Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer

Abstract

The PAX3-FKHR fusion protein is present in a majority of alveolar rhabdomyosarcomas associated with increased aggressiveness and poor prognosis. To better understand the molecular pathogenesis of PAX3-FKHR, we carried out the first, unbiased genome-wide identification of PAX3-FKHR binding sites and associated target genes in alveolar rhabdomyosarcoma. The data shows that PAX3-FKHR binds to the same sites as PAX3 at both MYF5 and MYOD enhancers. The genome-wide analysis reveals that the PAX3-FKHR sites are (a) mostly distal to transcription start sites, (b) conserved, (c) enriched for PAX3 motifs, and (d) strongly associated with genes overexpressed in PAX3-FKHR-positive rhabdomyosarcoma cells and tumors. There is little evidence in our data set for PAX3-FKHR binding at the promoter sequences. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common coregulation for many target genes. We also provide the first direct evidence that FGFR4 and IGF1R are the targets for PAX3-FKHR. The map of PAX3-FKHR binding sites provides a framework for understanding the pathogenic roles of PAX3-FKHR, as well as its molecular targets to allow a systematic evaluation of agents against this aggressive rhabdomyosarcoma.

Figure 1

ChIP-seq analysis of PAX3-FKHR binding sites in rhabdomyosarcoma cells. A, immunoblots show that PFM2 recognizes PAX3-FKHR protein in RMS cell lines positive for the translocation (RMS13 and Rh30). B, quantitative PCR analysis shows that PFM2 selectively immunoprecipitates the MYF5 enhancer from PAX3-FKHR⁺ Rh4 cells, not from the negative RD cells. C, distribution of PAX3-FKHR binding sites in chromosome 11. Peak height represents the number of sequence tags. D, a PAX3-FKHR site mapped within the MYF5 enhancer. The maximum signal aligns with a known PAX3 recognition site at this PAX3-dependent enhancer.

Figure 2

PAX3-FKHR binding sites as putative enhancers. A, distribution of PAX3-FKHR sites relating to the transcription start sites, exons, and introns. Enrichment or suppression PAX3-FKHR sites are shown. Unit of 1 represents the average of number of sites across the genome. B, degree of conservation of PAX3-FKHR sites compared with that of Pol-II ChIP-seq sites and randomly generated sequence tags. C, gene ontology analysis of genes associated with PAX3-FKHR sites with Ingenuity IPA. D, the association of PAX3-FKHR sites with genes up-regulated by PAX3-FKHR in RD cells (Table S3), and genes up-regulated in a panel of PAX3/7-FKHR positive RMS tumors (Table S4).

Figure 3

PAX3 recognition sequence in the MYOD core enhancer and the co-enrichment of PAX3 and E-box motifs in the binding sites. A, the identification of a novel PAX3 recognition sequence at the MYOD core enhancer and site-direct mutagenesis study of the site in transfected A204 RMS cells. B, enrichment of transcription factor motifs in PAX3-FKHR sites and clustering the top enriched motifs. C, results of a de novo motif search in which 9 of the top 10 motifs were shown to be related to either E-box or PAX3 motifs (see Table S6 for details). D, co-enrichment of PAX3 and E-box motifs among PAX3-FKHR sites. Either PAX3B or LBP1 was anchored, and the relative enrichment of other sites within 100 bp of the anchor is shown.

Figure 4

Fine mapping of a PAX3-FKHR-dependent enhancer for FGFR4 gene. A, ChIP-seq results identified two PAX3-FKHR binding sites downstream of FGFR4. The peak of the FGFR4.E2 binding signal is associated with a conserved PAX3 sequence. B, enhancer activity both for both binding sites in PAX3-FKHR (P3F) positive and negative cells; and with co-transfection PAX3 or PAX3-FKHR expression plasmid in RD cells. C, site-direct mutagenesis of PAX3 recognition sequence FGFR4.E2 enhancer resulted in the loss of trans-activation by PAX3 or PAX3-FKHR in RD cells. D, RT-qPCR data showing down-regulation of PAX3-FKHR with a lentiviral shRNA (s2) vector led to reduced ALK and FGFR4 mRNA in Rh4.

Figure 5

IGF1R as a target for PAX3-FKHR transactivation. A, identification of a PAX3-FKHR binding site in the 2^nd intron of IGF1R. ChIP-seq results of input and PFM2 IP DNA from two experiments are shown. B, PAX3 and PAX3-FKHR-dependent activation of the IGF1R enhancer in co-transfected RD cells. C, immunoblots of IGF1R, MYCN, MET, and PAX3-FKHR in a panel of RMS cell lines. D, shRNA lentiviral vectors against PAX3-FKHR results in the down-regulation of IGF1R, MYCN, and MET in Rh4.