Protein Interactions

homothorax (hth) is required for the Hox genes to pattern the body of Drosophila. hth is necessary for the nuclear localization of an essential HOX cofactor, Extradenticle (Exd), and encodes a homeodomain protein that shares extensive identity with the product of Meis1, a murine proto-oncogene. Meis1 is able to rescue hth mutant phenotypes and can induce the cytoplasmic-to-nuclear translocation of Exd in cell culture and Drosophila embryos. In all cells where hth is expressed, Exd is localized to nuclei. Conversely, in most cells (but not all), where Exd is nuclear, hth is expressed. For example, during embryogenesis, Exd is cytoplasmic in the labial segment and in the limb primordia cells that express the gene Distal-less. For both of these cell types, hth is not expressed. Meis1/Hth also specifically binds to Exd with high affinity in vitro. Conditional expression of Meis1 in cultured Drosophila cells shifts Exd's subcellular localization within an hour. Hth can induce Exd's nuclear localization even when Asn-51 of the Hth homeodomain (implicated in DNA binding of other homeodomain proteins) has been mutated to Ala. These data suggest a novel and evolutionarily conserved mechanism for regulating HOX activity in which a direct protein-protein interaction between Exd and Hth results in Exd's nuclear translocation (Rieckhof, 1997).

homothorax (hth) is a Drosophila member of the Meis family of homeobox genes. hth function is required for the nuclear localization of the Hox cofactor Extradenticle (Exd). There is also a post-transcriptional control of Hth by exd: exd activity is required for the apparent stability of the Hth protein. To determine whether the lack of Hth in exd- clones is a result of transcriptional or post-transcriptional regulation, the expression of an hth enhancer trap was examined. In contrast to Hth protein, lacZ expression from the hth enhancer trap is maintained and in many cases upregulated in exd- clones. This suggests that the loss of Hth protein occurs post-transcriptionally, perhaps by protein degradation. Thus, the activities of hth and exd are intimately associated with one another: removing hth function results in cytoplasmic and presumably non-functional Exd, and removing exd function results in the loss of detectable Hth protein (Abu-Shaar, 1998).

Nuclear localization of the Extradenticle (EXD) and PBX1 proteins is regionally restricted during Drosophila and mammalian development. The subcellular localization of EXD, PBX, and their partners Homothorax (HTH) and PREP1, have been studied in different cell contexts. HTH and PREP1 are cytoplasmic and require association with EXD/PBX for nuclear localization. EXD and PBX1 are nuclear in murine fibroblasts but not in Drosophila Schneider cells, in which the proteins are actively exported to the cytoplasm. Coexpression of EXD/PBX with HTH/PREP1 causes nuclear localization of their heterodimers in both cell contexts. It is proposed that heterodimerization with HTH/PREP induces nuclear translocation of EXD and PBX1 in specific cell contexts by blocking their nuclear export (Berthelsen, 1999).

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle (Exd). For Exd to bind DNA, it must be in the nucleus, and its nuclear localization requires a third homeodomain protein, Homothorax (Hth). A conserved N-terminal domain of Hth directly binds to Exd in vitro, and is sufficient to induce the nuclear localization of Exd in vivo. However, mutating a key DNA binding residue in the Hth homeodomain abolishes many of its in vivo functions. Hth binds to DNA as part of a Hth/Hox/Exd trimeric complex, and this complex is essential for the activation of a natural Hox target enhancer. Using a dominant negative form of Hth evidence is provided that similar complexes are important for several Hox- and exd-mediated functions in vivo. These data suggest that Hox proteins often function as part of a multiprotein complex, composed of Hth, Hox, and Exd proteins, bound to DNA (Ryoo, 1999).

Exd directly binds to Hth and to the mammalian Hth homolog, MEIS1 (Rieckhof, 1997), suggesting that Exd interacts with a domain that is conserved between these two proteins. Hth and MEIS1 have two highly conserved domains: the HM (Homothorax-Meis) domain near the N terminus, and the homeodomain near the C terminus. In addition, based on sequence comparisons with the related vertebrate protein PREP1, the HM domain can be considered to have two subdomains, HM A and HM B , that are more highly conserved. A glutathione S-transferase (GST) pull-down assay was used to determine which part of Hth interacts with Exd. GST-Hth and GST-HM are both able to interact with Exd protein in vitro. In contrast, neither GST-(HM B +HD), which begins in the middle of the HM domain and extends to the end of the protein, nor GST-HD, which spans the homeodomain, interacts with Exd. These results demonstrate that the HM domain of Hth is necessary and sufficient for the interaction with the PBC-A domain of Exd [EXD (amino acids 144-376) which is necessary for the HTH-EXD interaction]. Further, these results are consistent with the interaction domains defined in the vertebrate proteins MEIS1 and PBX1 (Ryoo, 1999).

