Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos.

Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities.     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Regulatory role for a conserved motif adjacent to the homeodomain of Hox10 proteins

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.     

The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities.

The Arabidopsis Athb-1 and -2 proteins are characterized by the presence of a homeodomain (HD) with a closely linked leucine zipper motif (Zip). We have suggested that the HD-Zip motif could, via dimerization of the leucine zippers, recognize dyad-symmetric DNA sequences. Here we report an analysis of the DNA binding properties of the Athb-1 homeodomain-leucine zipper (HD-Zip-1) domain in vitro. DNA binding analysis performed using random-sequence DNA templates showed that the HD-Zip-1 domain, but not the Athb-1 HD alone, binds to DNA. The HD-Zip-1 domain recognizes a 9 bp dyad-symmetric sequence [CAAT(A/T)ATTG], as determined by selecting high-affinity binding sites from random-sequence DNA. Gel retardation assays demonstrated that the HD-Zip-1 domain binds to DNA as a dimer. Moreover, the analysis of the DNA binding activity of Athb-1 derivatives indicated that a correct spatial relationship between the HD and the Zip is essential for DNA binding. Finally, we determined that the Athb-2 HD-Zip domain recognizes a distinct 9 bp dyad-symmetric sequence [CAAT(G/C)ATTG]. A model of DNA binding by the HD-Zip proteins is proposed.     

Embryonic expression patterns of the Hox genes of the crayfish Procambarus clarkii (Crustacea, Decapoda)

SUMMARY Higher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods. The superorders Eucarida and Peracarida, two large groups that separated over 350 million years ago, encompass most malacostracan diversity. Recently, the Hox genes of the peracarid woodlouse Porcellio scaber (Isopoda) were shown to be expressed in domains that coincide with morphological boundaries of body tagmata, which differ from those in insects ( Abzhanov and Kaufman 1999a,b ). Moreover, observed changes in Hox expression domains during ontogeny correlate with morphological remodeling, such as a transformation of the first thoracic leg into mouthpart maxillipeds, which occurs in the trunk of the embryo. Decapods have a different modification of the malacostracan bodyplan, with up to three pairs of maxillipeds and extensive fusion and cephalization of the thorax. Here we describe expression patterns of the trunk Hox genes Scr, Antp, Ubx, abd-A and cad in the eucarid crayfish Procambarus clarkii (Decapoda). We find that the crayfish expression patterns, for the most part, resemble those of the woodlouse Porcellio scaber (Isopoda), but are more modulated and complex . Nevertheless, as in Porcellio the boundaries of the Hox expression domains do correlate with morphological features and their modulations to transformations in the embryo. Thus we propose that the trunk Hox genes were likely important in the evolution of and currently play an essential role in the development of the complex decapod bodyplan.     

Expression and function of the homoeotic genes Antennapedia and Sex combs reduced in the embryonic midgut of Drosophila

Drosophila homoeotic genes control the formation of external morphological features of the embryo and adult, and in addition affect differentiation of the nervous system. Here we describe the morphogenetic events in the midgut that are controlled by the homoeotic genes Sex combs reduced (Scr) and Antennapedia (Antp). The midgut is composed of two cell layers, an inner endoderm and an outer visceral mesoderm that surround the yolk. Scr and Antp are expressed in the visceral mesoderm but not in the endoderm. The two genes are required for different aspects of the midgut morphogenesis. In Scr null mutant embryos the gastric caeca fail to form. Scr is expressed in the visceral mesoderm cells posterior to the primordia of the gastric caeca and appears to be indirectly required for the formation of the caeca. Antp is expressed in visceral mesoderm cells that overlie a part of the midgut where a constriction will form, and Antp null mutant embryos fail to form this constriction. An ultrastructural analysis of the midgut reveals that the visceral mesoderm imposes the constriction on the endoderm and the yolk. The mesodermal tissue contracts within the constriction and thereby penetrates the layer of the midgut endoderm. Microtubules participate in the morphological changes of the visceral mesoderm cells. The analysis of the expression of Scr in Antp mutant embryos revealed a case of tissue-specific regulation of Scr expression by Antp. In the epidermis, Antp has been shown to negatively regulate Scr, but it positively regulates Scr in the visceral mesoderm.     

Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex.

Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.