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. 1999 Jul;19(7):5134-42.
doi: 10.1128/MCB.19.7.5134.

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

Y Jacobs  1 , C A Schnabel, M L Cleary
Affiliations

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

Y Jacobs et al. Mol Cell Biol. 1999 Jul.

Abstract

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-amino-acid 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.

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Figures

FIG. 1
FIG. 1
The amino termini of Pbx and Meis proteins are necessary and sufficient for their heterodimerization in solution. (A) Schematic illustrations of Pbx and Meis proteins. The PBC-A and PBC-B domains consist of portions conserved in both the Pbx and Meis protein families (solid boxes) and portions conserved only within the Pbx or Meis subfamilies (lightly shaded areas). N-terminal and C-terminal deletion constructs are indicated by brackets. HD, homeodomain. (B) In vitro-produced proteins (indicated in schematic illustration above the gel lanes) were incubated together, immunoprecipitated with anti-Pbx1b antibodies (αPbx1b) or anti-Meis antibodies (αMeis) (indicated beneath the gel lanes), and then fractionated by SDS-PAGE. Coprecipitation of Meis-Pbx complexes was dependent on the amino termini of each protein and was not disrupted by the addition of increasing amounts of Hoxb1. CT, C terminus; NT, N terminus.
FIG. 2
FIG. 2
Pbx and Meis heterodimers bind DNA without stringent half-site orientation and spacing requirements. (A) In vitro-synthesized Pbx1 and Meis1 proteins were subjected to EMSA with DNA probes (schematically illustrated above the gel lanes). Dimers that formed irrespective of half-site spacing and orientation are indicated to the left. P, Pbx half site; M, Meis half site; N, inserted nucleotides between half sites; ×, mutant half site. Arrows indicate half-site orientations. (B) Schematic depictions of Pbx-Meis heterodimers binding to various configurations of DNA half sites.
FIG. 3
FIG. 3
Both the Hoxb2 r4 and Hoxb1 ARE enhancers contain Meis sites which are upstream and in reverse orientation with respect to Pbx-Hox sites. Nucleotide sequences are shown for HOX response elements employed as DNA probes for EMSA. Boxes and arrows indicate protein binding sites and their relative orientations. Mutations introduced into the Meis or Pbx-Hox sites of the Hoxb2 r4 element are shown below.
FIG. 4
FIG. 4
A trimeric Pbx1-Meis1-Hoxb1 DNA binding complex requires an intact Meis site for its formation of the Hoxb2 r4 element. (A) In vitro-synthesized proteins (indicated above the gel lanes) were subjected to EMSA with a radiolabeled Hoxb2 r4 element (20) containing an intact (lanes 1 to 3) or a mutant (lanes 4 to 6) Meis site. A trimeric complex that formed in the presence of all three proteins is indicated by an arrow. (B) In vitro-translated proteins were incubated in DNA binding reaction mixtures in the presence of a radiolabeled probe and then subjected to EMSA. Antibodies were added to selected binding reaction mixtures as indicated above lanes 2 to 4. ss, antibody-protein complexes resulting from supershift analyses. (C) EMSA was performed with the Hoxb2 r4 element and Hoxb1, Pbx1a, and Meis1a (lane 1). In lanes 2 to 4, a mutant protein (identity indicated above gel lanes) was substituted for the respective wild-type protein.
FIG. 5
FIG. 5
Trimeric Hoxb1-Pbx1-Meis1 DNA binding complexes preferentially bind to a subportion of the Hoxb1 ARE that contains consensus Meis and Pbx-Hox sites. EMSA was performed on DNA binding complexes containing various combinations of Pbx1, Meis1, or Hoxb1 proteins (indicated above the gel lanes). The DNA probes consisted of radiolabeled oligonucleotides containing subfragments of the Hoxb1 ARE (32), indicated schematically at the top. r1 to r3 were previously identified by their similarity to the Pbx consensus site. Migrations of dimer and trimer complexes are indicated to the left. Arrow heads denote trimeric complexes. Free DNA probes are not shown in the middle panels because they are smaller and migrate faster than the full-length ARE. The probe used in the rightmost panel consisted of a multimerized Pbx-Hox site from the Hoxb2 r4 enhancer (20).
FIG. 6
FIG. 6
Transcriptionally active trimeric DNA binding complexes display Meis site-dependent activity on the Hoxb2 enhancer. Luciferase activity was assayed from transiently transfected COS-7 cells. Cotransfection assays were performed in the presence (+) or absence (−) of the indicated expression plasmids encoding Pbx1a, Hoxb1, or Meis1 with reporter plasmids indicated at the tops of the respective panels. Reporter constructs contained a single 30-bp Hoxb2 enhancer element with Meis-Pbx-Hox sites (left panel) or the Hoxb1 ARE (right panel). The mutant Hoxb2 enhancer differed from the wild type by four nucleotide substitutions in the Meis site, as shown in Fig. 3. Data are expressed as the fold difference in luciferase activity obtained in comparison to activities obtained with a parental expression plasmid that did not contain coding sequences and a reporter plasmid that did not contain the enhancer element. Bars represent the means (plus standard deviations) of three to five independent experiments performed in duplicate.
FIG. 7
FIG. 7
Both the Meis and Pbx-Hox consensus sites are required for Hoxb2 r4 enhancer function in the hindbrain. (A) The Hoxb2 5′ flanking region is shown above, and transgene expression constructs are shown below. Krox20 sites are shown as dark filled ovals, Meis sites are shown as shaded ovals, and Pbx-Hox consensus sites are shown as open ovals. The domain(s) and frequency of expression for each construct are provided at the right (exp, expressing; trg, transgenic). The number of embryos with detectable expressions of the transgene are indicated along with the total number of embryos examined by lacZ staining. (B) Dorsal and lateral views of lacZ staining patterns in transgenic embryos. Constructs are indicated beneath the panels. Arrows indicate branchial arches. ov, otic vesicle. (C) Western blot analysis demonstrates the expression of Meis and Prep1 proteins in the hindbrain at embryonic day 9.5. In vitro translates of Meis and Prep1-Pknox1 are shown in the first two lanes, respectively. Extracts of COS-7 cells and microdissected hindbrain are shown in the right lanes. Immunoreactive proteins were detected with a commercial rabbit antiserum against Prep1 that also cross-reacts with Meis proteins.
FIG. 8
FIG. 8
Schematic representations of TALE homeoprotein trimeric complexes on various enhancer elements. Different trimeric complexes containing Hox, Pbx, and Meis-Prep1 components are depicted on enhancer elements whose in vivo functions have been reported in previous studies. There are two contrasting types of complexes: those in which the Meis-Prep1 component is simply tethered without binding to DNA and those in which DNA binding by the Meis component is essential.
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