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Ewing Sarcoma 11;22 Translocation Produces a Chimeric Transcription Factor That Requires the DNA-Binding Domain Encoded by FLII for Transformation WILLIAM A

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Ewing Sarcoma 11;22 Translocation Produces a Chimeric Transcription Factor That Requires the DNA-Binding Domain Encoded by FLII for Transformation WILLIAM A Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5752-5756, June 1993 Medical Sciences Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLII for transformation WILLIAM A. MAY*, MIKHAIL L. GISHIZKYtt, STEPHEN L. LESSNICK§, LYNN B. LUNSFORD*, BRIAN C. LEWIS*, OLIVIER DELATTRE11, JESSICA ZUCMAN1, GILLES THOMAS¶, AND CHRISTOPHER T. DENNY*§ *Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, Division of Hematology/Oncology, and Jonsson Comprehensive Cancer Center, School of Medicine, and tDepartment of Microbiology and Molecular Genetics and the Howard Hughes Medical Institute, and Molecular Biology Institute, University of California, Los Angeles, CA 90024; and lLaboratoires de Genetique des Tumeurs, Institut Curie, 26 Rue d'Ulm, 75231 Paris Cedex 5, France Communicated by Victor A. McKusick, March 18, 1993 ABSTRACT The 11;22 chromosomal translocation specif- related structural motifs in their DNA-binding regions, usu- icaOly linked to Ewing sarcoma and primitive neuroectodermal ally located at their carboxyl terminus (8). Divergent tran- tumor results in a chimeric molecule fusing the amino- scriptional activation domains at the amino terminus partic- terminal-encoding portion of the EWS gene to the carboxyl- ipate in protein-protein interactions with other transcription terminal DNA-binding domain encoded by the FLII gene. We molecules to activate gene expression at particular targets. have isolated a fourth EWS-FLII fusion cDNA that is struc- The 11;22 translocationjoins the 5' portion ofthe EWS locus turally distinct from the three forms previously described. To to the 3' (DNA-binding) region of the FLII gene and results determine the transforming activity of this gene, alternative in the replacement of the transcription activation domain of forms of the EWS-FLI1 fusion were transduced into NIH 3T3 FLII with EWS sequences. This chimeric product has the cells. CeOls expressing either ype 1 or type 4 fusion constructs potential to promote tumorigenesis by acting as an aberrant formed foci in culture and colonies in soft agar, indicating that transcription factor. We demonstrate here that (i) EWS-FLII EWS-FLI) is a transforming gene. EWS-FLII deletion mu- is a transforming gene and (ii) that both EWS and FLII tants were created to map functionally the critical regions domains of the chimera are necessary for transforming ac- within the chimera. Deletion of either the EWS domain or the tivity. FLII corresponding to the DNA-binding domain totally abro- gated the ability for EWS-FLII to hansform 3T3 cells. These MATERIALS AND METHODS data indicate that the oncogenic effect ofthe 11;22 translocation PNET cDNA Library Construction and Isolation of EWS- is caused by the formation of a chimeric transcription factor. FL!) Chimeras. TC-32, a PNET tumor cell line containing the Formation of chimeric transcription factors has now been 11;22 translocation, was grown in RPMI medium/10% fetal demonstrated to promote tumors of both neuroectodermal and calf serum, as described (5). Total RNA was harvested by hematopoietic origin, suggesting that this may be a common lysis with guanidine isothiocyanate and purified over cesium mechanism in human carcinogenesis. chloride (9). Poly(A)+ RNA was obtained by using columns packed with oligo(dT)-cellulose (Collaborative Research) Structural alteration or aberrant expression of transcription and used for construction of cDNA libraries. factors is often a critical event in neoplastic transformation A TC-32 cDNA library was made according to previously (for review, see ref. 1). Indeed, many known transcription published procedures (10). Briefly, first-strand synthesis was factors were initially identified as a result of their activation accomplished using methyl mercury-denatured poly(A)+ by oncogenic retroviruses of lower vertebrates. Insertion of RNA primed with oligo(dT) and murine leukemia virus retroviral enhancers in proximity to transcription factor reverse transcriptase (GIBCO/BRL). Second-strand synthe- genes can result in inappropriate expression and cellular sis was done by using RNase H and polymerase I (GIBCO/ transformation. Alternatively, replication-deficient retrovi- BRL), and synthesized products were purified over a Seph- ruses that harbor structurally altered transcription factors adex G100 column (Pharmacia). cDNAs were blunted by directly result in the expression of oncogenic proteins. using T4 polymerase (GIBCO/BRL) and ligated to a molar Structural alteration or aberrant expression of transcrip- excess of EcoRI adaptors (Invitrogen, San Diego). The tion factors is also common in human malignancies but adaptor ends were phosphorylated with T4 polynucleotide usually results through somatic genomic mutation (for re- kinase (United States Biochemical), and cDNAs were frac- views, see refs. 2 and 3). Karyotypic analyses have revealed tionated over a 6% acrylamide gel. DNA species >600 bp a tumor-specific t(11;22)(q24;ql2) chromosomal transloca- were recovered from gel slices by electroelution, purified tion in 86% of both Ewing sarcoma and primitive neuroec- over an Elutip-D column (Schleicher & Schuell), ligated into todermal tumor (PNET), suggesting that the product of this A GT-10 vector, and packaged (Gigapack; Stratagene). rearrangement is necessary for the formation of both these Approximately 700,000 primary phage were screened with malignancies (4, 5). Recently it has been shown that this a cDNA fragment from the carboxyl-terminal region of rearrangement juxtaposes the FLII gene on chromosome 11 murine Fli-i (amino acids 259-450, see ref. 7). Three positive with a previously uncharacterized gene ofunknown function, clones were identified ranging from 1.5 to 2.8 kb. The longest termed EWS, on chromosome 22 (6). cDNA contained a t(11;22) fusion point and was subcloned FLU (named for Friend leukemia integration site 1) is a into pBluescript KS(+) (Stratagene), and its coding regions member of the ETS family of transcription factors (7). These were sequenced by using reagents and instruction prepared proteins directly bind to target DNA sequences through Abbreviations: PNET, primitive neuroectodermal tumor; FL11, The publication costs ofthis article were defrayed in part by page charge Friend leukemia integration site 1. payment. This article must therefore be hereby marked "advertisement" tPresent address: Sugen, Inc., 515 Galveston Drive, Redwood City, in accordance with 18 U.S.C. §1734 solely to indicate this fact. CA 94063. 5752 Downloaded by guest on September 29, 2021 Medical Sciences: May et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5753 by United States Biochemical. In similar fashion, the 5' end A glutathione-S-transferase-EWS-FLI1 polypeptide was of this EWS-FLIJ cDNA was used as a probe to isolate created to be used as an immunogen to generate antibodies germ-line EWS clones from the TC-32 cDNA library. against EWS-FLI1. A 189-bp BamHI-Pvu II fragment con- The EWS-FLIl(alt) construct junction point was isolated taining the EWS-FLIl fusion point (see Fig. 3) was subcloned by reverse transcriptase PCR amplification of TC-32 mRNA into the vector pGEX-2T (Pharmacia), and the protein was and contained an additional 66 bp of FLII. First-strand expressed in Escherichia coli. The glutathione-S-transferase synthesis was accomplished using epi-1 (5'-TCTCGAAT- fusion polypeptide was purified from bacterial cell lysates TCTAAGCGTAATCTGGCACATCGTATGGGTAG- using glutathione-agarose (Sigma), as described (15). Purified TAGCTGCCTAAGTGTGA) or ESBP2 (5'-CCGTTGCTCT- protein was injected s.c. into rabbits to create specific GTATTCTTACTGA) oligonucleotides to prime poly(A)+ antisera. TC-32 RNA with murine leukemia virus reverse transcriptase EWS-FLI1 proteins were detected by sequential immu- (GIBCO/BRL), according to manufacturer's recommenda- noprecipitation with two different antibodies. [35S]Methio- tions. Portions ofthese reactions were amplified by Pfu DNA nine-labeled whole-cell lysates were prepared from the same polymerase (Stratagene) with ESBP1 (5'-CGACTAGTTAT- cell populations described in the RNA analysis plus EWS- GATCAGAGCAGT) and ESBP2 primers in a thermocycler FLI1 polypeptide translated in vitro using rabbit reticulocyte using the following parameters: denature, 94°C; anneal, 65°C; lysate (Promega). The first immune precipitation was done by 35 The resultant fragment was digested using monoclonal antibody 12CA5, which was raised to the extend, 72°C; cycles. hemagglutinin epitope placed at the carboxyl terminus of with BamHI and EcoRl, gel-purified, subcloned into pBlue- EWS-FLI1-encoding constructs (11). Immune complexes script KS(+) (Stratagene), and sequenced. were recovered by using protein G-Sepharose (Pharmacia), Retroviral Constructs and Agar Assays. EWS-FLIJ con- denatured, and hybridized with rabbit heteroantisera gener- structs and their derivatives were made as follows. ated to glutathione-S-transferase-EWS-FLI1 polypeptides. EWS-FLIL. A full-length EWS-FLIl cDNA was con- Secondary immune complexes were precipitated with protein structed by ligating the 5' end of a germ-line EWS cDNA to A-Sepharose, fractionated by SDS/PAGE, and autoradio- the chimeric t(11;22) cDNA at an overlapping Pst I site. graphed. Digestion with EcoRI and Hindlll yielded a 1.4-kb fragment that contained 123 bp of 5' untranslated sequence and no 3' untranslated sequence, which was subcloned into pBlue- RESULTS script KS (Stratagene). This construct was tagged with a Multiple Forms oftheEWS-FLII Are Expressed
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