The EWS Protein Is Dispensable for Ewing Tumor ... - Cancer Research

[CANCER RESEARCH 61, 5992–5997, August 15, 2001]

Advances in Brief

The EWS Protein Is Dispensable for Ewing Tumor Growth1 Heinrich Kovar,2 Gunhild Jug, Claudia Hattinger, Laura Spahn, Dave N. T. Aryee, Peter F. Ambros, Andreas Zoubek, and Helmut Gadner Children’s Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse 6, 1090 Vienna, Austria

Abstract EWS encodes a ubiquitously expressed RNA binding protein with largely unknown function. In Ewing sarcoma family tumors (EFT), one allele is rearranged with an ETS gene. This is the first description of an EFT with a complete EWS deficiency in the presence of two copies of a rearranged chromosome 22 carrying an interstitial EWS-FLI1 translocation. Absence of EWS protein suggested that it is dispensable for EFT growth. By sequencing of EWS cDNA from unrelated EFTs, we excluded inactivation of EWS as a general mechanism in EFT pathogenesis. Rather, EWS was found to be uniformly expressed in two splicing variants of similar abundancy, EWS␣ and EWS␤, which differ in a single amino acid. Three EWS negative cell lines were established, which will serve as valuable models to study normal and aberrant EWS function upon reintroduction into the tumor cells.

Introduction

mation has been demonstrated (reviewed in Ref. 4). This function was largely dependent on the transcriptional activity of the protein. However, a transcription-independent contribution of the EWS NH2 terminus has been suggested based on deletion analysis (14) and by an EWS-FLI1 DNA-binding mutant (15). Protein interaction studies revealed communication between the RNA polymerase II component hsRPB7 and the EWS NH2 terminus only in the context of the EWS-FLI1 fusion protein or COOH-terminally truncated EWS but not with germ-line EWS (6, 16), indicating distinct context-dependent functions of the EWS NH2 terminus. To investigate the role of normal EWS and of the EWS component of oncogenic fusion proteins, EWS knock-out cells would be of great advantage. Here, we report on EWS-negative cell lines that will serve as a valuable tool in future studies of EWS and its oncogenic derivatives. Materials and Methods

The EWS gene on chromosome 22q12 (1) has been identified as a target of tumor-specific chromosomal translocations in EFTs3, malignant melanoma of soft parts, desmoplastic small round cell tumor, myxoid chondrosarcoma, and myxoid liposarcoma. The respective gene rearrangements result in the production of chimeric DNA binding oncoproteins to which EWS contributes a strong transcriptional activation domain. Besides the rearranged gene, the normal EWS allele is always active in both tumor cells and normal tissues (2). Because of its GC rich promoter structure lacking a TATA sequence and its abundant ubiquitous expression, EWS is assumed a housekeeping gene (3). Whereas tumor-derived chimeric EWS proteins function as sequence-specific transcription factors (reviewed in Ref. 4), the role of normal EWS is largely unknown. The COOH-terminal portion contains a RNA-binding motif and a putative zinc-finger domain (5). The EWS protein, primarily localized in the nucleus, has been found to associate with components of the basal transcriptional machinery (6 – 8) and RNA splicing factors (7, 9), as well as to partition into the ribosome-dense fraction of the cytoplasm, in particular, upon G protein coupled receptor signaling (10). The most recently described exposure of extensively arginine methylated EWS on the cell surface (11) and phosphorylation by protein kinase C (12), as well as interaction with the tyrosine kinase Pyk2 and Bruton’s tyrosine kinase (10, 13), indicate that EWS may be the target and/or part of as yet undefined signal transduction pathways. In EFT, one EWS allele is consistently rearranged with an ETS transcription factor gene, predominantly FLI1. For the corresponding fusion protein, a rate-limiting role in cell proliferation and transforReceived 5/9/01; accepted 6/29/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants 13708-GEN and 14299-GEN of the Austrian Science Foundation (to H. K.) 2 To whom requests for reprints should be addressed, at Children’s Cancer Research Institute, St. Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 43 (1) 40470, extension 409; Fax: 43 (1) 4087230; E-mail: [email protected]. 3 The abbreviations used are: EFT, Ewing sarcoma family tumor; FISH, fluorescence in situ hybridization; EWSR, Ewing’s sarcoma breakpoint region; RT-PCR, reverse transcription-PCR; RACE, rapid amplification of cDNA ends.

