Ewing's sarcoma and related subtypes of primitive neuroectodermal tumours share a recurrent and specific t(11;22) (q24;q12) chromosome translocation, the breakpoints of which have recently been cloned. Phylogenetically conserved restriction fragments in the vicinity of EWSR1 and EWSR2, the genomic regions where the breakpoints of chromosome 22 and chromosome 11 are, respectively, have allowed identification of transcribed sequences from these regions and has indicated that a hybrid transcript might be generated by the translocation. Here we use these fragments to screen human complementary DNA libraries to show that the translocation alters the open reading frame of an expressed gene on chromosome 22 gene by substituting a sequence encoding a putative RNA-binding domain for that of the DNA-binding domain of the human homologue of murine Fli-1.

A subgroup of small-round-cell tumors identified as belonging to the Ewing family of tumors can be defined according to a specific molecular genetic lesion that is detectable by a rapid, reliable, and efficient method. This approach can be applied to small specimens obtained by fine-needle biopsies.

Balanced translocations involving band q12 of human chromosome 22 are the most frequent recurrent translocations observed in human solid tumours. It has been shown recently that this region encodes EWS, a protein with an RNA binding homologous domain. In Ewing's sarcoma and malignant melanoma of soft parts, translocations of band 22q12 to chromosome 11 and 12 result in the fusion of EWS with the transcription factors FLI‐1 and ATF1, respectively. The present analysis of 89 Ewing's sarcomas and related tumours show that in addition to the expected EWS‐FLI‐1 fusion, the EWS gene can be fused to ERG, a transcription factor closely related to FLI‐1 but located on chromosome 21. The position of the chromosome translocation breakpoints are shown to be restricted to introns 7‐10 of the EWS gene and widely dispersed within introns 3‐9 of the Ets‐related genes. This heterogeneity generates a variety of chimeric proteins that can be detected by immuno‐precipitation. On rare occasions, they may be associated with a truncated EWS protein arising from alternate splicing. All 13 different fusion proteins that were evidenced contained the N‐terminal domain of EWS and the Ets domain of FLI‐1 or ERG suggesting that oncogenic conversion is achieved by the linking of the two domains with no marked constraint on the connecting peptide.

The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAF II 68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF II s). In vitro binding studies revealed that both EWS and hTAF II 68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAF II 68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAF II 68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAF II 68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS-FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS-FLI-1 may play different roles in Pol II transcription.Structural alteration or aberrant expression of transcription factors is often a critical event in tumorigenic transformation (13,19, 22). Karyotypic analysis has revealed a tumor-specific t(11;22)(q24;q12) chromosomal translocation in 86% of both Ewing sarcoma and primitive neuroectodermal tumor, suggesting that the product of this rearrangement is involved in the formation of these malignancies (34). This chromosomal translocation fuses the EWS gene on chromosome 22 to the FLI-1 gene on chromosome 11 (8). EWS is a protein with unknown function containing an RNA-binding motif and an activation domain(s) (18,24,25). In the EWS-FLI-1 fusion protein, the RNA-binding motif containing the C-terminal half of EWS is replaced by the DNA-binding domain (DBD) of the FLI-1 protein. FLI-1 is a member of the ETS family of transcription factors which activate specific target genes by binding to their cognate DNA sequences through their DNA-binding regions, usually located at their carboxyl termini (2, 37). The replacement of the native transcription activation domain(s) of FLI-1 by the N-terminal region of EWS converts the nontransforming activator, FLI-1, into a transforming protein with new transcriptional activation potential. In the EWS-FLI-1 fusion protein, both the N-terminal domain of EWS and the DBD of FLI-1 are necessary for the transforming activity (20). Recently, the EWS gene was also shown to be involved in tumorigenesis by chromosomal translocation with other genes encoding either other members of the ETS family (Erg, ETV1, E1A-F, and FEV) or other transcription factors, including ATF-1, WT1, and the nuclear orphan receptor TEC1 (...