Anti-Barx2 polyclonal antibody

The downregulation of Pax7 expression is an essential requirement for adult muscle progenitor cell differentiation during muscle regeneration. We previously found that canonical Wnt signals drive myogenic differentiation by positively regulating the expression of myogenic target genes, and also by negatively regulating Pax7 expression. To better understand how Wnt signals repress Pax7 expression, we screened for Wnt-regulated microRNAs (miRNAs) that could target the Pax7 3 ' untranslated region (UTR). Using wild-type and beta-catenin null primary mouse myoblasts, we identified several Wnt/beta-catenin regulated miRNAs, the most abundant of which were miR-133b and miR-206. While miR-206 was previously identified as Pax7 regulator, miR-133b has never been shown to regulate the Pax7 transcript. We show here that miR-133b is a more potent inhibitor of Pax7 expression than miR-206, and that it acts via a site adjacent to the miR-206 binding site in the Pax7 3 ' UTR. The primary transcript encoding miR-133b/miR-206 is specifically induced by Wnt/beta-catenin, while the miR-1/miR-133a transcript is not; moreover, Wnt signals increase the secretion of mature miR-133b/miR-206 into exosomes. Overall, we conclude that miR-133b and to a lesser degree miR-206, but not miR-1 or miR-133a, are key components of the canonical Wnt-mediated pathway that allows differentiation to proceed by relieving Pax7-mediated repression of the myogenic program. Secretion of these miRNAs into exosomes may allow them to directly control the differentiation of neighboring cells.

Barx1 and Barx2 are homeodomain proteins originally identified using regulatory elements of genes encoding certain cell adhesion molecules (CAMs). In the present study, we characterize regions of Barx2 that bind to regulatory elements of genes encoding three CAMs, L1, neuron-glia CAIM (Ng-CAM), and neural CAM (N-CAM), and identify domains of Barx2 that regulate N-CAM transcription. The homeodomain of Barx2 was sufficient for binding to homeodomain binding sites (HBS) from all three CAM genes. The presence of a 17-amino acid Barx basic region resulted in a 2-fold decrease in binding to HBS sequences from the Ng-CAM and L1 genes, whereas it led to a 6.5-fold increase in binding to the HBS from the N-CAM promoter. Thus, the Barx basic region influences the strength and specificity of Barx2 binding to DNA In co-transfection experiments, Barx2 repressed N-CAM promoter activity. A 24-residue N-terminal region of Barx2 was essential for repression. When this region was absent, Barx2 activated the N-CAM promoter. A 63-residue C-terminal domain was required for this activation, In GST pull-down experiments, Barx2 bound to proteins of the CREB family, CREB1 and ATF2. Overall, these findings provide a framework for understanding developmental and physiological contexts that influence repressor or activator functions of Barx2.