Processing of Tumor Samples and Cell Lines. Cell lines were expanded from single cell suspensions of primary tumor material after mechanical disaggregation. All cell lines used in this study have been described previously (17, 18). Double-color FISH on metaphase spreads and dynamic molecular combing for FISH on DNA fibers were achieved as recently reported (19). Cosmid probes G9 and F7 (20), flanking the EWSR1 on chromosome 22, and 1p3 and 1d1 from the EWSR2 region on chromosome 11 (21) were kindly supplied by Olivier Delattre (Institut Curie, Paris, France). DNA Analysis. The integrity of the EWS gene was analyzed on genomic Southern blots. Localizations along the EWSR1 region of probes HP.5, 5.5SAC, RP.8, RX.3 (3), kindly supplied by O. Delattre (Institut Curie), and int9 (22) are depicted in Fig. 1A. Allelotyping of variable numbers of dinucleotide repeats on chromosome 22 was performed by PCR using the following oligonucleotide combinations. D22S258 localized 3.4 Mb proximal of EWS: forward 5⬘-GCCTGAAATTATTCCAGCTG-3⬘, reverse 5⬘-AATAGTAGAGTTTGCCTTTC-3⬘; IL2RB localized 8.1 Mb distal of EWS: forward 5⬘-GAGAGGGAGGGCCTGCGTTC3⬘, reverse 5⬘-CACCCAGGGCCAGATAAAGA-3⬘; D22S299 localized 10.9 Mb distal of EWS: forward 5⬘-TGACAACAACCATCAAGTCCA-3⬘, reverse 5⬘-GGAGCTGCATGTACTAGCTGG-3⬘. RNA and Protein Analysis. Expression analysis of EWS-derived transcripts was performed on Northern blots probed with a full-length EWS cDNA. RT-PCR for the detection of EWS-FLI1 chimeric transcripts was performed as described (1). Protein expression was studied on Western blots sequentially probed with polyclonal antisera to the EWS NH2 terminus (139-2, kindly provided by Christopher T. Denny, University of California at Los Angeles, Los Angeles, CA), and SE680 was directed to the EWS COOH-terminus (a gift of Olivier Delattre). For the detection of wild-type and chimeric FLI1 proteins, the polyclonal antibody C19 (Santa Cruz Biotechnology, Santa Cruz, CA) was used. Cloning of Wild-type and Truncated EWS cDNAs. The coding region of full-length EWS was amplified and cloned using the EWS 5⬘-primer 5⬘GGCCGAATTCATGGCGTCCACGGATTACAGT-3⬘ and the EWS 3⬘primer 5⬘-GCGCCTCGAGAAAATAAATCTGGTAGTCAATGCAGCTC3⬘. RACE for the characterization of truncated EWS transcripts was achieved using the Marathon cDNA Amplification kit (CLONTECH, Palo Alto, CA). For 5⬘-RACE, adapter-ligated double-stranded cDNA from STA-ET-7.2 and IARC-EW2 cells was amplified with adapter-specific primer AP1 and the EWS 3⬘-primer and, in a second step, with adapter-specific forward primer AP2 and exon 16-specific reverse primer 5⬘-CCACCTCTGTCTCCACCACG-3⬘ or

