A Hox-Eya-Pax complex regulates early kidney developmental gene expression

During embryonic development, the anterior-posterior body axis is specified in part by the combinatorial activities of Hox genes. Given the poor DNA binding specificity of Hox proteins, their interaction with cofactors to regulate target genes is critical. However, few regulatory partners or downstream target genes have been identified. Herein, we demonstrate that Hox11 paralogous proteins form a complex with Pax2 and Eya1 to directly activate expression of Six2 and Gdnf in the metanephric mesenchyme. We have identified the binding site within the Six2 enhancer necessary for Hox11-Eya1-Pax2-mediated activation and demonstrate that this site is essential for Six2 expression in vivo. Furthermore, genetic interactions between Hox11 and Eya1 are consistent with their participation in the same pathway. Thus, anterior-posterior-patterning Hox proteins interact with Pax2 and Eya1, factors important for nephrogenic mesoderm specification, to directly regulate the activation of downstream target genes during early kidney development.     

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity are poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA-binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA-binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins.