Faculty Bibliography

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs,the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

The Hox genes are involved in patterning along the A/P axes of animals. The clustered organization of Hox genes is conserved from nematodes to vertebrates. During evolution, the number of Hox genes within the ancestral complex increased, exemplified by the five-fold amplification of the AbdB-related genes, leading to a total number of thirteen paralogs. This was followed by successive duplications of the cluster to give rise to the four vertebrate HOX clusters. A specific subset of paralogs was subsequently lost from each cluster, yet the composition of each cluster was likely conserved during tetrapod evolution. While the HOXA, HOXC and HOXD clusters contain four to five AbdB-related genes, only one gene (Hoxb-9) is found in the HOXB complex. We have identified a new member of paralog group 13 in human and mouse, and shown that it is in fact Hoxb-13. A combination of genetic and physical mapping demonstrates that the new gene is found approx. 70 kb upstream of Hoxb-9 in the same transcriptional orientation as the rest of the cluster. Despite its relatively large distance from the HOX complex, Hoxb-13 exhibits temporal and spatial colinearity in the main body axis of the mouse embryo. The onset of transcription occurs at E9.0 in the tailbud region. At later stages of development, Hoxb-13 is expressed in the tailbud and posterior domains in the spinal cord, digestive tract and urogenital system. However, it is not expressed in the secondary axes such as the limbs and genital tubercle. These results indicate that the 5' end of the HOXB cluster has not been lost and that at least one member exists and is highly conserved among different vertebrate species. Because of its separation from the complex, Hoxb-13 may provide an important system to dissect the mechanism(s) responsible for the maintenance of colinearity.

Nearly all metazoan homeodomains (HDs) possess DNA binding targets that are related by the presence of a TAAT sequence. We use an in vitro genetic DNA binding site selection assay to refine our understanding of the amino acid determinants for the recognition of the TAAT site. Superimposed upon the conserved ability of metazoan HDs to recognize a TAAT core is a difference in their preference for the bases that lie immediately 3' to it. Amino acid position 50 of the HD has been shown to discriminate among these base pairs, and structural studies have suggested that water-mediated hydrogen bonds and van der Waals contacts underlie for this ability. Here, we show that each of six amino acids tested at position 50 can confer a distinct DNA binding specificity.

We have cloned a Drosophila homologue (D-gsc) of the vertebrate homeobox gene goosecoid (gsc). In the Gsc proteins, the pressure for conservation has been imposed on the homeodomain, the functional domain of the protein: sequence homology is limited to the homeodomain (78% identity) and to a short stretch of 7 aminoacids also found in other homeoproteins such as Engrailed. Despite this weak homology, D-gsc is able to mimic gsc function in a Xenopus assay, as shown by its ability to rescue the axis development of a UV-irradiated embryo. Moreover, our data suggest that the position of insect and vertebrate gsc homologues within a regulatory network has also been conserved: D-gsc expression is controlled by decapentaplegic, orthodenticle, sloppy-paired and tailless whose homologues control gsc expression (for BMP4 and Otx-2), or are expressed at the right time and the right place (for XFKH1/Pintallavis and Tlx) to be interacting with gsc during vertebrate development. However, the pattern of D-gsc expression in ectodermal cells of the nervous system and foregut cannot easily be reconciled with that of vertebrate gsc mesodermal expression, suggesting that its precise developmental function might have diverged. Still, this comparison of domains of expression and functions among Gsc proteins could shed light on a common origin of gut formation and/or on basic cellular processes. The identification of gsc target genes and/or other genes involved in similar developmental processes will allow the definition of the precise phylogenetic relationship among Gsc proteins.

A Drosophila Stat gene (D-Stat) with a zygotic segmental expression pattern was identified. This protein becomes phosphorylated on Tyr-704 when coexpressed in Schneider cells with a Drosophila janus kinase (JAK), Hopscotch (HOP). The phosphorylated protein binds specifically to the consensus sequence TTCCCGGAA. Suppressor mutations of hopTum-I, a dominant hyperactive allele of hop whose phenotype is hematocyte overproduction and tumor formation, were selected. One of these mutants, statHJ, mapped to the same chromosomal region (92E) as does D-Stat, had an incompletely penetrant pair rule phenotype, and exhibited aberrant expression of the pair rule gene even skipped (eve) at the cellular blastoderm stage. Two D-STAT-binding sites were identified within the eve stripe 3 enhancer region. Mutations in either of the STAT-binding sites greatly decreased the stripe 3 expression in transgenic flies. Clearly, the JAK-STAT pathway is connected to Drosophila early development.

The Drosophila gene buttonhead (btd) encodes a zinc-finger protein related to the human transcription factor Sp1. btd is expressed in the syncytial blastoderm embryo in a stripe covering the anlagen of the antennal, intercalary and mandibular head segments. btd has been characterized as a head gap gene, since these segments are deleted in btd mutant embryos. We report here that the cis-acting elements required for btd head stripe expression are contained in a 1 kb DNA fragment, located about 3 kb upstream of the promoter. The four maternal coordinate systems are necessary for correct btd head stripe expression, likely by acting through the 1 kb cis-acting control region. Expression of the btd head stripe depends on the anterior morphogen encoded by the gene bicoid (bcd). bcd-dependent activation also involves the activity of the morphogens of the posterior and dorsoventral systems, hunchback and dorsal, respectively, which act together to control the spatial limits of the expression domain. Finally, the terminal system takes part in the regulation of btd head stripe expression by enhancing activation at low levels of activity and repression at high levels of activity.

The crystal structure of the paired homeodomain bound to DNA as a cooperative dimer has been determined to 2.0 A resolution. Direct contacts between each homeodomain and the DNA are similar to those described previously. In addition, an extensive network of water molecules mediates contacts between the recognition helix and the DNA major groove. Several symmetrical contacts between the two homeodomains underlie the cooperative interaction, and deformations in the DNA structure are necessary for the establishment of these contacts. Comparison with structures of homeodomains bound monomerically to DNA suggests that the binding of a single paired homeodomain can introduce these DNA distortions, thus preparing a template for the cooperative interaction with a second homeodomain. This study shows how the paired (Pax) class homeodomains have achieved cooperativity in DNA binding without the assistance of other domains, thereby enabling the recognition of target sequences that are long enough to ensure specificity.

The 2.5 A resolution structure of a cocrystal containing the paired domain from the Drosophila paired (prd) protein and a 15 bp site shows structurally independent N-terminal and C-terminal subdomains. Each of these domains contains a helical region resembling the homeodomain and the Hin recombinase. The N-terminal domain makes extensive DNA contacts, using a novel beta turn motif that binds in the minor groove and a helix-turn-helix unit with a docking arrangement surprisingly similar to that of the lambda repressor. The C-terminal domain is not essential for prd binding and does not contact the optimized site. All known developmental missense mutations in the paired box of mammalian Pax genes map to the N-terminal subdomain, and most of them are found at the protein-DNA interface.

The product of the Drosophila extradenticle gene interacts cooperatively with homeodomain proteins encoded by homeotic selector genes, and may account in part for their distinct regulatory properties.