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EWS LOSS IN A EWING TUMOR

Fig. 1. Analysis of EWS gene status by genomic Southern blotting. A, schematic representation of EWSR1 indicating restriction sites for EcoRI (R) and XbaI (X) and localization of probes used for Southern blot analysis. Breakpoint localizations for IARC-EW2 (A), STA-ET-7.2 (B), and STA-ET-2.2 (C) as deduced from the results shown in C are indicated by arrows. B, detection of an EWS rearrangement and of absence of the second EWS allele in an EFT and derived cell lines using probe HP.5 on EcoRI-digested genomic DNA. BM, patient’s bone marrow; Tu, patient’s primary tumor; 1, STAET-7.1; 2, STA-ET-7.2; 3, STA-ET-7.3; 4, unrelated EFT cell line STA-ET-8. C, restriction analysis of the EWSR1 for STA-ET-7.2 (B) in comparison to two unrelated EFT cell lines: IARCEW2 (A) and STA-ET-2.2 (C). Arrows, positions of germ-line EWS-derived bands; 夞, bands derived from the rearranged EWS allele; ⌿, hybridization of probes HP.5 and 5.5SAC to an EWS pseudogene (30).

exon 15-specific reverse primer 5⬘-GGGGGCGGAAAGGGTGGC-3⬘, respectively. For 3⬘-RACE, adapter-ligated cDNA was amplified with primer AP1 and the EWS exon 12-specific forward primer 5⬘-AAGACCCACCCACTGCCAAG-3⬘ in a first step and AP2 and the exon 13-specific forward primer 5⬘-CAGTATGCGGGGTGGTCT-3⬘ in a second step. Analysis of EWS Splicing Variants. The EWS exon 8/9 boundary was amplified from random hexamer primed cDNA using primers 5⬘-GTGGAGGCATGAGCAGAGG-3⬘ and 5⬘-GAAGCCACCTCGCTCTCCA-3⬘ in the presence of radioactive dCTP. The 73-bp PCR product was resolved on a 7.5% denaturing polyacrylamide gel.

Results An EFT with Two Cytogenetically Inconspicuous Chromosomes 22 Expresses EWS-FLI1. A 4-year-old girl was presented with a localized but rapidly progressing CD99-positive small round cell tumor of the chest wall. Three cell lines (STA-ET-7.1, STA-ET7.2, and STA-ET-7.3) were established from the primary tumor, a pleural effusion, and a metastasis, respectively. Despite the presence of ⱕ3 cytogenetically normal chromosomes 22 in an otherwise complex, near-triploid karyotype (18), routine RT-PCR analysis detected expression of a type 2 EWS-FLI1 chimeric transcript (EWS exon 7 fused to FLI1 exon 5) in the tumor and in the corresponding cell lines consistent with the diagnosis of EFT (data not shown). Evidence for an EWS-FLI1 Gene Rearrangement and Loss of the Second EWS Allele. Genomic Southern blotting using probe HP.5 covering EWS exon 7 (Fig. 1A) confirmed the presence of a rearranged EWS allele (Fig. 1B). Surprisingly, when compared with the patient’s bone marrow and to an unrelated EFT, the tumor and the three cell lines lacked the band for germ-line EWS indicative of a partial or complete deletion of the second EWS allele. Additional

analysis using probes downstream of HP.5 along the EWSR1 (20) was performed on STA-ET-7.2 to characterize rearrangement points more precisely. Only a single aberrant EWS band was detected with all probes 5⬘ of the EcoRI site in intron 8 except RP.8, which was found to be split in the cell line (Fig. 1C). Thus, a gene rearrangement delineated to the middle of an 800-bp region within intron 8 of one EWS allele and no evidence for the presence of an intact second EWS allele was obtained. The localization of the genomic EWS breakpoint downstream of exon 8 is compatible with the juxtaposition of EWS exon 7 with FLI1 exon 5 on RNA level, because an exon 8 fusion to FLI1 exon 5 would shift the reading frame, and consequently, exon 8 is frequently spliced-out from the chimeric transcript in EFT (23). Probes int9 and RX.3 detected an unrearranged band that may contain the second half of the translocated EWS allele and/or remnants of a partially deleted second EWS allele. Loss of the Normal and Duplication of the Rearranged Chromosome 22. Because two chromosomes 22 have been identified by karyotyping, we investigated the allelic status of these chromosomes by PCR of variable numbers of dinucleotide repeats within a 14-Mb region surrounding the EWS gene on chromosome 22. Loss of heterozygosity was detected for all markers, indicating that one chromosome 22 was lost, whereas the rearranged chromosome was duplicated (not shown). The EWS-FLI1 Gene Rearrangement Results from an Interstitial Translocation. To clarify the structure of these chromosomes, FISH was performed on cell lines STA-ET-7.1 and STA-ET-7.2 using probes flanking and overlapping EWSR1 and EWSR2 (Fig. 2A). Interestingly, probes G9 and F7 that flank the EWSR1 were not separated from each other by the EWS gene rearrangement (Fig. 2B).

