The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q1 4) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-te rminal forkhead (FKHR) sequences that contain a potent transcriptional acti vation domain. To determine which of these domains are required for cellula r transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally e xpressed in NIH 3T3 cells and evaluated for their capacity to promote ancho rage-independent cell growth. Mutational analysis revealed that both the th ird alpha-helix of the homeodomain and a small region of the FKHR transacti vation domain are absolutely required for efficient transformation by the P ax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potent ial equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for trans formation. Our results demonstrate that the integrity of the Pax3 homeodoma in recognition helix and the FKHR transactivation domain is necessary for e fficient cellular transformation by the Pax3-FKHR fusion protein.