title: The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.
abstract: Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The 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 using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.
authors.affiliation: Wistar Institute, University of Pennsylvania School of Medicine, Philadelphia 19104.
authors.forename: W J
authors.initials: WJ
authors.lastname: Fredericks
authors.forename: N
authors.initials: N
authors.lastname: Galili
authors.forename: S
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: G
authors.initials: G
authors.lastname: Rovera
authors.forename: J
authors.initials: J
authors.lastname: Bennicelli
authors.forename: F G
authors.initials: FG
authors.lastname: Barr
authors.forename: F J
authors.initials: FJ
authors.lastname: Rauscher
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D002882:Chromosomes, Human, Pair 13; D002889:Chromosomes, Human, Pair 2; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015870:Gene Expression; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion / Q000096:biosynthesis / Q000235:genetics*; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors; D014176:Protein Biosynthesis; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection; D014178:Translocation, Genetic
chemical_list: D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015345:Oligonucleotide Probes; D015514:Oncogene Proteins, Fusion; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D014157:Transcription Factors; C068686:Pax3 protein, mouse