Extradenticle protein modulates themorphological consequences of homeotic selector genes. Extradenticle proteinraises the DNA binding specificity of Ultrabithorax and Abdominal-A but not that of Abdominal-B.Extradenticle modulates the DNA binding activity of Engrailed. While a region N-terminal of the Extradenticle homeodomain is required forUltrabithorax and Abdominal-A cooperativity, Engrailed requires a domain C-terminal of theextradenticle homeobox (van Dijk, 1994).

A highly conserved region of the Engrailed protein, EH2, N-terminal of the homeodomain is required for interaction with Extradenticle and vertebrate homologues of EXD designated PBX1, PBX2 and PBX3. Two tryptophan residues present in the Drosophila and murine Engrailed EH2 domain are required for cooperativity with EXD and PBX. A related motif, present in Hox proteins, called the hexapeptide, is necessary for Hox interaction with PBX proteins. The EH2 domain is distinct from the hexapeptide present in Hox proteins with respect to the amount of conserved residues, but both contain conserved tryptophan residues and the length of the linker region separating the Pbx interaction motif from the homeodomain in both Hox and Engrailed proteins is important for cooperativity. Nevertheless the N-terminal flanking regions and homeodomains of En and Hox proteins cannot be interchanged, consistent with the idea that the Pbx interaction domain in Hox and Engrailed proteins have evolved with their associated homeodomains (Peltenburg, 1996).

Engrailed protein is posttranslationally modified in embryos. The primary embryonic protein kinase modifying En protein is Casein kinase II . Analysis of mutant proteins reveals that the in vitro phosphoacceptors are mainly clustered in a region outside the En homeodomain and has identified serines 394, 397, 401, and 402 as the targets for CkII phosphorylation. CkII-dependent phosphorylation of anN-truncated derivative of En protein purified from bacteria increases its DNA binding2-4-fold (Bourbon, 1995).

Engrailed is a specific repressor of activated transcription, and may repress transcription by interfering with interactions between transcriptional activators and the general transciption machinery. A minimal repression domain composed of 55 residues can function when fused to a heterologous DNA binding domain. Like repression domains identified in Even-skipped and Krüppel, the EN repression domain is rich in alanine residues (26%), but unlike these other domains, is moderately charged (six arginine and three glutamic acid residues) (Han, 1993).

A repressor domain from the Engrailed (En) homeodomain protein is Groucho dependent. HairyEn was created, a chimeric protein including Hairy1-286 fused to a repressor domain from the En segmentation protein (amino acids 168-298). HairyEn behaves as a strong repressor of Sex lethal. Previous results suggest that ftz is a direct target of repression by Hairy/Gro. Repression of fushi tarazu by HairyEn is Groucho dependent. The latter result correlates with an abilityof this En domain to bind to Gro in vitro. In contrast, repressor regions from the Even-skipped, Snail, Kruppel, and Knirps transcription factors are effective in the absence of Gro. A conserved element (called eh1), present in other homeodomain proteins (such as Goosecoid) plays an important role in the ability of En to bind to Gro. Mutants in Groucho lacking the WD repeat region suggest that the WD repeats plays a role in the interaction with Hairy, although it is clear that both halves of Gro are required for full interaction with Hairy and Engrailed.These results show that Gro is not generally required for repression, but acts as a specific corepressor for a fraction of negative regulators, including Hairy and En (Jimenez, 1997).

Active transcriptional repression has been characterized as a function of many regulatory factors. Repression facilitates combinatorialregulation of gene expression by allowing repressors to be dominant over activators under certain conditions. The Engrailed protein uses two distinct mechanisms to repress transcription. One is predominant undernormal transient transfection assay conditions in cultured cells. The second is predominant in an in vivo active repression assay. Two repression domains (region 4 immediately flanking the EN HD and region6) are more potent in transient transfections of cultured cells thanin vivo. The domain mediating the in vivo activity (region 3 or eh1, not closely associated with the HD inthe primary sequence) is highly conserved throughout several classes ofhomeoproteins and interacts specifically with the Groucho corepressor. When both regions 4 and 6 are deleted, very little activityremains in culture, while repression in vivo is still strong. Thus, in vivo, region 3 contributes thepredominant repression activity, while in transient transfection assays in culture, regions 4 and6 contribute the predominant activity. While eh1 shows only weak activity in transienttransfections, much stronger activity is seen in culture when an integrated target gene is used. In this assay, the relativeactivities of different repression domains closely parallel those seen in vivo, with eh1 showing the predominant activity.Reducing the amounts of repressor and target gene in a transient transfection assay also increases the sensitivity of theassay to the Groucho interaction domain, albeit to a lesser extent. This suggests that the Groucho interaction domain utilizes rate-limiting components that are relatively low in abundance. Since Groucho itself is abundant in these cells, the results suggest that a limiting component is recruited effectively by the repressor-corepressor complex only on the integrated target gene (Tolkunova, 1998).