To determine the function of the HM and homeo domains in vivo, mutant and wild-type Hth coding sequences were fused to green fluorescent protein (GFP), and these fusion genes were expressed in flies under the control of the yeast transcription factor Gal4. In wild-type Drosophila imaginal wing discs, Exd is cytoplasmic in cells that will generate the future wing blade, but is nuclear in cells surrounding the wing blade region. Exd is usually nuclear only in those cells where Hth is present, but when expressed at high levels or when fused to an additional nuclear localization sequence (NLS-Exd), Exd becomes partially nuclear. When GFP-Hth expression is driven in wing discs by the ptc:Gal4 driver line (which is expressed in a stripe of cells that bisects the wing blade), the endogenous Exd is shifted into the nucleus in GFP-Hth-expressing cells. To test if the Hth homeodomain is required for Exd’s nuclear localization, two mutant proteins were tested: GFP-HM and GFP-Hth 51A (which has Asn 51 of the Hth homeodomain mutated to alanine). Asn 51 is conserved in all known homeodomains and makes essential DNA contacts. GFP-Hth 51A is able to induce the nuclear localization of Exd in wing pouch cells, suggesting that the Hth homeodomain does not need to bind to DNA for this function. GFP-HM is also able to induce the nuclear localization of Exd, demonstrating that the HM domain is sufficient for this activity. GFP-HD, which lacks the HM domain but contains an intact homeodomain, is unable to induce Exd’s nuclear localization. These data suggest that hth does not induce the nuclear localization of Exd by transcriptionally regulating a third factor. Instead, together with the in vitro interaction data, they suggest that Hth induces the nuclear localization of Exd via a direct interaction between the Hth HM domain and the Exd PBC-A domain (Ryoo, 1999).

During leg development, expression of the homeobox gene Distal-less, which is required for ventral limb development, is mutually antagonistic with Hth/Exd function: Dll is a repressor of hth and Hth can also repress Dll. Hth’s ability to repress Dll requires Hth's homeodomain. From ectopic expression assays, it is concluded that although the Hth homeodomain is not required to induce Exd’s nuclear localization, it is necessary for many Hth functions, including the regulation of specific target genes such as Dll. The one known exception is that all forms of Hth, including GFP-Hth 51A and GFP-HM, are able to interfere with distal leg development when expressed with the Dll:Gal4 driver. This phenotype, however, is also observed when wild-type Exd is expressed with this driver, and therefore does not require any Hth input. The different in vivo activities of Hth and Hth 51A indicate that Hth has functions in addition to localizing Exd to nuclei, and that these functions require Hth to bind DNA (Ryoo, 1999).

The tight interaction between Hth and Exd proteins, together with the requirement for the Hth homeodomain for many of Hth’s functions, suggested that Hth might be binding to the same target enhancers as Hox/Exd heterodimers. One well characterized Hox/Exd target is an autoregulatory enhancer from the labial (lab) gene, called lab550. A 48 bp fragment of lab550, lab48/95, is necessary for lab550 activity and, in one copy, is sufficient to direct a labial- and exd-dependent pattern of expression in endodermal cells. In lab48/95 there is a single Lab/Exd heterodimer binding site, TGATGGATTG; this binding site is necessary for the activity of lab550. Also in lab48/95 is a binding site that resembles a high affinity site for MEIS1: GACTGTCA, a murine Hth homolog. To test if this site is a bona fide Hth binding site, band shift experiments were performed with Lab, Hth, and Exd proteins on the wild-type lab48/95 oligo, and on an oligo with point mutations in the putative Hth binding site, GACTtatA (lab48/95 hth). Neither Lab, Exd, nor Hth are able to bind lab48/95 on their own. The combination of Exd plus Hth is able to weakly bind this DNA. Because binding is diminished on lab48/95 hth, these data suggest that Exd and Hth exhibit weak cooperative binding to lab48/95, consistent with previous studies with MEIS1 and PBX1. Lab cooperatively binds with Exd to lab48/95 and the binding of this heterodimer requires both the Exd and Lab half sites. In contrast, no complex formation is observed when Hth and Lab are combined. However, when increasing amounts of Hth are added to a constant amount of Lab plus Exd, the Lab/Exd band disappears and in its place a Hth/Lab/Exd trimeric complex is observed. The Hth/Lab/Exd band is more intense than the Lab/Exd band, suggesting that Hth contributes to the DNA binding affinity of the trimeric complex. Additonal tests show that the Hth/Lab/Exd complex requires the putative Hth binding site; use of truncated proteins show that protein-protein interaction between Hth and Exd is necessary for the formation of the Hth/LAB/Exd complex, but that DNA binding by the Hth homeodomain contributes to the stability of this complex. Also, the Hth binding site is required for lab48/95 activity in embryos. Thus a DNA bound Hth/LAB/Exd triple complex is capable of activating lab48/95-lacZ in vivo. This was confirmed by interfering with the stable assembly of this complex by expressing the HM domain, which binds to Exd and therefore competes with the interaction between Exd and Hth (Ryoo, 1999).

