Trimeric association of hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity

Pbx/exd proteins modulate the DNA binding affinities and specificities of H ox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodime rize and bind DNA with Meis and Pknox1-Prep1, additional members of the TAL E (three-aminoacid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complex es with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates th e cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA bindin g specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be require d for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cogn ate DNA sites is topologically facilitated by the ability of Pbx and Meis t o interact through their amino termini and bind DNA without stringent half- site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhance r-directed expression of a reporter transgene in the murine embryonic hindb rain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are me diated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, a s a pair of essential cofactors in a higher-order molecular complex, to med iate its transcriptional effects on an endogenous Hox response element.