In addition to their role as linkers between cadherins and the cytoskeleton, B-catenin and plakoglobin (gamma-catenin) regulate gene expression by binding to TCF-type HMG-box transcription factors. Catenin-TCF interactions are modulated by the canonical Wnt signaling pathway. Wnt signaling is essential at multiple points in development and defects in catenin regulation are associated with a number of human diseases, in particular cancer. Recently, we discovered an unexpected regulator of catenin-TCF activity, SOX-type HMG box transcription factors that compete with TCFs for binding to catenins. These SOXs can modulate cellular responses to Wnt-catenin-TCF signaling, and so play a critical role in Wnt/TCF-dependent events and pathogenic processes. In Xenopus dorsal-ventral axis determination is based on catenin-TCF interactions and provides an ideal system in which to test the role of catenin-binding SOXs in modulating catenin-TCFregulated gene expression. We propose to answer three specific questions. I. Do the catenin-binding SOXs compete effectively with XLEF1 and XTCF3 for binding to B-catenin in vivo and in vitro? We will use a combination of immunoprecipitation analysis and BlAcore plasmon resonance measurements to define relative and absolute affinities between these proteins. II. Do SOXs compete effectively with B-catenin/TCF for binding to specific regulatory sites in target genes? Four TCF-regulated target promoters, associated with dorsal-ventral axis determination and mesoderm differentiation in Xenopus have been identified, i.e. Siamois, Twin, Xnr3 and XBra. We will test whether SOXs expressed in the early embryo, and known to interfere with TCF signaling, bind directly to sites within these promoters and whether SOXs complete effectively with TCFs for binding to specific regulatory elements. Ill. Do maternal and zygotic SOXs modulate the expression of B-catenin/TCF regulated genes in vivo? Ectopic expression of catenin-binding XSOX3 and XSOX17B inhibits B-catenin-dependent dorsal axis formation. We will test whether decreasing the expression of specific SOXs using anti-sense reagents leads to the predicted expansion of TCF regulated target genes and/or increased sensitivity of embryonic cells to Wnt signaling components

R01GM054001

1995-08-01

2006-09-30