title: Co-expression of alternatively spliced forms of PAX3, PAX7, PAX3-FKHR and PAX7-FKHR with distinct DNA binding and transactivation properties in rhabdomyosarcoma.
abstract: PAX3 and PAX7 encode transcription factors implicated in the pathogenesis of rhabdomyosarcoma (RMS), including alveolar RMS in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR fusions. Previous studies of wild-type PAX3 and PAX7 identified alternative splicing events that modify the paired box and generate 2 isoforms of PAX3 (Q+ and Q-) and 4 isoforms of PAX7 (Q+GL+, Q+GL-, Q-GL+, Q-GL-). In our study, we investigated alternative splicing of the wild-type and fusion forms of PAX3 and PAX7 in alveolar and embryonal RMS and assessed the functional implications. For PAX3 and PAX3-FKHR, the Q+ and Q- isoforms were consistently co-expressed in RMS tumors with slightly higher levels of the Q+ isoform. For PAX7 and PAX7-FKHR, there was a consistent pattern of co-expression of the 4 isoforms in RMS tumors: Q+GL- > Q+GL+ >/= Q-GL- > Q-GL+. DNA binding analysis demonstrated that PAX3 and PAX3-FKHR Q- isoforms exhibit higher affinity than corresponding Q+ isoforms for class I sites and no difference for class II sites. For PAX7 and PAX7-FKHR, the relative affinity was Q-GL- > Q+GL- > Q-GL+ >/= Q+GL+ for class I sites and Q-GL-, Q+GL- > Q-GL+, Q+GL+ for class II sites. Finally, the transcriptional activities of the PAX3-FKHR and PAX7-FKHR isoforms on reporter plasmids varied over a 5-fold and 50-fold range, respectively, in accord with the differences in DNA binding activity. In conclusion, these studies reveal that PAX3, PAX7 and their fusions with FKHR are each expressed in RMS tumors as a consistent mixture of functionally distinct isoforms.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Shouying
authors.initials: S
authors.lastname: Du
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Lawrence
authors.forename: Donna
authors.initials: D
authors.lastname: Strzelecki
authors.forename: Prerna
authors.initials: P
authors.lastname: Rajput
authors.forename: Shujuan J
authors.initials: SJ
authors.lastname: Xia
authors.forename: Dina M
authors.initials: DM
authors.lastname: Gottesman
authors.forename: Frederic G
authors.initials: FG
authors.lastname: Barr
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry*; D005091:Exons; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D041681:NIH 3T3 Cells; D000072041:PAX3 Transcription Factor; D051763:PAX7 Transcription Factor; D051761:Paired Box Transcription Factors; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry*; 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; D018398:Homeodomain Proteins; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051763:PAX7 Transcription Factor; C497321:PAX7 protein, human; D051761:Paired Box Transcription Factors; C497322:Pax7 protein, mouse; D020033:Protein Isoforms; D014157:Transcription Factors; D004247:DNA