title: Pax3 target gene recognition occurs through distinct modes that are differentially affected by disease-associated mutations.
abstract: The paired box protein Pax3 is an essential regulator of muscle and neural crest-derived cell types, including melanocytes. Within this lineage, Pax3 has been shown to regulate the genes encoding microphthalmia-associated transcription factor (Mitf) and tyrosinase-related protein-1 (Trp-1), despite each having dissimilar Pax3 recognition sequences. We have, therefore, examined the structural requirements for Pax3 binding to the MITF and TRP-1 promoter elements, focusing on the contribution of the paired domain and homeodomain to Pax3 target site recognition. Unexpectedly, although the MITF element is characterized by suboptimal recognition motifs for the paired domain and homeodomain, it sustains a higher level of Pax3 binding than TRP-1, which contains a canonical paired domain site. The basis for this difference involves a context-dependent cooperative binding event requiring both the paired domain and homeodomain, while the paired domain alone is sufficient for TRP-1 recognition. Significantly, the analysis of Waardenburg syndrome mutations reveals marked disparity in their effects on MITF and TRP-1 binding that further underscores mechanistic differences in their interaction with Pax3. Importantly, these mutations also exert distinct effects on the ability of Pax3 to regulate reporter genes fused to either the MITF or TRP-1 promoters. Our results, therefore, establish that Pax3 can regulate target genes through alternate modes of DNA recognition that are differentially impacted by disease-causing mutations, which together have important implications for understanding Pax3-regulated gene networks.
authors.affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Gareth N
authors.initials: GN
authors.lastname: Corry
authors.forename: D Alan
authors.initials: DA
authors.lastname: Underhill
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics* / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000072041:PAX3 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014849:Waardenburg Syndrome / Q000235:genetics*
chemical_list: C497234:MITF protein, human; D008562:Membrane Glycoproteins; D051739:Microphthalmia-Associated Transcription Factor; D000072041:PAX3 Transcription Factor; C489409:PAX3 protein, human; D051761:Paired Box Transcription Factors; D011994:Recombinant Proteins; D004247:DNA; D010088:Oxidoreductases; D008156:Luciferases; C489799:TYRP1 protein, human