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Fig. 2. Detection of an interstitial chromosome 22 rearrangement by FISH. A, schematic representation of cosmid probe localizations relative to the EWSR1 and EWSR2 on chromosomes 22 and 11, respectively (21). B, simultaneous hybridization of probe G9 (green) with either F7, 1d1, or 1p3 (red) to STA-ET-7.2 metaphases as indicated. C, fine mapping of the interstitial translocation by fiber FISH. Cohybridization of probes G9 (green) and 1d1 (red); G9 (green), 1p3 (red), and 1d1 (mixed color); and F10 (red) and G9 (green), as indicated. D, summarizes schematically the spatial distribution of the used probes along the derivative chromosome 22 as deduced from all FISH experiments performed.

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Fig. 3. Detection of normal and rearranged EWS gene transcripts. A, Northern blot of total RNA from 13 EFT cell lines, including STA-ET-7.2 (arrowhead) and 6 neuroblastoma (Nb) cell lines hybridized to a full-length EWS cDNA. Positions of EWS-FLI1 and EWS RNA, as well as of an apparently truncated EWS transcript (⌬EWS), in STA-ET-7.2 are indicated. B, schematic representation of the interstitial EWS-FLI translocation and derived transcripts. Boxes, exons as indicated; blank areas, untranslated regions.

However, juxtaposition of G9 with 1d1 distal to the EWSR2, as well as with the EWSR2 overlapping probe 1p3 on chromosome 22, suggested an interstitial translocation as underlying the EWS-FLI1 fusion. The probe G9 was consistently associated with a chromosome 11 probe confirming the absence of a normal EWS allele. These data were additionally corroborated by fiber FISH on combed DNA of the cell line STA-ET-7.2, which allowed for exact length measurements of hybridization signals (Fig. 2C). The spatial order of probes along the rearranged DNA fiber was found to be F10 –G9-1p3-1d1. The hybridizing region for 1p3 was significantly shortened in line with rearrangement of the FLI-1 gene within EWSR2. Although FISH indicated no separation of probes G9 and F7 on chromosome 22, F7 was never seen on the same DNA fiber with any of the other probes used. On the basis of the limits of microscopic resolution of fiber FISH, these data suggest that the chromosome 11 region inserted into the EWSR1 comprised ⬎300 kb. The combined results from FISH and fiber FISH are summarized schematically in Fig. 2D. Absence of EWS Gene Expression. Despite the absence of a germ-line EWS allele, RT-PCR using primers to EWS exons 12 and 16 consistently gave positive results for all tumor materials of our patient (data not shown). We, therefore, performed Northern blot analysis with a full-length cDNA as a probe to identify all EWS-related transcripts in 12 EFT cell lines, including STA-ET-7.2 and, for control, 6 neuroblastoma cell lines. EFT-specific expression of EWSFLI1 and abundant expression of germ-line EWS in all cell lines, except STA-ET-7.2, were confirmed. Here, a variant EWS-related transcript smaller than 18S rRNA was identified (⌬EWS; Fig. 3A). RACE experiments using EWS exon 15- and exon 16-specific primers were performed on the EFT cell lines STA-ET-7.2 and, for control, IARC-EW2 to establish the structure of EWS-derived transcripts. As expected, in IARC-EW2, the predominant product corresponded to full-length EWS. In contrast, the longest transcript identified in STAET-7.2 cells corresponding in size to ⌬EWS was found to be initiated in intron 9, 52 nucleotides upstream of EWS exon 10, and comprised entirely by EWS sequences, suggesting that it was derived from the 3⬘