If GFP-HM is interfering with Hth and Exd function in vivo, its over-expression should be able to phenocopy other hth or exd mutant phenotypes. One function of hth is to direct antennal development; in the absence of either hth or exd activities, antennal structures are autonomously transformed into leg identities. Consistent with GFP-HM acting as a dominant negative, its expression in the Dll domain transforms distal antenna into distal leg. The antenna to leg transformations observed in GFP-HM-expressing animals show bristles with bracts, typical of a distal leg identity. In contrast, expression of GFP-Hth 51A does not generate this transformation. Together with the noted effect on the reporter genes, these data suggest that GFP-HM, but not GFP-Hth 51A, interferes with hth function. This would indicate that GFP-HM has dominant negative activity whereas GFP-Hth 51A behaves as a hypomorph. GFP-HM can also alter the segment identity of the adult abdomen which, unlike antennal development, requires input from both exd and Hox genes. In wild-type male abdomens, posterior tergites have darker pigmentation and a lower density of small hairs (trichomes) than anterior tergites. hth minus clones, like exd minus clones, in the second or third tergite of a male fly show an increase in pigmentation and a decrease in trichome density, consistent with a transformation into a more posterior abdominal identity. When GFP-HM is expressed using pnr-Gal4, an increase in pigmentation in anterior tergites results, consistent with an anterior-to-posterior transformation of abdominal segment identity. However, no effect on trichome density is observed following GFP-HM expression, suggesting that this transformation is incomplete. In contrast, expression of wild-type GFP-Hth using pnr-Gal4 results in a decrease in pigmentation and an increase in trichome density in tergites 5 and 6, consistent with a posterior-to-anterior shift in cell fate. Expression of GFP-Hth 51A generates a weak version of this transformation. These results suggest that interfering with hth function by expressing the HM domain can interfere with a Hox-dependent function, such as tergite identity in the adult abdomen. Moreover, they suggest that different amounts of hth activity in the abdomen contribute to differences in tergite identity (Ryoo, 1999).

Drosophila Homothorax (Hth) and Extradenticle (Exd) are two homeoproteins required in a number of developmental processes. Exd can function as a cofactor to Hox proteins. Its nuclear localization is dependent on Hth. Evidence is presented of in vivo physical interaction between Hth and Exd, mediated primarily through an evolutionarily conserved MH domain in Hth. This interaction is essential for the mutual stabilization of both proteins, for Exd nuclear localization, and for the cooperative DNA binding of the Exd-Hth heterodimer. Some in vivo functions require both Exd and Hth in the nucleus, suggesting that the Exd-Hth complex may function as a transcriptional regulator (Jaw, 2000).

To assess the ability of Hth in inducing Exd nuclear localization, the Hth mutant constructs were expressed using the UAS-GAL4 system in two different cell types: the eye field of larval eye disc, and the larval salivary gland. Two Hth deletion constructs were tested: deltaMH deletes residues 31-312, which include the conserved MH domain (residues 91-219), and deltaHD deletes residues 299-459, which include the HD (residues 368-428). deltaHD is still capable of inducing Exd nuclear localization, indicating that the region deleted is not required for this function. deltaMH has no ability to induce Exd nuclear localization, suggesting that the MH domain is required for Exd nuclear localization. A GST pull-down assay shows that the region deleted in deltaMH is required for the physical interaction with Exd, and the region deleted in deltaHD is not essential for the interaction. In salivary gland, both deltaMH and deltaHD proteins are located in the nucleus. The observation that deltaMH is itself nuclear indicates that Hth can enter the nucleus independent of Exd. Since only the 30 N-terminal residues (1-30) and the 28 C-terminal residues (460-487) are shared between deltaMH and deltaHD, it is possible that a nucleus localization signal (NLS) is located in one of these two regions. However, no sequence in these two regions fits the canonical NLS motif. Therefore it is likely that Hth has two independent NLS, one located within residues 91-219 and another in residues 368-428. The two best match of NLS motif are indeed found in these two regions: KRDK (residues 91-94, at the N-terminal end of MH) and KKNQKKR (residues 363-369, at the N-terminal end of HD). The eye field deltaMH, although still retaining the putative NLS in HD, is primarily cytoplasmic. The different distribution in different cells suggests that the remaining NLS function in deltaMH is weak and is influenced by other factors (Jaw, 2000).

Ectopic expression of the full length Hth (driven by dpp-GAL4) causes several major phenotypes in adult flies: eyes are absent or very small, the arista of the antennae are missing, the third antennal segments are occasionally duplicated, and the distal leg segments are deleted, malformed, and occasionally bifurcated. Whether the mutant Hth constructs can cause any of these phenotypes in transgenic flies was examined. deltaMH causes no effect on antenna, eye and leg morphology. Since deltaMH fails to induce Exd nuclear localization, it appears that nExd is required to affect antenna, eye and leg development. Although deltaHD can induce Exd nuclear localization, it caused no effect on antenna and eye development, suggesting that the Hth HD is required to affect eye and antenna development and nExd alone is not sufficient. deltaHD caused leg defects similar to those induced by full length Hth, indicating that the Hth HD is not required to affect leg development. When a NLS-Exd construct is expressed, the FLAG-tagged NLS-Exd is located in the nucleus in the absence of Hth. When NLS-Exd expression is driven by the dpp-GAL4, the eyes and antennae are not affected, but the femur and tibia leg segments are deformed. These results confirm that nExd alone is not sufficient to affect eye and antenna development, while part of its effect on leg development does not require Hth. The leg phenotypes caused by deltaHD and NLS-Exd are not the same: deltaHD affects the distal segments and NLS-Exd affects the medial segments. Even when induced at 29 degrees C, NLS-Exd did not affect the distal segments. The difference in phenotype suggests that some effect on leg development also requires the contribution from Hth, but does not require its HD. It is concluded that some functions require both nExd and nHth, while some require only nExd (Jaw, 2000).