The fact that multiple domains contribute to repression activity in the two assays and the likelihood that they utilize distinct mechanisms suggests that theevolution of En has involved strong selection for repression function. This possibility is reinforced by the observation that none of the deletion derivativesshow significant activation function on appropriate reporter genes in culture, either alone or in combination with other activators, even when all identified repression domains are removed. Indeed, preliminary data suggest that even the En homeodomain contributes to repression activity in thenormal En molecule, since single domain deletions that significantly affect repression activity in the context of the Ftz homeodomain do not affect therepression activity of En itself to the same degree. The idea that En might be primarily a repressor in vivo conflicts, on thesurface, with results from ectopic expression assays in embryos, in which En has been shown to induce expression of its own gene, as well as withthe positive regulatory action of En on hedgehog. These interactions might be indirect, through repression of a repressor; this is suggested by theresults presented here. However, it remains possible that protein-protein interactions allow En to have a net positive regulatory effect on some direct target genes. It isworthy of note in this context that a similar positive autoregulatory effect of Even-skipped, a strong repressor in both cell culture assays andin vitro, has been attributed to indirect effects in vivo, involving repression of other repressors (Tolkunova, 1998).

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).

Exd cooperates with En to repress target genes and to pattern embryos.Loss of exd function has been shown to result in a loss of en expression at later embryonic stages. Because en function is required to maintain its own expression, the loss of en expression could be a downstream effect of a loss of en function, or it could be due to some other consequence of the lack of exd. This ambiguity concerning the role of exd in en function led to an investigation of whether the activities of ectopically expressed En are dependent on exd function. En was ectopically expressed in two ways: from a heat-shock promoter and using a patterned Gal4 'driver' transgene. An advantage of the former approach is that one can often distinguish between immediate and secondary downstream effects based on how rapidly they occur following heat induction. Advantages of the second approach include having normal and altered expression in parts of the same embryo, providing a rigorous internal control. Both of these approaches led to similar conclusions, that exd function is important for the repression by En of its direct target gene slp, that wg also shows a strong dependence on exd function for its repression by En and that the ability of En to alter the pattern of embryonic cuticles is sensitive to the gene dosage of exd. Further, in each set of experiments, the observed dependence of repression on exd was accompanied by a residual repression activity when exd function was removed both maternally and zygotically. This residual exd-independent repression activity might be due to the ability of En to bind to target sites independently of exd but with a reduced affinity, or it could be accounted for by the existence of two classes of binding sites, one exd dependent and the other exd independent. This possibility is paralleled by the relationship of Exd with Ubx, which has been shown to function either co-operatively with Exd or alone on multiple binding sites in target genes. Alternatively, exd might be exerting an indirect effect on repression by En. However, because Exd forms complexes with En in yeast and in vitro, and because it appears to facilitate repression by En directly in cultured cells, it seems likely that the dependence of En on exd function in vivo is due at least in part to the direct action of En-Exd complexes. Confirmation of this model will require the analysis of specific regulatory sites, which have not yet been identified, in target genes such as slp. If this model is correct then these results suggest that the repression activity of Exd-En complexes might come exclusively from En repression domains, because Exd has been shown to act as a cofactor in the activation of target genes in vivo in conjunction with Hox 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).

The identification of slp as a direct target gene of En has implications for the mechanism by which En helps to maintain the activity of its own and other genes, including hedgehog, within its domains of expression, the posterior compartments. The slp locus produces two closely related, coordinately regulated gene products (Slp1 and Slp2), which have essentially indistinguishable functions. They are forkhead-domain transcription factors that repress en expression, and both contain a conserved motif (homology region II) that is similar to the Groucho-binding domain of En. Slp1 has also been shown to bind the Groucho co-repressor in vitro, suggesting that it is a repressor and therefore that its action on the en gene is likely to be direct. Thus, the mechanism of en autoregulation, as well as the ability of En to activate other target genes, is likely to be due, at least in part, to an indirect effect of repression of slp expression. In addition, En might activate target genes indirectly by repressing other repressors that are also normally excluded from its expression domain, such as Odd-skipped and the repressor form of Cubitus interruptus (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).

engrailed:Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Developmental Biology | Effects of mutation | References

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