SPECIFIC RESIDUES IN THE PBX HOMEODOMAIN DIFFERENTIALLY MODULATE THE DNA-BINDING ACTIVITY OF HOX AND ENGRAILED PROTEINS

Two classes of homeodomain proteins, Hox and Engrailed, have been show n to act in concert with the atypical homeodomain proteins Pbx and ext radenticle. We now show that specific residues located within the Pbx homeodomain are essential for cooperative DNA binding with Hox and Eng railed gene products. Within the N-terminal region of the Pbx homeodom ain, we have identified a residue that is required for cooperative DNA binding with three Hox gene products but not for cooperativity with E ngrailed-2 (En-2). Furthermore, there are similarities between heterod imeric interactions involving the yeast mating type proteins MATa1 and MAT alpha 2 and those that allow the formation of Pbx/Hox and Pbx/En- 2 heterodimers. Specifically, residues located in the al homeodomain t hat were previously shown to form a hydrophobic pocket allowing the al pha 2 C-terminal tail to bind, are also required for Pbx/Hox and Pbx/E n-2 cooperativity. Furthermore, we show that three residues located in the turn between helix 1 and helix 2, characteristic of many atypical homeodomain proteins, are required for cooperative DNA binding involv ing both Hox and En-2. Replacement of the three residues located in th e turn between helix 1 and helix 2 of the Pbx homeodomain with those o f the atypical homeodomain proteins controlling cell fate in the basid iomycete Ustilago maydis, bE5 and bE6, allows cooperative DNA binding with three Hox members but abolishes interactions with En-2. The data suggest that the molecular mechanism of homeodomain protein interactio ns that control cell fate in Saccharomyces cerevisiae and in the basid iomycetes may well be conserved in part in multicellular organisms.