PBX MODULATION OF HOX HOMEODOMAIN AMINO-TERMINAL ARMS ESTABLISHES DIFFERENT DNA-BINDING SPECIFICITIES ACROSS THE HOX LOCUS

Pbx cofactors are implicated to play important roles in modulating the DNA-binding properties of heterologous homeodomain proteins, includin g class I Hox proteins. To assess how Pbx proteins influence Hox DNA b inding specificity, we used a binding-site selection approach to deter mine high-affinity target sites recognized by various Pbx-Hox homeopro tein complexes, Pbx-Hox heterodimers preferred to bind a bipartite seq uence 5'-ATGATTNATNN-3' consisting of two adjacent half sites in which the Pbx component of the heterodimer contacted the 5' half (ATGAT) an d the Hox component contacted the more variable 3' half (TNATNN). Bind ing sites matching the consensus mere also obtained for Pbxl complexed with HoxA10, which lacks a hexapeptide but requires a conserved trypt ophan-containing motif for cooperativity with Pbx. Interactions vvith Pbx were found to play an essential role in modulating Hox homeodomain amino-terminal arm contact with DNA in the core of the Hox half site such that heterodimers of different compositions could distinguish sin gle nucleotide alterations in the Hox half site both in vitro and in c ellular assays measuring transactivation. When complexed with Pbx, Hox proteins B1 through B9 and A10 showed stepwise differences in their p references for nucleotides in the Hox half site core (TTAT to TGAT, 5' to 3') that correlated with the locations of their respective genes i n the Hox cluster. These observations demonstrate previously undetecte d DNA-binding specificity for the amino-terminal arm of the Hox homeod omain and suggest that different binding activities of Pbx-Hox complex es are at least part of the position-specific activities of the Hox ge nes.