THE PAX3-FKHR FUSION PROTEIN CREATED BY THE T(2 13) TRANSLOCATION IN ALVEOLAR RHABDOMYOSARCOMAS IS A MORE POTENT TRANSCRIPTIONAL ACTIVATOR THAN PAX3/

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translo cation of chromosomes 2 and 13 [t(2;1 3) (q35;q14)]. The genes on each chromosome involved in this transloca tion have been identified as the transcription factor-encoding genes P AX3 and FKHR, The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chr omosome 13-derived FKHR gene, a novel member of the forkhead DNA-bindi ng domain family. To determine the role of the fusion protein in trans criptional regulation and oncogenesis, we identified the PAX3-FKHR fus ion protein and characterized its function(s) as a transcription facto r relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor c ell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKH R sera demonstrated expression of a 97-kDa PAX3 FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified tha t a single polypeptide contains epitopes derived from each protein, Th e PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays u sing a canonical PAX binding site (e5 sequence), we found that DNA bin ding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains, How ever, the PAX3-FKHR fusion protein was a much more potent transcriptio nal activator than PAX3 as determined by transient cotransfection assa ys using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.