REGULATION (part 3/3)

Protein Interactions

The homeodomain proteins encoded by the Drosophila extradenticle gene and its mammalian homologues, the pbx genes, contribute to HOX specificity by cooperatively binding to DNA with HOX proteins. For example, the HOX protein labial cooperatively binds with extradenticle protein to a 20-bp oligonucleotide identified in the 5' region of the mouse Hoxb-1 (the mammalian homolog of Drosophila labial) gene that is sufficient to direct a labial-like autoregulated expression pattern in Drosophila embryos. Labial and Extradenticle, their binding sites separated by only 4 bp, bind DNA as a heterodimer in a head-to-tail orientation. Mutations in base pairs predicted to contact the HOX N-terminal arm results in a change in HOX preference in the heterodimer, from Labial to Ultrabithorax. These results demonstrate that Extradenticle prefers to bind cooperatively with different HOX proteins depending on subtle differences in the heterodimer binding site (Chan, 1996a).

Outside of their homeodomains, HOX protein have no sequence similarities except for two short peptides: 'MXSYF', at their N-termini, and the hexapeptide (also called the 'YPWM' motif or pentapeptide) N-terminal to their homeodomains. A 20 bp human Hoxb-1 promoter oligonucleotide is sufficient to direct an expression pattern in the fly that is very similar to the endogenous labial pattern. This expression pattern requires lab and extradenticle. Labial proteins with mutations in the hexapeptide bind DNA in the absence of EXD and have an increased ability to activate transription in vivo. Proteolysis experiments suggest that EXD can induce a conformational change in LAB. Apparently, LAB hexapeptide inhibits LAB function by inhibiting DNA binding, and an EXD-induced conformational change in LAB relieves this inhibition, promoting highly specific interactions with biologically relevant binding sites (Chan, 1996b).

Phenotypic suppression

In the absence of the eight genes of the homeotic cluster (HOM-C), which specify the identity of head, thoracic and abdominal segments, thoracic and abdominal structures develop a 'ground' pattern which includes cephalic structures called sclerotic plates. These plates are specified by empty spiracles. EMS has the potential to induce sclerotic plates, but this potential is suppressed by the HOM-C genes, including the labial gene, the most anteriorly expressed homeotic gene.

When labial is expressed without the other HOM-C genes a stereotyped arch-shaped cephalic structure forms in every thoracic and abdominal segment. This head piece probably represents the contribution of the lab gene to head development. The suppression of the head piece by all other HOM-C genes is the basis of the argument that labial is hierarchically the lowest ranking homeotic gene.

The suppression phenomenon which does not occur at the transcriptional or translational level is called phenotypic suppression. The phenomenon is used to explain posterior dominance, the observation that a homeotic gene product will have an effect only in the body region anterior to the normal domain of the gene. Posterior dominance is given an evolutionary context by the argument that the primordial segment pattern is thoracic-like and that head structures are formed by modifying an archetypal thoracic-like pattern (Macías, 1996).

Recognition of distinct target sites by a unique Labial/Extradenticle/Homothorax complex

Hox genes encode evolutionarily conserved transcriptional regulators, which define regional identities along the anteroposterior axis of multicellular animals. In Drosophila, Hox proteins bind to target DNA sequences in association with the Extradenticle (Exd) and Homothorax (Hth) co-factors. The current model of Hox-binding selectivity proposes that the nucleotide sequence identity defines the Hox protein engaged in the trimeric complex, implying that distinct Hox/Exd/Hth complexes select different binding sites and that a given Hox/Exd/Hth complex recognizes a consensus DNA sequence. The regulation of a newly identified Lab target gene does not rely on the previously established consensus Lab/Exd/Hth-binding site, but on a strongly divergent sequence. Thus Lab, and most probably other Hox proteins, selects different DNA sequences in regulating downstream target genes. These observations have implications with regard to the current model of Hox-binding selectivity (Ebner, 2005).

Understanding how Hox proteins trigger diversified morphogenesis requires the identification of the mechanisms underlying appropriate target gene selection as well as appropriate target gene regulation, which relies on controlling Hox transregulatory properties. At present, most studies have focused on how Hox proteins cooperate with two classes of co-factors, Exd/Pbx and Hth/Meis, to reach DNA-binding selectivity. Although not valid for the regulation of all Hox target genes, the Hox-binding selectivity model is a useful conceptual framework for understanding how Hox proteins, which as monomers display similar DNA-binding properties, reach specificity in target site recognition by interacting with a single co-factor, Exd. This model implies that distinct Hox/Exd complexes select different binding sites for three reasons, each of which has been well documented: (1) in vitro studies have shown that the prototypical TGAT[NN]ATNN Hox/Exd site recruits Lab or Ubx, depending on the identity of the two central NN nucleotides: GG selects Lab/Exd, while TT or TA recruits a Ubx/Exd complex; (2) the Distalless regulatory element that mediates repression by Ubx contains a Hox/Exd site where the two central nucleotides are TT; (3) switching the identity of these two central nucleotides from GG to TA, within the context of repeat3, leads to the recruitment of a Dfd/Exd complex instead of Lab/Exd, and to transformation of the Lab-responsive enhancer into a Dfd-responsive enhancer, as revealed by an in vivo test. Similar DNA binding preferences were also observed with the vertebrate Hox and Pbx homologues (Ebner, 2005 and references therein).

