, Averboukh L, Pardee AB. Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization. Nucleic Acids Res. 1993 Jul 11;21(14):3269–3275. [PMC free article] [PubMed]
  • Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992 Aug 14;257(5072):967–971. [PubMed]
  • Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 1991 Jan 25;64(2):327–336. [PubMed]
  • Lisitsyn N, Lisitsyn N, Wigler M. Cloning the differences between two complex genomes. Science. 1993 Feb 12;259(5097):946–951. [PubMed]
  • Lisitsyn NA, Lisitsina NM, Dalbagni G, Barker P, Sanchez CA, Gnarra J, Linehan WM, Reid BJ, Wigler MH. Comparative genomic analysis of tumors: detection of DNA losses and amplification. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):151–155. [PubMed]
  • Lisitsyn NA, Segre JA, Kusumi K, Lisitsyn NM, Nadeau JH, Frankel WN, Wigler MH, Lander ES. Direct isolation of polymorphic markers linked to a trait by genetically directed representational difference analysis. Nat Genet. 1994 Jan;6(1):57–63. [PubMed]
  • Mao X, Miesfeldt S, Yang H, Leiden JM, Thompson CB. The FLI-1 and chimeric EWS-FLI-1 oncoproteins display similar DNA binding specificities. J Biol Chem. 1994 Jul 8;269(27):18216–18222. [PubMed]
  • Matrisian LM, Bowden GT. Stromelysin/transin and tumor progression. Semin Cancer Biol. 1990 Apr;1(2):107–115. [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. [PubMed]
  • May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB, Hromas R, Denny CT. The Ewing's sarcoma EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful transforming gene than FLI-1. Mol Cell Biol. 1993 Dec;13(12):7393–7398. [PMC free article] [PubMed]
  • Mignatti P, Robbins E, Rifkin DB. Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell. 1986 Nov 21;47(4):487–498. [PubMed]
  • Mizukami Y, Sumimoto H, Isobe R, Minakami S. Omega-hydroxylation of lipoxin B4 by human neutrophil microsomes: identification of omega-hydroxy metabolite of lipoxin B4 and catalysis by leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega). Biochim Biophys Acta. 1993 May 20;1168(1):87–93. [PubMed]
  • Mizukami Y, Sumimoto H, Isobe R, Minakami S, Takeshige K. omega-Oxidation of lipoxin B4 by rat liver. Identification of an omega-carboxy metabolite of lipoxin B4. Eur J Biochem. 1994 Sep 15;224(3):959–965. [PubMed]
  • Moll R, Lee I, Gould VE, Berndt R, Roessner A, Franke WW. Immunocytochemical analysis of Ewing's tumors. Patterns of expression of intermediate filaments and desmosomal proteins indicate cell type heterogeneity and pluripotential differentiation. Am J Pathol. 1987 May;127(2):288–304. [PubMed]
  • Nordeen SK. Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques. 1988 May;6(5):454–458. [PubMed]
  • Ohno T, Ouchida M, Lee L, Gatalica Z, Rao VN, Reddy ES. The EWS gene, involved in Ewing family of tumors, malignant melanoma of soft parts and desmoplastic small round cell tumors, codes for an RNA binding protein with novel regulatory domains. Oncogene. 1994 Oct;9(10):3087–3097. [PubMed]
  • Ohno T, Rao VN, Reddy ES. EWS/Fli-1 chimeric protein is a transcriptional activator. Cancer Res. 1993 Dec 15;53(24):5859–5863. [PubMed]
  • Oshima RG. Intermediate filament molecular biology. Curr Opin Cell Biol. 1992 Feb;4(1):110–116. [PubMed]
  • Panagopoulos I, Mandahl N, Ron D, Höglund M, Nilbert M, Mertens F, Mitelman F, Aman P. Characterization of the CHOP breakpoints and fusion transcripts in myxoid liposarcomas with the 12;16 translocation. Cancer Res. 1994 Dec 15;54(24):6500–6503. [PubMed]
  • Rabbitts TH. Chromosomal translocations in human cancer. Nature. 1994 Nov 10;372(6502):143–149. [PubMed]
  • Rabbitts TH, Forster A, Larson R, Nathan P. Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet. 1993 Jun;4(2):175–180. [PubMed]
  • Rivera RR, Stuiver MH, Steenbergen R, Murre C. Ets proteins: new factors that regulate immunoglobulin heavy-chain gene expression. Mol Cell Biol. 1993 Nov;13(11):7163–7169. [PMC free article] [PubMed]
  • Schultz RM, Silberman S, Persky B, Bajkowski AS, Carmichael DF. Inhibition by human recombinant tissue inhibitor of metalloproteinases of human amnion invasion and lung colonization by murine B16-F10 melanoma cells. Cancer Res. 1988 Oct 1;48(19):5539–5545. [PubMed]
  • Seth A, Robinson L, Panayiotakis A, Thompson DM, Hodge DR, Zhang XK, Watson DK, Ozato K, Papas TS. The EndoA enhancer contains multiple ETS binding site repeats and is regulated by ETS proteins. Oncogene. 1994 Feb;9(2):469–477. [PubMed]
  • Shinoda M, Tsutsumi Y, Hata J, Yokoyama S. Peripheral neuroepithelioma in childhood. Immunohistochemical demonstration of epithelial differentiation. Arch Pathol Lab Med. 1988 Nov;112(11):1155–1158. [PubMed]
  • Sorensen PH, Lessnick SL, Lopez-Terrada D, Liu XF, Triche TJ, Denny CT. A second Ewing's sarcoma translocation, t(21;22), fuses the EWS gene to another ETS-family transcription factor, ERG. Nat Genet. 1994 Feb;6(2):146–151. [PubMed]
  • Spaete RR, Mocarski ES. Regulation of cytomegalovirus gene expression: alpha and beta promoters are trans activated by viral functions in permissive human fibroblasts. J Virol. 1985 Oct;56(1):135–143. [PMC free article] [PubMed]
  • Sumimoto H, Isobe R, Mizukami Y, Minakami S. Formation of a novel 20-hydroxylated metabolite of lipoxin A4 by human neutrophil microsomes. FEBS Lett. 1993 Jan 11;315(3):205–210. [PubMed]
  • Takemoto Y, Fujimura Y, Matsumoto M, Tamai Y, Morita T, Matsushiro A, Nozaki M. The promoter of the endo A cytokeratin gene is activated by a 3' downstream enhancer. Nucleic Acids Res. 1991 May 25;19(10):2761–2765. [PMC free article] [PubMed]
  • Wasylyk C, Gutman A, Nicholson R, Wasylyk B. The c-Ets oncoprotein activates the stromelysin promoter through the same elements as several non-nuclear oncoproteins. EMBO J. 1991 May;10(5):1127–1134. [PubMed]
  • Wasylyk C, Wasylyk B. Oncogenic conversion alters the transcriptional properties of ets. Cell Growth Differ. 1992 Sep;3(9):617–625. [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, 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. [PubMed]

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