Engrailed cooperates with Extradenticle and Homothorax to repress target genes in Drosophila

Engrailed is a key transcriptional regulator in the nervous system and in the maintenance of developmental boundaries in Drosophila, and its vertebrate homologs regulate brain and limb development. The functions of both of the Hox cofactors Extradenticle and Homothorax play essential roles in repression by Engrailed. Mutations that remove either of them abrogate the ability of Engrailed to repress its target genes in embryos, both cofactors interact directly with Engrailed, and both stimulate repression by Engrailed in cultured cells. A model is suggested in which Engrailed, Extradenticle and Homothorax function as a complex to repress Engrailed target genes. These studies expand the functional requirements for Extradenticle and Homothorax beyond the Hox proteins to a larger family of non-Hox homeodomain proteins (Kobayashi, 2003).

As a first step in determining the mechanisms whereby exd and hth contribute to repression by En in vivo, the possibility of direct interaction was examined. En can bind co-operatively with Exd in vitro to artificial DNA sites. Whether a direct En-Exd interaction could also occur in other contexts was examined using yeast two-hybrid and in vitro assays. Whether En could interact similarly with Hth was also examined. En appears to interact robustly with Exd in the yeast two-hybrid system, because the signal strength observed with both isolated colonies and colony streaks is consistently higher than that seen with some positive controls, including the functionally important interaction between En and Groucho. This signal was also comparable to that seen with Exd and the mouse homolog of Hth, Meis1. En also gives a somewhat weaker, but apparently specific, signal in combination with either Hth or Meis1 (Kobayashi, 2003).

In vitro, En also interacts specifically with both Exd and Meis1. En fused with GST effectively pulls down either Exd or Meis1. Meis1 was used in these studies because of the high level of non-specific interaction observed with in-vitro-translated Hth, perhaps owing to the heterologous nature of the translation system. In this system, it is unlikely that the interactions are due to co-operative binding to DNA, and these results are interpreted to mean that these interactions can occur in solution. Furthermore, Meis1 appears to interact more strongly with En in the presence of Exd, suggesting that the three proteins form a co-complex (Kobayashi, 2003).

In cultured Drosophila cells, Exd and Hth cooperate with En to repress transcription. Using a co-operative binding site for Exd and En to construct an En-responsive target gene, it was found that both Exd and Hth are required for full repression activity. When a mutation is introduced into an Exd consensus binding sequence that eliminates co-operative binding, co-operative repression is largely eliminated, whereas mutating the En consensus binding sequence eliminates repression. This, along with the fact that RNA interference directed against Exd mRNA also largely eliminates co-operative repression, suggests that a complex containing Exd and En is responsible for the co-operative repression caused by coexpression of Hth and En (Exd is constitutively expressed in these cells). Because Hth regulates the nuclear localization of Exd, it can allow Exd-En repression complexes to form in the nucleus. In addition, the observed molecular interactions suggest that the fully active repression complex might include all three proteins (Kobayashi, 2003).

The effects of eliminating exd function on repression by En appear to be different in the abdomen and the more-anterior regions: En is less dependent on exd in the abdomen (parasegments 6-12). One possible explanation is that hth can provide the observed exd-independent activity. However, in exd mutants, Hth levels are reduced, probably because Hth protein is less stable without Exd. Nevertheless, these data are consistent with the possibility that, on their own, either Hth or Exd might provide partial cofactor activity, whereas both together might be required for full activity. The latter possibility is suggested by the observation that maximal repression activity in S2 cells requires all three gene products (Kobayashi, 2003).

An additional possibility to account for the residual exd- and hth-independent repression activity of En in the abdomen is that other cofactors assist En in binding to its target genes in the abdomen. If there are other cofactors at work, it is likely that their activity (or expression) is dependent, either directly or indirectly, on the Hox genes Ubx and abd-A, because these genes are responsible for all known aspects of differential segment identity in this region of the embryo (Kobayashi, 2003).

It is noteworthy that the difference in the dependence of En on exd in the abdomen versus the thorax is seen only after stage 9, when the levels of Hth, and the consequent nuclear concentration of Exd, have declined in the abdomen. Thus, the dependence of En on exd parallels the nuclear concentration of Exd, and might reflect an evolutionary adaptation to the changing levels of Exd in different regions of the embryo (Kobayashi, 2003).

Hth has been shown to act in part through its facilitation of the nuclear localization of Exd, and strong hth and exd mutants have very similar phenotypes. Although Hth can also interact with En independently of Exd, transfection assays in cultured cells suggest that Hth might depend entirely on Exd for its ability to increase repression by En, at least from artificial En-Exd co-operative binding sites. Because Hth forms complexes with En in these cells, in addition to increasing its repression activity, a simple model is that maximal repression activity is due to complexes containing En, Exd and Hth. However, the possibility cannot be ruled out that Hth acts solely by making Exd available to interact with En on target sites, through its ability to bring Exd into the nucleus (Kobayashi, 2003).

