Richard J. Davis¹, Jeannette L. Bennicelli¹, Roberto A. Macina³, Lynn M. Nycum⁴, Jaclyn A. Biegel^2,4 and Frederic G. Barr^1,*

¹Departments of Pathology and Laboratory Medicine 37th Street and Hamilton Walk, Philadelphia, PA 19104-6082 ²Departments of Pediatrics, University of Pennsylvania School of Medicine 37th Street and Hamilton Walk, Philadelphia, PA 19104-6082 ³The Wistar Institute 3601 Spruce Street, Philadelphia, PA 19104 ⁴Division of Human Genetics and Molecular Biology, The Children’s Hospital of Philadelphia Philadelphia, PA 19104, USA

^*To whom correspondence should be addressed

Received August 23, 1995; Revised September 25, 1995; Accepted September 25, 1995

The FKHR gene, which contains a forkhead DNA-binding motif, is fused to either PAX3 or PAX7 by the t(2;13) or t(1;13) translocation in alveolar rhabdo-myosarcoma, respectively. These tumors express chimeric transcripts encoding the N-terminal portion of either PAX protein fused to the C-terminal portion of FKHR. To understand the structural basis and functional consequences of these translocations, we characterized the wild-type FKHR gene and its rearrangement in alveolar rhabdomyosarcomas. By isolating and analyzing phage, cosmid and YAC clones, we determined that FKHR consists of three exons spanning 140 kb and that several highly similar loci are present in other genomic regions. Exon 1 encodes the N-terminus of the forkhead domain and is embedded within a demethylated CpG island. RNA analyses reveal FKHR transcripts initiate from a TATA-less promoter within this island. Exon 2 encodes the C-terminus of the forkhead domain and a transcriptional activation domain, whereas exon 3 encodes a large 3′ untranslated region. The intron 1—exon 2 boundary precisely matches the FKHR fusion point in the chimeric transcripts found in alveolar rhabdo-myosarcomas. Using pulsed-field and fluorescence in situ hybridization analyses, we demonstrate that the 130 kb FKHR intron 1 is rearranged in t(2;13)-containing alveolar rhabdomyosarcomas. Our findings indicate that FKHR intron 1 provides a large target for DNA rearrangement. Rearrangement of this intron with PAX3 produces two important functional consequences: in-frame fusion of N-terminal PAX3 sequences to the FKHR transcriptional activation domain and disruption of the FKHR DNA binding domain.

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