portion of the split EWS gene on the derivative chromosome 22, as depicted schematically in Fig. 3B. Although potentially encoding a truncated protein of 35 kDa, no EWS-related product was identified in STA-ET-7.2 cells by antibody SE680 directed to the EWS COOH

Fig. 4. Lack of EWS protein expression in STA-ET-7.2 cells. Western blots were probed with the polyclonal antibodies (pAb) to the FLI1 COOH terminus (C19), the EWS NH2 terminus (139-2), or the EWS COOH terminus (SE680) as indicated. 1, EWS-FLI1negative neuroblastoma cell line SJ-NB-7; 2, germ-line FLI1 expressing T-ALL cell line MOLT4; 3– 6, EFT cell lines SK-N-MC (EWS-FLI1 exon 7/6 fusion), RDES (EWS-FLI1 exon 7/5 fusion), STA-ET-2.2 (EWS-FLI1 exon 9/4 fusion), and STA-ET-7.2 (EWS-FLI1 exon 7/5 fusion), respectively.

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Fig. 5. Predominant EWS splicing variants. A, schematic representation. The region of the EWS gene spanning exons 7–11 with the exonintron boundaries flanking intron 8 are shown on top, and mRNA sequences and corresponding protein sequences for the two isoforms EWS␣ and EWS␤ are shown on the bottom, with the discriminating three nucleotides and the EWS␣specific amino acid Ser325 indicated in bold. B, relative expression levels. The exon 8/9 boundary was amplified from cDNA and PCR products resolved on a sequencing gel. Lanes 1 and 2, cloned EWS␣ and EWS␤, respectively; Lane 3, fibroblasts; Lane 4, keratinocytes; Lanes 5–9, neuroblastoma cell lines; Lane 10, STA-ET-7.2; Lanes 11–18, unrelated EFT cell lines; Lane 19, MCF7 breast cancer cells. C, localization of ectopically expressed EWS␣ and ␤ isoforms in STA-ET-7.2 cells by immunofluorescence using antibody SE680.

terminus (Fig. 4). Also, consistent with the Southern and Northern blot data, antibody 139-2 specific for the EWS NH2 terminus failed to detect full-length EWS expression in STA-ET-7.2. EWS-FLI1 expression, which was unambiguously demonstrated using a FLI1-specific antibody in all EFT, is generally undetectable by EWS-specific antibodies on Western blots, presumably attributable to comparably low steady-state levels. Thus, although highly unlikely, the presence of minute amounts of a truncated EWS protein in STA-ET-7.2 cannot be ruled out completely. EWS Is Not Generally Inactivated in EFT but Rather Expressed as Two Splicing Variants. Formation of an oncogenic gene fusion is considered to be the primary aberration affecting EWS in EFT. However, loss of heterozygosity and disruption of the remaining EWS allele by rearrangement in our case may indicate that loss of EWS function contributes to EFT pathogenesis more generally, if inactivating aberrations would be identified in the unrearranged EWS allele in other EFTs too. We, therefore, cloned EWS cDNA from three unrelated EFT cell lines and compared the sequences to EWS from normal peripheral blood. Half of the cDNA clones obtained for each sample were found to lack 3 bp at the exon 8/9 boundary leading to a loss of serine residue 325. This variation presumably resulted from alternative splicing at the tandemly duplicated splice acceptor site, as depicted in Fig. 5A and mentioned previously by Plougastel et al. (24). The two isoforms, subsequently named EWS␣ and EWS␤, were apparently equally expressed with only little variation, not only in EFT but also in neuroblastoma cells, MCF7 breast cancer cells, fibroblasts, and keratinocytes as depicted in Fig. 5B. Upon transient transfection into STA-ET-7.2 cells, both variants localized to the nucleus (Fig. 5C). Otherwise, no mutations were identified in the cell