Whether the repression activity of ectopically expressed En in vivo is dependent on hth function was tested using assays similar to those used for exd. In each case, a close similarity was observed to results with exd mutants. En activity shows a strong dependence on hth function, although residual activity remains in hth mutants. In addition, En activity shows a sensitivity to the hth gene dose. All of these results are consistent with the effects of Hth being exerted through its effect on Exd nuclear localization, provided that the nuclear targeting of Exd is necessary for its ability to function with En. However, Hth might also increase the effectiveness of En repression directly, by forming complexes with En and/or as part of En-Exd complexes. A detailed analysis of a number of in vivo target sites will be necessary to distinguish among these possibilities (Kobayashi, 2003).

Exd and Hth are essential to the correct regulation of target genes by the homeodomain proteins of the Hox clusters. However, their functional interactions have not previously been shown to extend beyond the highly restricted subset of homeodomain proteins that are found within the Hox clusters (the Antp, Abd-B and Labial classes). The identification of functional interactions with En suggests that exd and hth might provide functional specificity in conjunction with other non-Hox-class homeodomain proteins (Kobayashi, 2003).

Although there have been previous suggestions that Exd and Hth might participate in active repression as well as activation complexes, most of the well-characterized direct Exd-Hth-Hox target genes are activated in an exd- or hth-dependent fashion. In fact, these observations raised the question of whether Exd and Hth might be dedicated to gene activation. Recently, Hth and Exd have been shown to act directly with Ubx to repress the Hox target gene Distalless in the Drosophila abdomen. The partnership with En in repression further argues that these cofactors can increase the target site discrimination of homeodomain proteins without restricting the resulting transcriptional activity to activation alone. Based on these results, it is suggested that Hth and Exd increase the target-site discrimination of several classes of homeodomain proteins and that they do so without defining the transcriptional activity of the resulting protein complex (Kobayashi, 2003).

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).

Patterning function of homothorax/extradenticle in the thorax of Drosophila

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. This study investigates the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single Hox independent functional unit. Their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and ß subdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. Evidence is presented for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain (Aldaz, 2005).

This study deals with a novel hth/exd function: its patterning role in the notum. It is not related to the specification of notum identity because notum identity is not affected by alterations of hth/exd activity. For example, in the absence of hth/exd, the cells still differentiate as notum, if an abnormal one. Conversely, high and uniform Hth levels also produce notum tissue but with abnormal pattern. This function is only required in part of the notum and is therefore linked to the modulation of hth expression during the development of the disc. The final result of this modulation is the appearance of the alpha and ß subdomains of hth that is reported in this study. These two subdomains differentiate distinct notum patterns, suggesting that Hth/Exd interact with other localised products to generate these patterns (Aldaz, 2005).

Thus, there are two principal aspects in the patterning function of hth/exd: (1) the spatial regulation, that eventually results in the restriction of its expression to the alpha and ß subdomains, and (2) the local interactions of Hth/Exd with other products in either of the subdomains (Aldaz, 2005).

Although hth and exd form a single functional unit, their mode of regulation is different: exd is expressed ubiquitously but is regulated at the subcellular level by hth, which promotes Exd nuclear transport. Therefore, the key element of hth/exd regulation is the transcriptional control of hth (Aldaz, 2005).

Originally, hth is expressed in all the notum cells and later becomes restricted to the alpha and ß subdomains. Consequently, the principal aspect of hth regulation is the mechanism(s) leading to its repression in the regions outside the alpha and ß subdomains. Two negative regulators have been identified, the eyg gene and the Dpp pathway, which probably acts through some unidentified downstream gene. In the notum hth behaves as a downstream target of both the Dpp pathway and eyg (Aldaz, 2005).

The role of Eyg as a negative regulator of hth is based on the following observations: (1) the beginning of the modulation of hth expression in the notum at the early third instar coincides with the initiation of eyg expression; (2) in eyg mutants the hth domain is expanded, extending to most of the notum; (3) mutant eyg clones show hth derepression in the inter-subdomains region, and conversely, ectopic eyg activity in the ß subdomain represses hth. The fact that this ectopic activity fails to affect hth in the alpha subdomain was expected since eyg and hth are normally co-expressed in this subdomain. In conclusion, eyg suppresses hth in the inter-subdomains region and also acts as a barrier for hth in the eyg/ß-hth border (Aldaz, 2005).

The role of the Dpp pathway as a negative regulator of hth is based on results showing that Mad - mutant clones in the inter-subdomains region show activation of hth. This is in contrast to the behaviour of those clones in the alpha subdomain, where they have no effect, or in the ß subdomain, where they show suppression of hth. It is believed that the reason for the latter effect is that eyg is up regulated in those clones, and in turn Eyg suppresses hth. The lack of effect of Mad - clones in the alpha subdomain is probably due to the low activity of Dpp in that region. In principle, the observation that the high activity levels generated in the TkvQD clones suppress hth in this subdomain supports this view. Expectedly, TkvQD clones do not affect hth expression in the ß subdomain, because it normally possesses high Dpp activity levels (Aldaz, 2005).

