extradenticle: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - extradenticle

Synonyms -

Cytological map position - 14A1-B1

Function - transcription factor

Keyword(s) - cofactor with homeodomain transcription factors, oncogene

Symbol - exd

FlyBase ID:FBgn0000611

Genetic map position - 14A1-B1

Classification - homeodomain PBX class

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

extradenticle behaves like a homeotic gene, causing transformation of segmental identities. EXD is the best described example of how other proteins cooperatively interact with homeotic proteins to increase the specificity of homeotic protein binding to DNA. EXD acts as a cofactor with homeotic genes in transcriptional activation. This can be shown phenotypically by generating clones of mutant exd cells in flies and observing the resultant adult patterns. Such mutants show ectopic transformation of head structures and legs, similar to those found in homeotic gene mutants (Gonzalez-Crespo, 1995, Rauskolb, 1995 and Mann, 1995).

One area of research investigates how transcription factors interact to achieve specific binding to DNA and consequent transcriptional activation. This information is important because of the pervasive influence of homeotic proteins on morphogenesis. Homeobox genes recognize more or less the same consensus DNA sequence. With low sequence recognition specificity, how does specific DNA recognition take place? Thus there is a problem in explaining the specificity of homeobox binding to DNA. Cofactors such as EXD increase the binding specificity of homeotic genes to DNA, and thus provide a mechanism that explains the specific effects of homeobox genes.

The biochemical basis of EXD interaction with homeotic genes has been studied intensively. Ultrabithorax is one EXD partner in gene activation. The best characterized enhancer element DNA binding site for EXD-UBX interaction is the parasegment 7 enhancer responsible for activation of decapentaplegic in the visceral mesoderm. The interaction between EXD and UBX requires three surface-exposed homeodomain residues of UBX and the UBX C-tail, adjacent to the C-terminal end of the homeodomain (Chan, 1994).

Binding sites for the two transcription factors EXD and UBX on a dpp midgut enhancer fragment are close enough to partially overlap. On this fragment EXD and UBX bind cooperatively, with UBX binding increasing 6 to 30 fold in the presence of EXD. Dissociation studies reveal that EXD stabilizes the DNA-bound form of UBX. Although ANTP binds on the fragment, EXD does not work cooperatively with ANTP (Chan, 1994).

One effect of homeotic gene function, phenotypic suppression (described in detail at the labial site), resembles these results. When two homeotic genes are expressed in the same segment, the resulting segment identity is usually governed by only one of the two. Ubiquitous expression of Ubx does not activate dpp posterior to PS7 because UBX cannot override repression by ABD-A and ABD-B. In effect UBX is phenotypically suppressed by ABD-A and ABD-B. Homeotic genes may compete for an interaction with EXD or the binding of some homeotic proteins may block EXD binding (Chan, 1994).

The homeodomain proteins encoded by the Hox complex genes do not bind DNA with high specificity. In vitro, Hox specificity can be increased by binding to DNA cooperatively with the homeodomain protein Extradenticle or its vertebrate homologs, the PBX proteins (when considered together, known as the PBC family). One of the best characterized Hox-PBC binding sites is present in a 20 bp oligonucleotide repeat 3, which was identified in the 5' promoter region of the mouse Hoxb-1 gene. Hoxb-1 protein or its Drosophila ortholog Labial are both able to bind cooperatively with Exd to the binding site whereas other Hox proteins, such as Ultrabithorax or Hoxb-4 cannot. A two basepair change in a Hox-PBC binding site, from GG to TA, switches the Hox-dependent expression pattern generated in vivo from labial to Deformed. The change in vivo correlates with an altered Hox binding specificity in vitro. Similar Deformed-PBC binding sites were identified in the Deformed and Hoxb-4 genes. The Deformed sites include well characterized epidermal (EAE) and neural (NAE) autoregulatory enhancers. Two repeats containing TA sequence binding sites were found in the 2.7 kb EAE and two were found in the 600 bp NAE. These sites generate Deformed or Hoxb-4 expression patterns in Drosophila and mouse embryos, respectively. These results suggest a model in which Hox-PBC binding sites play an instructive role in Hox specificity by promoting the formation of different Hox-PBC heterodimers in vivo. Thus, the choice of Hox partner, and therefore Hox target genes, depends on subtle differences between Hox-PBC binding sites (Chan, 1997).

To regulate their target genes, the Hox proteins of Drosophila often bind to DNA as heterodimers with the homeodomain protein Extradenticle. 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; 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).


GENE STRUCTURE

Bases in 5' UTR - 206

Introns - none

Bases in 3' UTR - 1541


PROTEIN STRUCTURE

Amino Acids - 376

Structural Domains

Exd has a homeodomain of the pbx class. Exd is homologous to human proto-oncogene PBX1 and two other family members, PBX2 and PBX3 (Rauskolb, 1993).


extradenticle: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

date revised: 20 APR 97 

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