lines, suggesting that a loss of normal EWS function as observed in the described case is not a general phenomenon in EFT. Discussion The EFT described here is remarkable in several aspects: the EWS-FLI1 rearrangement was found to be the result of an interstitial translocation. The normal chromosome 22 was lost, whereas the rearranged copy was duplicated. Both aberrations were undetectable by classical cytogenetics and by FISH with cosmids flanking the EWSR1. The insertion of ⱖ300 kb of chromosome 11 material into the derivative chromosome 22 split the EWS gene locus within intron 8. The 5⬘ portion of the disrupted gene was fused to FLI1, resulting in the production of a chimeric EWS-FLI1 transcript characteristic of EFT. In addition, the EWS 3⬘ portion gave rise to a truncated EWS RNA which did not encode any detectable amounts of protein. No normal EWS gene product was present in this case, whereas a normal gene dosage of other chromosome 22-encoded loci should be retained because of the duplication of the whole derivative chromosome 22. Thus, the EFT described here constitutes an in situ EWS knockout. Because absence of germ-line EWS was observed already in the primary tumor, our data indicate that EWS expression is dispensable for EFT growth in vivo and in vitro. We have demonstrated previously the presence of a p53 mutation (p53Arg273:Cys) in this case (number 33 and cell lines I1 and I2 in Ref. 17). Consequently, secondary genetic aberrations may have supported EWS independence of tumor cell growth. Whereas rearrangement of one EWS allele is highly associated with EFT, we demonstrate that complete loss of EWS function is not a general feature in this disease. EWS belongs to a growing family of closely related genes, including two members that

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take part in structurally similar translocations with transcription factor genes, TLS (FUS) and TAFII68 (TAF2N and RPB56). In fact, the NH2-terminal portions of EWS and TLS, as well as of EWS and TAFII68, appear to be functionally interchangeable (reviewed in Ref. 4). The normal roles of the three putative RNA-binding EWS family members is not well defined, but functions within the transcriptional and splicing apparatus have been suggested (6, 7, 16, 21, 25–27). Cellular knockout systems for the study of EWS family proteins have thus far been established for TLS only (28). Here, disruption of the TLS gene was associated with genomic instability. A complex karyotype was observed in our case too, although the karyotype of EFT is usually relatively simple with only very few structural and numerical chromosome aberrations. It is tempting to speculate that, similar to the TLS knockout, this cytogenetic feature may be related to the loss of EWS. Alternatively, other destabilizing genetic aberrations present in the tumor, such as mutant p53, may have contributed to the aberrant karyotype. Despite the complexity of genetic aberrations present, the three EWS-negative cell lines should provide excellent tools to study the function of normal and aberrant EWS proteins in a human system. It is noteworthy that EWS is normally expressed in several isoforms. Thus far, four such variants with widespread expression have been observed. In addition to EWS␣ and EWS␤ described here, which differ in a single amino acid, a putative casein kinase II phosphorylation site (29), a splicing variant (EWS-b) completely lacking exons 8 and 9 and a brain-specific variant carrying an additional 18-bp exon between exons 4 and 5 have been reported (5, 31). For the first time, it should be possible to investigate the role of alternatively spliced EWS variants individually after reintroduction into one of our EWSnegative cell lines. Additionally, studies on the specific function of EWS-FLI1 in EFT will be greatly facilitated because agents directed to the EWS moiety of the fusion product, such as antisense RNA, will not be competed out by much more abundantly expressed normal EWS. Thus, although probably constituting an extremely rare case, the tumor and derived cell lines described here will greatly promote our understanding of EFT pathogenesis and normal and aberrant EWS function. Acknowledgments

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8. 9. 10.