Taking all the results together, the following model of hth regulation is proposed. Since hth is originally expressed in all trunk embryonic cells and in all the notum cells in the early disc, the regulation of hth during wing disc development essentially reflects local repression in specific parts of the disc. The basic idea is that hth is repressed by the joint contribution of eyg and high/moderate levels of the Dpp pathway. Neither of these elements can repress hth individually. Although eyg appears to act uniformly in its domain, the repressing activity of Dpp is concentration dependent. Within the eyg domain, the hth alpha subdomain is located in the anterior region, in which the Dpp levels are too low to be effective and Eyg alone cannot repress hth/exd. In the inter-subdomains region the Dpp levels are high enough to repress hth, since here it acts together with Eyg. The ß subdomain is outside the eyg domain and therefore in the absence of Eyg even the high Dpp levels are not capable of repressing hth/exd. The model is also supported by the experiments of overexpressing eyg. The eyg-expressing clones in the ß subdomain suppress hth because the two repressors are active in the clones, while they have no effect in the alpha subdomain because it normally contains high eyg levels. In principle the experiments overexpressing the Dpp pathway (TkvQD clones) appear to support the model. These clones have no effect in the ß subdomain, which normally possesses high Dpp activity levels, but they suppress hth in the alpha subdomain. However, these clones are known to suppress eyg and therefore hth should not be repressed according to this model. It is possible that in certain circumstances the very high Dpp activity levels induced by these clones may be sufficient to down regulate hth, even in the absence of eyg (Aldaz, 2005).

The presence of two distinct repressors may suggest that the hth promoter region contains binding sites for Eyg and for Mad/Medea that would be responsible for the transcriptional repression. The ubiquitous expression in the absence of these two repressors may be due to a constitutive promoter (Aldaz, 2005).

The second aspect of the late patterning function of hth/exd arises from the observation that the alpha and ß subdomains form different patterns with similar levels of hth. This suggests the existence of interactions between Hth/Exd and products specifically localised to the different subdomains. In the case of the alpha subdomain, the obvious candidate for the interaction is Eyg. The joint activity of hth/exd and eyg specifies a notum pattern that is different from those specified by each of these genes alone (Aldaz, 2005).

The finding that the Eyg and Hth proteins associate to form a complex in vitro suggests a mechanism to achieve the pattern difference between the alpha and the ß subdomains. As it has been shown to be the case for the in vivo specificity of the Hox genes, the association of Hth/Exd with the different Hox products results in higher affinity and specificity for target sites. Here, the formation of an Eyg/Hth/Exd complex in the alpha subdomain may result in a constellation of gene activity different from that in the ß subdomain where Eyg is not present. In the latter subdomain hth/exd may act alone, for after all the two genes encode transcription factors. Alternatively, the Hth/Exd products may interact with some other yet unidentified co-factor (Aldaz, 2005).

An interesting aspect of the interaction of hth/exd and eyg is that it acts in two different ways. At the gene regulation level, eyg participates in the spatial control of hth/exd activity, but where the two genes are co-expressed their proteins interact, presumably to contribute to the in vivo affinity and specificity for target genes (Aldaz, 2005).

Distinct functions of homeodomain-containing and homeodomain-less isoforms encoded by homothorax

The homothorax (hth) gene of Drosophila is required for executing Hox functions, for head development, and for forming the proximodistal (PD) axis of the appendages. Alternative splicing of hth generates two types of protein isoforms, one that contains a DNA-binding homeodomain (HthFL) and one that does not contain a homeodomain (HDless). Both types of Hth isoforms include the evolutionarily conserved HM domain, which mediates a direct interaction with Extradenticle (Exd), another homeodomain protein. Although both HthFL and HDless isoforms of Hth can induce the nuclear localization of Exd, they carry out distinct sets of functions during development. Surprisingly, many of hth’s functions, including PD patterning and most Hox-related activities, can be executed by the HDless isoforms. In contrast, antennal development shows an absolute dependency on the HthFL isoform. Thus, alternative splicing of hth results in the generation of multiple transcription factors that execute unique functions in vivo. It is further demonstrated that the mouse ortholog of hth, Meis1, also encodes a HDless isoform, suggesting that homeodomain-less variants of this gene family are evolutionarily ancient (Noro, 2006: full text of article).

hth includes 16 annotated exons distributed over >100 kb of genomic DNA. All functionally characterized isoforms of hth include both the HM domain, encoded by exons 2–6, and the HD, encoded by exons 11–13. In addition, hth encodes at least two additional alternatively spliced variants that have an intact HM domain but no HD. Both alternatively spliced mRNAs code for two almost identical HM-containing proteins that are largely derived from the first six coding exons. Both of these HDless isoforms have an additional 24 amino acids at their C termini encoded by alternate exons. One of these variants (the 7' isoform) uses an alternative exon 7 (exon 7'). Sequence comparisons between D. melanogaster and Anopheles gambiae hth genes enabled identification of a second hth splice variant that is also missing the HD. This isoform (the 6' isoform) is generated when the splice site at the 3' end of exon 6 is not used, generating an extended ORF. The existence of both 6' and 7' isoforms in vivo was confirmed by sequencing ESTs and performing RT–PCR on mRNA isolated from embryonic and larval tissues. The presence of 6' and 7' isoforms raised the possibility that HDless variants of Hth might carry out distinct functions, suggesting a functional diversification of the hth gene that depends on alternative splicing (Noro, 2006).

