Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (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 complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding 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 required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to 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 enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, 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 mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.Hox proteins make critical contributions to cell fate and segmental patterning during embryonic development (30). As targets of oncogenic mutations in human and murine leukemias, they are also implicated in cancer pathogenesis (3,4,21,34,35), which likely reflects perturbations of their roles in normal hematopoietic cell differentiation (23). In these capacities, they are presumed to function as transcription factors whose DNA binding activities are mediated through a conserved motif known as the homeodomain, which is structurally related to the bacterial helix-turn-helix motif (48). However, at a molecular level, the contributions of Hox proteins to developmental processes and disease pathogenesis are inadequately explained, given their disappointingly poor in vitro DNA binding affinities and specificities as monomeric proteins. This has led to the proposal that additional factors are required to modulate the DNA binding and transcriptional properties of Hox proteins (13), which would be consistent with models for achievement of specificity by other classes of transcriptional proteins.Genetic and biochemical studies support the argument for a role for members of the Pbx, exd, and ceh-20 subfamily (5) of divergent homeodomain proteins as potential Hox cofactors. In Drosophila melanogaster, exd is required for the execution of genetic programs that are also dependent on Hox proteins for appropriate segment-specific expression (40,45,46). A similar role for mammalian Pbx proteins is suggested by genetic analyses demonstrating that sequence elements with features of Pbx-Hox consensus sites are required for appropriate expression of murine Hoxb-1 in th...

Pbx1 is a member of the TALE (three-amino acid loop extension) class of homeodomain transcription factors, which are components of hetero-oligomeric protein complexes thought to regulate developmental gene expression and to maintain differentiated cell states. In vitro studies have shown that Pbx1 regulates the activity of Ipf1 (also known as Pdx1), a ParaHox homeodomain transcription factor required for the development and function of the pancreas in mice and humans. To investigate in vivo roles of Pbx1 in pancreatic development and function, we examined pancreatic Pbx1 expression, and morphogenesis, cell differentiation and function in mice deficient for Pbx1. Pbx1-/- embryos had pancreatic hypoplasia and marked defects in exocrine and endocrine cell differentiation prior to death at embryonic day (E) 15 or E16. In these embryos, expression of Isl1 and Atoh5, essential regulators of pancreatic morphogenesis and differentiation, was severely reduced. Pbx1+/- adults had pancreatic islet malformations, impaired glucose tolerance and hypoinsulinemia. Thus, Pbx1 is essential for normal pancreatic development and function. Analysis of trans-heterozygous Pbx1+/- Ipf1+/- mice revealed in vivo genetic interactions between Pbx1 and Ipf1 that are essential for postnatal pancreatic function; these mice developed age-dependent overt diabetes mellitus, unlike Pbx1+/- or Ipf1+/- mice. Mutations affecting the Ipf1 protein may promote diabetes mellitus in mice and humans. This study suggests that perturbation of Pbx1 activity may also promote susceptibility to diabetes mellitus.