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We thank O. Delattre (Institut Curie) for supplying hybridization probes and EWS antibody SE680 and C. Denny (University of California at Los Angeles) for providing the polyclonal antiserum 139-2.

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References

25.

1. Delattre, O., Zucman, J., Plougastel, B., Desmaze, C., Melot, T., Peter, M., Kovar, H., Joubert, I., De Jong, P., Rouleau, G., Aurias, A., and Thomas, G. Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature (Lond.), 359: 162–165, 1992. 2. Aman, P., Panagopoulos, I., Lassen, C., Fioretos, T., Mencinger, M., Toresson, H., Hoglund, M., Forster, A., Rabbitts, T. H., Ron, D., Mandahl, N., and Mitelman, F. Expression patterns of the human sarcoma-associated genes FUS and EWS and the genomic structure of FUS. Genomics, 37: 1– 8, 1996. 3. Plougastel, B., Zucman, J., Peter, M., Thomas, G., and Delattre, O. Genomic structure of the EWS gene and its relationship to EWSR1, a site of tumor-associated chromosome translocation. Genomics, 18: 609 – 615, 1993. 4. Kovar, H. Progress in the molecular biology of Ewing tumors. Sarcoma, 2: 3–17, 1998. 5. Ohno, T., Ouchida, M., Lee, L., Gatalica, Z., Rao, V. N., and Reddy, E. S. 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, 9: 3087–3097, 1994. 6. Bertolotti, A., Melot, T., Acker, J., Vigneron, M., Delattre, O., and Tora, L. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions

26. 27.

28.

29. 30.

31.