This study addresses the functional relevance of alternatively spliced isoforms of Hth, a transcription factor involved in a wide variety of developmental programs that are critical for the construction of the D. melanogaster body plan. Analysis of hth100-1 mutant tissues during both embryonic and larval stages have demonstrated a strict requirement for the HD in a surprisingly small subset of developmental functions, such as the instruction of antennal identity and the correct patterning of the wing hinge. In contrast, partial loss of function of HDless forms, resulting either from siRNA injection against the 6' and 7' isoforms or from the ectopic expression of HthFL, suggest that these forms carry out crucial functions in vivo. Intriguingly, the data further suggest that HthFL is apparently unable to substitute for at least a subset of HDless functions. This idea rests primarily on the observation that 6' + 7' siRNA-injected embyros exhibit hth loss-of-function phenotypes yet still express HthFL. However, the possibility that the injected siRNAs might have off-target effects cannot be excluded, even though the specificity of the observed phenotypes suggests that it is unlikely. In future experiments, it may be possible to more definitively test this idea by generating hth mutant alleles that are unable to express the 6' and 7' isoforms (Noro, 2006).

In contrast, the generally weak phenotypes observed in hth100-1 embryos and adults support the hypothesis that HDless/Exd dimers work as bona fide transcription factors that are essential for the correct regulation of many hth-dependent functions. In some respects, these findings are reminiscent of reports showing that an artificially truncated and HDless version of the segmentation protein Fushi tarazu (Ftz) retains many of the activities of full-length Ftz. These earlier findings provide additional support to the idea that HDless forms of some homeoproteins retain biological activity, probably due to their ability to assemble stable protein complexes in vivo. What is unique to the current results is that hth normally expresses HDless isoforms and that there is a division of labor between HDless and HthFL isoforms. This is best exemplified by the finding that HthFL isoforms are essential for antennal development but largely dispensable for proximal leg development. Based on these observations, it is suggested that distinct Hth/Exd dimers may bind to partially overlapping sets of target genes in vivo, and that the presence or absence of the DNA-binding HD expands the range of target genes that Hth/Exd can select and regulate (Noro, 2006).

Hth is composed of two conserved modules: the HM domain that mediates an interaction with Exd and the DNA-binding HD. Since the 6' and 7' isoforms do not have a HD, they are unable to directly interact with DNA. However, the presence of the HM domain allows them to complex with Exd, whose HD can mediate DNA binding, as demonstrated by the formation of cooperative HDless/Exd/Hox complexes on the fkh[250] and lab48/95 elements. Consistent with these results, Meis has also been shown to form trimeric complexes with Pbx and Hox without binding directly to the DNA (Noro, 2006).

The absence of the Hth HD has several implications for the transcriptional properties of HDless/Exd complexes. (1) It is likely that HDless/Exd and HthFL/Exd complexes have distinct DNA-binding specificities because the latter complex contains two HDs, while the former contacts DNA exclusively through Exd's HD. It is imagined that the two types of complexes regulate partly overlapping sets of target genes by decoding different cis-regulatory architectures, possibly in the same cells. For example, the HthFL isoform appears to be unable to carry out some hth functions since the 6' + 7' siRNA-injected embryos exhibit hth loss-of-function phenotypes. This observation suggests that the presence of the HD might be incompatible with a subset of the cis-regulatory architectures that bind HDless/Exd. (2) HDs can also be protein interactions motifs, raising the possibility that the absence of the Hth HD from HDless/Exd could influence its ability to contact other transcription factors, coactivators, and/or corepressors. The Exd and Pbx TALE HD mediate direct interactions with Hox factors, and with the HD-containing transcription factor Engrailed (En). The HD of Hth, which is also of the TALE family, is also likely to interact with other transcription factors, including Hox proteins. Thus, through alternative splicing, the modular architecture of Hth is exploited to produce unique transcription factor complexes that are likely to have distinct protein and DNA-binding properties (Noro, 2006).

Given that HthFL and HDless isoforms have some unique functions during development, it is tempting to suggest some generalizations about which functions require the Hth HD and which do not require this domain. Insect body plans are made up of repeated units that develop into diverse body parts in the adult due to the activity of selector genes, transcription factors that instruct morphological identities by regulating unique sets of target genes. Legs and antennae in Drosophila represent an example of serially homologous appendages that develop from a leg-like ground-state in response to different selector activities: Hox factors select for legs while Hth/Exd select for antenna. The demonstration that a hth100-1 mutant antenna is completely transformed toward a ground-state leg-like appendage demonstrates that the antennal selector function of Hth is absolutely dependent on its HD (Noro, 2006).

