Heterodimerization of Msx and Dlx homeoproteins results in functional antagonism

Protein-protein interactions are known to be essential for specifying the transcriptional activities of homeoproteins, Here we show that representative members of the Msx and Dir homeoprotein families form homo- and heterodimeric complexes, We demonstrate that dimerization by MSK and Dir proteins is mediated through their homeodomains and that the residues required for this interaction correspond to those necessary for DNA binding. Unlike most other known examples of homeoprotein interactions, association of Msx and Dir proteins does not promote cooperative DNA binding; instead, dimerization and DNA binding are mutually exclusive activities, In particular, we show that Msx and Dir proteins interact independently and noncooperatively with homeodomain DNA binding sites and that dimerization is specifically blocked by the presence of such DNA sites, We further demonstrate that the transcriptional properties of Msx and Dir proteins display reciprocal inhibition. Specifically, Msx proteins act as transcriptional repressors and Dir proteins act as activators, while in combination, Msx and Dir proteins counteract each other's transcriptional activities, Finally, we show that the expression patterns of representative Msx and Dir genes (Msx1, Msx2, Dlx2, and Dlx5) overlap in mouse embryogenesis during limb bud and craniofacial development, consistent with the potential for their protein products to interact in vivo. Based on these observations, we propose that functional antagonism through heterodimer formation provides a mechanism for regulating the transcriptional actions of Msx and Dir homeoproteins in vivo.