Molecular analysis of the chromosomal translocations associated with the pediatric solid tumor alveolar rhabdomyosarcoma

The t(2;13)(q35;q14) and t(1;13)(p36;q14) chromosomal translocations are specifically associated with the pediatric solid tumor alveolar rhabdomyosarcoma (ARMS). The t(2;13) fuses PAX3, a paired box transcription factor family member on chromosome 2, with FKHR, a fork head transcription factor family member on chromosome 13. As a result of gene fusion, t(2;13) ARMS express fusion gene products encoding the N-terminus of PAX3 fused in-frame with the C-terminus of FKHR. Involvement of 13q14 in the t(1;13) suggests that FKHR is fused to a novel gene on chromosome 1. Using reverse-transcriptase PCR and Southern blot analyses, we demonstrate that the t(1;13) rearranges another paired box family member, PAX7 on chromosome 1, and fuses it to FKHR on chromosome 13. Thus, either translocation fuses homologous DNA-binding domains of PAX3 or PAX7 to identical FKHR sequences to create potentially similar chimeric transcription factors. Fusion of PAX3 and PAX7 to identical FKHR sequences suggests that the ARMS translocations rearrange the same intron in wild-type FKHR. By isolating and analyzing genomic clones, we find that FKHR consists of three exons spanning 140 kb and that the intron 1-exon 2 boundary precisely matches the fusion point in the fusion transcripts. Using pulsed-field and fluorescence in situ hybridization analyses, we demonstrate that FKHR intron 1 is rearranged in t(2;13) ARMS. Rearrangement of intron 1 with PAX3 produces two important functional consequences: in-frame fusion of PAX3 sequences to the FKHR transcriptional activation domain and disruption of the FKHR DNA binding domain. Finally, RNase protection and protein immunoprecipitation analyses demonstrate that these translocations are associated with fusion product overexpression. By analysing gene copy number, RNA stability and transcription levels for the wild-type and fusion genes, we demonstrate that PAX3-FKHR and PAX7-FKHR overexpression occurs by distinct mechanisms. Transcription of PAX3-FKHR is increased relative to wild-type PAX3 by a copy number-independent process. In contrast, PAX7-FKHR overexpression results from fusion gene amplification. We postulate that these overexpression mechanisms ensure a critical level of gene product for the oncogenic effects of these fusions