An in silico approach based on the Hox DNA-binding selectivity model was used to find novel Lab target genes. Although the approach identified 40 putative target sequences for the Lab/Exd/Hth complex, expression analysis of half of them only identified a single novel Lab target, CG11339. This suggests that sequences mediating Lab regulatory function in vivo are insufficiently well defined, which is further supported by the finding that the regulation of CG11339 does not rely on the consensus Lab/Exd/Hth-binding site used for the in silico approach, but on a strongly divergent sequence. These results have implications both with regard to the mode of Lab DNA-binding and more generally to the Hox-binding selectivity model (Ebner, 2005).

Previous work proposed that Lab is very peculiar among all other Hox proteins, in the sense that it does not bind DNA as a monomer, but does so in association with the co-factor Exd. Mutation of the hexapeptide (HX), a short motif upstreaam of the homodomain, confers to Lab the capacity to bind DNA in the absence of Exd. Accordingly, it was proposed that the HX exerts an inhibitory effect on Lab DNA binding, which is neutralized when interaction occurs with Exd. This conclusion was reached by studying the DNA-binding properties of Lab on the mouse repeat3 enhancer. The current study observed that this conclusion does not hold on another target sequence, the EVIII enhancer of CG11339, indicating that the previous conclusion could reflect a specialisation of Lab activity with regard to its autoregulation, rather than a general feature that distinguishes the mode of Lab DNA binding from that of other Hox proteins (Ebner, 2005).

The Hox-binding selectivity model also implies that a given Hox/Exd complex should recognize a consensus nucleotide sequence in downstream target genes; owing to the lack of well characterised Hox target sequences, this still remains to be experimentally validated. The sequence responsible for Lab-mediated regulation of CG11339 is TGAT[CA]ATTA, which diverges from the TGAT[GG]ATTG site mediating lab autoregulation, at the two central positions that are predicted to define the choice of the Hox protein recruited with Exd. The fact that Lab can recognize target sequence differing at the central NN nucleotide is also observed upon mutation of these nucleotides from GG to TA in the lab550 autoregulatory enhancer. Thus, Lab can form a complex with Exd and activates transcription in vivo on at least three sequences that differ with regard to the identity of the central NN nucleotides: GG in repeat3, TA in the mutated lab enhancer and CA in CG11339 (Ebner, 2005).

Since altering the GG identity of the central NN nucleotides in repeat3 to TA or TT alleviates Lab/Exd complex assembling, the readout of the nucleotide identity at the central NN positions most probably depends upon neighbouring nucleotides that are different in repeat3, lab48/95 and CG11339. Examination of the three sites shows that the Exd half sites are conserved, while the Hox half site differs at the most 3' end. In support for a role of nucleotides lying in the Hox half site in the readout of the identity of the central NN nucleotides, it was found that loss of Lab/Exd complex assembly following mutations at the 3' end of the Hox half site can be reversed by modifying the two central positions. This compensatory effect might result from subtle changes in contacting helix 3 of the HD, which in turn might modify the sequence requirement at the central NN position for efficient Lab/Exd recruitment. The importance of the Hox half site 3' end sequences is further supported by the observation that Scr and Dfd both bind in vitro and act in vivo on a prototypical Hox/Exd site that shares a TA at the central NN position, but differs in the identity of nucleotides at the 3' end of the Hox half site: GA for Dfd and CT for Scr (Ebner, 2005).

Variability in the sequence and spacing of the Hth-binding site might also influence the choice of the Hox protein that will preferentially form a complex with Exd and Hth. In any case, this study clearly shows that one Hox/Exd complex can recognize divergent sequences in two different regulated target genes. Although the two central nucleotides play a crucial role in assembling a specific Hox/Exd complex, added complexity to the Hox-binding selectivity model needs to be considered, and the nature of these two base pairs will not necessarily predict which Hox protein will selectively bind with the co-factor Exd (Ebner, 2005).

Finally, the data might also open perspectives on the mechanisms underlying the establishment of complex and distinct transcriptional patterns downstream of Hox genes. Hox transcription factors are usually expressed in broad domains, yet downstream target genes are often activated or repressed only in part of the Hox expression domain. It has previously been shown that regulatory regions of downstream target genes integrate signalling inputs, which provides additional positional information to restrict downstream target gene activation. These observations highlight the importance of the environment of the Hox/Exd-binding sequence in mediating transcriptionally distinct outputs. This study shows that Lab responsive enhancers that bear Lab/Exd-binding sites drive distinct expression patterns, both with regard to spatial and temporal characteristics. It suggests that in addition to environmental cues, the identity of the Hox/Exd sites might also be instructive (Ebner, 2005).

labial: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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