The Oncogenic Potential of the Pax3-FKHR Fusion Protein Requires the Pax3 Homeodomain Recognition Helix but Not the Pax3 Paired-Box DNA Binding Domain

Abstract

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third α-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

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The Oncogenic Potential of the Pax3-FKHR Fusion Protein Requires the Pax3 Homeodomain Recognition Helix but Not the Pax3 Paired-Box DNA Binding Domain

ACKNOWLEDGMENTS

We thank Peter Gruss, Max Planck Institute, for the murine Pax3 cDNA, pBH3.2; Charles Sawyers, UCLA Medical Center, for the pSRα(ΔHindIII)-tk-Neo retroviral vector; Martyn Goulding, the Salk Institute for Biological Studies, for the pTK-CAT vector; David Baltimore, California Institute of Technology, for the 293T cells; and Christopher Denny, University of California at Los Angeles, for the NIH 3T3 cells. We also thank Carol Bockhold, Craig McPherson and Rose Mathew for their expert technical assistance. Finally, we thank Suzanne Baker, Thomas Curran, Gerard Grosveld, and Susan Watson for their critical reading of the manuscript.

This work was supported, in part, by NIH grants CA-56819 and PO1-CA-71907 (M.F.R.), the Cancer Center (CORE) support grant CA21765, and the American Lebanese Syrian Associated Charities (ALSAC) of St. Jude Children’s Research Hospital.