A group 13 homeodomain is neither necessary nor sufficient for posterior prevalence in the mouse limb

Posterior prevalence is the general property attributed to HOX proteins describing the dominant effect of more posterior HOX proteins over the function of anterior orthologs in common areas of expression. To explore the HOX group 13 protein domains required for this property, we used the mouse Prx-1 promoter to drive transgenic expression of Hox constructs throughout the entire limb bud during development. This system allowed us to conclusively demonstrate a hierarchy of Hox function in developing limbs. Furthermore, by substituting the HOXD11 or HOXA9 homeodomain for that of HOXD13, we show that a HOXD13 homeodomain is not necessary for posterior prevalence. Proximal expression of these chimeric proteins unexpectedly caused defects consistent with wild-type HOXD13 mediated posterior prevalence. Moreover, group 13 non-homeodomain residues appear to confer the property as proximal expression of HOXA9 containing the HOXD13 homeodomain did not result in limb reductions characteristic of HOXD13. These data are most compatible with models of posterior prevalence based on protein-protein interactions and support examination of the N-terminal non-homeodomain regions of Hox group 13 proteins as necessary agents for posterior prevalence.     

Lock and key binding of the HOX YPWM peptide to the PBX homeodomain

HOX homeodomain proteins bind short core DNA sequences to control very specific developmental processes. DNA binding affinity and sequence selectivity are increased by the formation of cooperative complexes with the PBX homeodomain protein. A conserved YPWM motif in the HOX protein is necessary for cooperative binding with PBX. We have determined the structure of a PBX homeodomain bound to a 14-mer DNA duplex. A relaxation-optimized procedure was developed to measure DNA residual dipolar couplings at natural abundance in the 20-kDa binary complex. When the PBX homeodomain binds to DNA, a fourth alpha-helix is formed in the homeodomain. This helix rigidifies the DNA recognition helix of PBX and forms a hydrophobic binding site for the HOX YPWM peptide. The HOX peptide itself shows some structure in solution and suggests that the interaction between PBX and HOX is an example of "lock and key" binding. The NMR structure explains the requirement of DNA for the PBX-HOX interaction and the increased affinity of DNA binding.