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between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol. Cell. Biol., 18: 1489 –1497, 1998. Yang, L., Chansky, H. A., and Hickstein, D. D. EWS/Fli-1 fusion protein interacts with hyperphosphorylated RNA polymerase II and interferes with serine-arginine protein-mediated RNA splicing. J. Biol. Chem., 275: 37612–37618, 2000. Rossow, K. L., and Janknecht, R. The Ewing’s sarcoma gene product functions as a transcriptional activator. Cancer Res., 61: 2690 –2695, 2001. Knoop, L. L., and Baker, S. J. The splicing factor U1C represses EWS/FLI-mediated transactivation. J. Biol. Chem., 275: 24865–24871, 2000. Felsch, J. S., Lane, W. S., and Peralta, E. G. Tyrosine kinase Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS. Curr. Biol., 9: 485– 488, 1999. Belyanskaya, L. L., Gehrig, P. M., and Gehring, H. Exposure on cell surface and extensive arginine methylation of EWS protein. J. Biol. Chem., 276: 18681–18687, 2001. Deloulme, J. C., Prichard, L., Delattre, O., and Storm, D. R. The prooncoprotein EWS binds calmodulin and is phosphorylated by protein kinase C through an IQ domain. J. Biol. Chem., 272: 27369 –27377, 1997. Guinamard, R., Fougereau, M., and Seckinger, P. The SH3 domain of Bruton’s tyrosine kinase interacts with Vav, Sam68 and EWS. Scand. J. Immunol., 45: 587–595, 1997. Lessnick, S. L., Braun, B. S., Denny, C. T., and May, W. A. Multiple domains mediate transformation by the Ewing’s sarcoma EWS/FLI-1 fusion gene. Oncogene, 10: 423– 431, 1995. Jaishankar, S., Zhang, J., Roussel, M. F., and Baker, S. J. Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity. Oncogene, 18: 5592–5597, 1999. Petermann, R., Mossier, B. M., Aryee, D. N., Khazak, V., Golemis, E. A., and Kovar, H. Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II. Oncogene, 17: 603– 610, 1998. Kovar, H., Auinger, A., Jug, G., Aryee, D., Zoubek, A., Salzer Kuntschik, M., and Gadner, H. Narrow spectrum of infrequent p53 mutations and absence of MDM2 amplification in Ewing tumours. Oncogene, 8: 2683–2690, 1993. Hattinger, C. M., Rumpler, S., Strehl, S., Ambros, I. M., Zoubek, A., Potschger, U., Gadner, H., and Ambros, P. F. Prognostic impact of deletions at 1p36 and numerical aberrations in Ewing tumors. Genes Chromosomes Cancer, 24: 243–254, 1999. Hattinger, C. M., Rumpler, S., Kovar, H., and Ambros, P. F. Fine-mapping of cytogenetically undetectable EWS/ERG fusions on DNA fibres of Ewing tumors. Cytogenet. Cell Genet., in press, 2001. Zucman, J., Delattre, O., Desmaze, C., Plougastel, B., Joubert, I., Melot, T., Peter, M., De Jong, P., Rouleau, G., Aurias, A., and G. Thomas. Cloning and characterization of the Ewing’s sarcoma and peripheral neuroepithelioma t(11;22) translocation breakpoints. Genes Chromosomes Cancer, 5: 271–277, 1992. Desmaze, C., Zucman, J., Delattre, O., Melot, T., Thomas, G., and Aurias, A. Interphase molecular cytogenetics of Ewing’s sarcoma and peripheral neuroepithelioma t(11;22) with flanking and overlapping cosmid probes. Cancer Genet. Cytogenet., 74: 13–18, 1994. Sezer, O., Jugovic, D., Blohmer, J. U., Turzynski, A., Thiel, G., Langelotz, C., Possinger, K., and Kovar, H. CD99 positivity and EWS-FLI1 gene rearrangement identify a breast tumor in a 60-year-old patient with attributes of the Ewing family of neoplasms. Diagn. Mol. Pathol., 8: 120 –124, 1999. Zucman, J., Melot, T., Desmaze, C., Ghysdael, J., Plougastel, B., Peter, M., Zucker, J. M., Triche, T. J., Sheer, D., Turc Carel, C., Ambros, P., Combaret, V., Lenoir, G, Aurias, A, Thomas, G., and Delattre, O. Combinatorial generation of variable fusion proteins in the Ewing family of tumours. EMBO J., 12: 4481– 4487, 1993. Plougastel, B., Mattei, M. G., Thomas, G., and Delattre, O. Cloning and chromosome localization of the mouse Ews gene. Genomics, 23: 278 –281, 1994. Bertolotti, A., Lutz, Y., Heard, D. J., Chambon, P., and Tora, L. hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J., 15: 5022– 5031, 1996. Calvio, C., Neubauer, G., Mann, M., and Lamond, A. I. Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry. RNA, 1: 724 –733, 1995. Yang, L., Embree, L. J., Tsai, S., and Hickstein, D. D. Oncoprotein TLS interacts with serine-arginine proteins involved in RNA splicing. J. Biol. Chem., 273: 27761– 27764, 1998. Hicks, G. G., Singh, N., Nashabi, A., Mai, S., Bozek, G., Klewes, L., Arapovic, D., White, E. K., Koury, M. J., Oltz, E. M., Van Kaer, L., and Ruley, H. E. Fus deficiency in mice results in defective B-lymphocyte development and activation, high levels of chromosomal instability and perinatal death. Nat. Genet., 24: 175–179, 2000. Pinna, L. A. Casein kinase 2: an ’eminence grise’ in cellular regulation? Biochim. Biophys. Acta, 1054: 267–284, 1990. Bovee, J. V., Devilee, P., Cornelisse, C. J., Schuuring, E., and Hogendoorn, P. C. Identification of an EWS-pseudogene using translocation detection by RT-PCR in Ewing’s sarcoma. Biochem. Biophys. Res. Commun., 213: 1051–1060, 1995. Melot, T., Dauphinot, L., Sevenet, N., Radvanyi, F., and Delattre, O. Characterization of a new brain-specific isoform of the EWS oncoprotein. Eur. J. Biochem., 268: 3483–3489, 2001.