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We report the genome-wide maps of PAX3-FKHR binding sites. Chromatin immunoprecipitation was performed against PAX3-FKHR positive (Rh4) and PAX3-FKHR negative (RD) rhabdomyosarcoma cells with a monoclonal antibody (pFM2) specific for the fusion region of PAX3-FKHR. We obtained 4 million sequence tags for both input and ChIP DNA that aligned to the human genome. We identified 1,463 binding sites from ChIP-seq of Rh4 cells, none of which appeared from ChIP-seq of fusion negative RD cells. The PAX3-FKHR binding sites were found to associate with 1,072 genes in RMS cells. The data shows that PAX3-FKHR binds to the same sites as PAX3, at the enhancers for MYF5, FGFR4, and the MYOD core enhancer previously shown to be regulated by PAX3. Moreover, our dataset has the precision for rapid identification and validation of novel and specific sequences required for the enhancer activity of MYOD and FGFR4. The genome wide analysis reveals that the PAX3-FKHR sites are: 1) mostly distal to transcription start sites; 2) conserved; 3) enriched for PAX3 motifs; and 4) strongly associated with genes over-expressed in PAX3-FKHR positive RMS cells and tumors. There is little evidence in our dataset for PAX3-FKHR binding at the promoters. In one instance, we show two intronic enhancer elements for MET, rather than at the previously described promoter. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common co-regulation for many target genes. The map of PAX3-FKHR binding sites provides new links for PAX3 and PAX3-FKHR functions and new targets for RMS therapy. Examination of PAX3-FKHR binding sites in translocation-positive rhabdomyosarcoma cells via ChIP-seq with an antibody specific for the fusion protein.

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