Lymphoid enhancer factor-1 mediates loading of Pax3 to a promoter harbouring lymphoid enhancer factor-1 binding sites resulting in enhancement of transcription

The transcription factor, Pax3, alters transcription by binding directly to promoter regions harbouring sequences recognized by either its paired domain or its homeodomain. We demonstrated previously that the promoter regions of many of the genes whose expression was altered during a Pax3-induced mesenchymal-to-epithelial transition harboured sequences recognized by lymphoid enhancer factor-1 (Lef1). Given the apparent lack of DNA-binding consensus sequences for Pax3 in these promoters, it was hypothesized that Pax3 might alter transcriptional activity of promoters harbouring Lef1-binding sites independent of Pax3 binding to DNA. We describe here a novel mode of Pax3-dependent regulation of transcription that is mediated through DNA-independent binding to Lef1. Specifically, we demonstrate that Pax3 binds to Lef1, determined in binding assays and co-immunoprecipitation of endogenous Pax3 and Lef1. Binding assays employing deletion mutants of Pax3 and Lef1 determined that association was mediated through the homeodomain of Pax3 and the first half of the Lef1 DNA-binding domain. The significance of this association was demonstrated in transcriptional assays using a luciferase reporter gene downstream of a model promoter harbouring Lef1 DNA-binding consensus sites. Pax3 augmented Lef1-dependent transactivation from this promoter. This increase in transcriptional activity occurred in the absence and presence of added β-catenin. Chromatin immunoprecipitation assays demonstrated further that Pax3-association to complexes bound to DNA harbouring Lef1 consensus sequences was dependent on Lef1. These data reveal a novel mode of transcriptional regulation by Pax3. This mode of transcriptional regulation suggests further that Pax3 activity may directly effect the expression of factors regulated by signal transduction pathways dependent on Lef1.