In contrast to its antennal selector role, the data suggest that the Hth HD is largely dispensable for at least some of the Hox-cofactor functions of Hth/Exd. This surprising conclusion is based in part on the cuticle phenotypes of hth100-1 and 6' + 7' siRNA-injected larvae. Specifically, hth100-1 larvae show no or very weak transformations of segmental identity, whereas 6' + 7' siRNA-injected larvae show clear posterior-directed transformations. Consistently, hth100-1 mutant embryos still express two directly activated Hox/Exd/Hth targets, fkh[250] and lab550. Repression of Distalless (Dll), which also requires direct Hox/Exd/Hth input, also occurs normally in hth100-1 mutant embryos. Thus, from these diverse observations it is concluded that the Hth HD is largely dispensable for the Hox-cofactor function of Hth/Exd. However, it is noted that there are exceptions to this generalization. Although activation of lab550 does not require the Hth HD, activation of a weakened derivative of this enhancer, lab48/95, does require the Hth HD. Similarly, mutation of the Hth-binding site in the Dll repressor element, DllR, results in weak abdominal derepression. Taken together, these data suggest that the transcription factor complexes binding to the lab and Dll regulatory elements contain the Hth HD, but that its presence is only required when the activity of these elements is compromised or weakened (Noro, 2006).

A third well-characterized function of Hth/Exd is its role in the establishment of the PD axis in both ventral (legs) and dorsal (wings and halteres) appendages. The experiments suggest that the Hth HD is not required for PD axis formation or for specifying proximal identities in the legs. In the wing, the Hth HD is also apparently dispensable for forming a correct PD axis (in particular, repression of wg at the DV boundary) but is partially required for specifying proximal (hinge) fates. Notably, both functions in which the Hth HD is largely dispensable (PD axis formation and Hox cofactor activity) appear to be evolutionarily ancient. Like Hth/Exd, Meis/Pbx are Hox cofactors and are also instrumental for establishing the PD axis of the vertebrate limb. In contrast, the antennal-specifying activity of Hth/Exd, which requires the Hth HD, is not known to have a vertebrate correlate. Thus, it is tempting to speculate that, in Drosophila, the Hth HD is more essential for executing evolutionarily recent, invertebrate-specific Hth functions and plays a less crucial, supplemental role in evolutionarily ancient Hth/Meis activities. Consistent with the idea that the HDless activities of Hth are ancient is the identification of an analogous HDless isoform made by Meis1 in Mus musculus, which underscores the functional relevance of HDless isoforms for the fulfillment of Hth/Meis-dependent functions during both invertebrate and vertebrate development. Interestingly, Prep2, another vertebrate gene related to hth, also appears to encode both HD-containing and HDless isoforms. Although the functions of these isoforms are not known, the results suggest that there may be a similar division of labor of HD-containing and HDless isoforms encoded by the Meis1 and Prep2 genes of vertebrates (Noro, 2006).

In summary, these results strongly support the idea that alternative splicing of Hth and its vertebrate orthologs is an evolutionarily conserved mechanism to expand the architectural diversity of Hth/Exd and Meis/Pbx transcriptional complexes. It is proposed that by excluding or including the HD of Hth, Hth/Exd complexes acquire distinct DNA-binding and protein interaction properties, which allow them to regulate different sets of target genes and execute unique developmental programs in vivo (Noro, 2006).

Hox proteins display a common and ancestral ability to diversify their interaction mode with the PBC class cofactors

Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. This study questioned the unique and general use of the HX for PBC (Extradenticle in Drosophila) recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. It was found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, it was revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, these results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes (Hudry, 2012).

In Drosophila, it is interesting to note that the only Hox proteins which were described to achieve their regulatory activities in absence of HD-containing isoforms of Hth were Lab and Scr. Accordingly, the formation of Lab/Exd/Hth or Scr/Exd/Hth complexes in vitro is more sensitive to the DNA-binding of Exd than to the DNA-binding of Hth. On the contrary, Ubx and AbdA are more sensitive to the loss of Hth DNA-binding for trimeric complex assembly in vitro and for regulating their respective physiological target enhancers in vivo. These data suggest that Meis DNA-binding could be more critical for Hox proteins displaying alternative PBC interaction modes than for Hox proteins displaying a unique HX-dependent interaction mode (Hudry, 2012).

The understanding of the molecular mechanisms by which Meis proteins could influence Hox-PBC interactions will require the resolution of Hox/PBC/Meis/DNA structures. It is speculated that Hox-PBC interactions that are strongly remodeled by Meis likely rely on PBC-Meis and Hox-Meis interactions. Although the formation of PBC/Meis complexes is well established, interactions between Hox and Meis proteins were rarely described. Meis proteins can form cooperative DNA-binding complexes with vertebrate Hox proteins of posterior paralog groups, but interactions with more anterior Hox proteins have only been described in a DNA-binding-independent context. In an EMSA experiments, no Hox-Meis DNA-binding complex was observed, except with the HX-mutated form of AbdB. Hox-Meis interactions could thus require the presence of the PBC cofactor to be stabilized, eventually leading to alternative Hox-PBC contacts. In that 'ménage-à-trois,' the existence of Hox-PBC and Hox-Meis interactions have the advantage to expand the range of molecular strategies that could be used by Hox proteins to assemble into a trimeric complex (Hudry, 2012).

homothorax: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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