Protein Interactions

The binding of vertebrate Lef-1 to ß-catenin is mediated by its N-terminal 56 amino acids (Huber, 1996). Two pan mutants encode PAN proteins with amino-acid substitutions in the corresponding domain. A test was made whether the wild-type or mutant N-terminal portions of PAN (amino acids 1-133) can bind to purified ß-catenin. The pan mutant proteins bind ß-catenin four-to-five fold less, as compared with wild type. These results indicate (1) that PAN can physically interact with the Armadillo homolog ß-catenin and (2) that mutations in this domain compromise wingless signaling in vivo and have a corresponding deleterious affect on the affinity of the binding interaction in vitro (Brunner, 1997).

Both Armadillo and ß-catenin are capable of coactivating transcription driven by Pangolin. The C-terminus of ß-Catenin is necessary for the effect, whereas its N-terminus is dispensible (van de Wetering, 1997).

The HMG-box protein Pangolin (Drosophila Tcf) can function as either an activator or a repressor of Wingless-responsive genes depending on the state of the Wingless signaling pathway and the availability of Armadillo, Pangolin's coactivator. Mutations of Tcf-binding sites in the promoters of Drosophila Ultrabithorax or Xenopus siamois reduce the level of gene expression in the normal expression domain of the animal, showing that Pangolin and its vertebrate homolog act as gene activators. In Drosophila, signal transduction from Wingless stabilizes cytosolic Armadillo, which then forms a bipartite transcription factor with Pangolin and activates expression of Wingless-responsive genes. In the absence of Armadillo, Pangolin acts as a transcriptional repressor of Wingless-responsive genes, and Groucho acts as a corepressor in this process. Reduction of Pangolin activity partially suppresses wingless and armadillo mutant phenotypes, leading to derepression of Wingless-responsive genes. wingless null mutants completely lose epidermal engrailed expression before stage 10, but in homozygous wg embryos that are heterozygous for pangolin, some cells maintain en expression. This corroborates a repressive role for Pangolin in cells in which the Wg signalling pathway is not active. Reduction of Armadillo levels causes Pangolin to act as a repressor. Dominant negative Pangolin, lacking the Armadillo-binding regions, acts as a constitutive repressor. Furthermore, overexpression of wild-type Pangolin enhances the phenotype of a weak wingless allele. Finally, mutations in the Drosophila groucho gene also suppress wingless and armadillo mutant phenotypes since Groucho physically interacts with Pangolin and is required for its full repressor activity. When the N-terminal region of Groucho is expressed in cultured cells, it localizes to the cytoplasm. Coexpression of either human Tcf-1 or Pangolin results in the localization of this truncated Groucho to the nucleus, consistent with a physical association between the proteins. Full-length Gro is constitutively nuclear, and as such, is not informative in this assay. The recruitment of truncated Groucho by Pangolin is very similar to the recruitment of beta-catenin, a known Tcf-binding partner. groucho mutations show dose-senstive interactions with both wg and arm. Reducing the dose of maternal Gro suppresses the wg null phenotype, whereas reduction of paternal Gro has no effect. Pangolin repression is shown to requires Groucho. Deletion analysis defines a minimal region in hTCF-1 (amino acids 176-359) that is capable of binding to Grg-5; this domain is separable from the Armadillo (Arm)-interaction domain (amino acids 4-63). XGrg-5, which lacks the C-terminal WD40 repeats of the longer Grg proteins, enhances the transcriptional activity of suboptimal amounts of Arm-XTcf-3 complexes. mGrg-5 has no intrinsic transactivation properties when fused to a Gal4 DNA-binding domain. The enhancement of transcription by XGrg-5 could probably be attributed to its interference with the repressive effects of endogenous Gro proteins. A deletion mutant of XTcf-3 that lacks the Grg-interaction domain is a tenfold more potent transcriptional activator than its wild-type counterpart, confirming the activity of endogenous corepressors of Tcf factors. Therefore, it is proposed that the balance between the activity of Gro and Arm controls cell-fate choice by the Wnt pathway in both vertebrates and invertebrates (Cavallo, 1998).

T-cell factor (TCF), a high-mobility-group domain protein, is the transcription factor activated by Wnt/Wingless signaling. When signaling occurs, TCF binds to its coactivator, beta-catenin/Armadillo, and stimulates the transcription of the target genes of Wnt/Wingless by binding to TCF-responsive enhancers. Inappropriate activation of TCF in the colon epithelium and other cells leads to cancer. It is therefore desirable for unstimulated cells to have a negative control mechanism to keep TCF inactive. Drosophila CREB-binding protein (dCBP: Nejire) binds to Drosophila TCF (Pangolin). dCBP mutants show mild Wingless overactivation phenotypes in various tissues. Consistent with this, dCBP loss-of-function suppresses the effects of armadillo mutation. Moreover, dCBP is shown to acetylate a conserved lysine in the Armadillo-binding domain of dTCF, and this acetylation lowers the affinity of Armadillo binding to dTCF. Although CBP is a coactivator of other transcription factors, these data show that CBP represses TCF (Waltzer, 1998).

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, that then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. These hypotheses were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with altered intracellular localizations were generated, and these were expressed in wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo’s nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners (Cox, 1999).

Data in vivo suggest that among Arm’s known partners, cadherins have the highest affinity, with APC and dTCF (Pangolin) having lower and lowest affinities, respectively. Thus, in embryos with reduced levels of Arm, the remaining Arm is exclusively associated with cadherins, as assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm binds to APC/Axin, leading to its destruction and thus preventing accumulation of free Arm. While APC levels, at least in mammalian cells, are low, relative to the total pool of beta catenin, Arm bound to APC is rapidly targeted for destruction, thus opening the way for the binding of additional Arm. Normally the destruction machinery can not only dispose of all non-junctional Arm, but its resources will not even be fully employed, since Arm synthesis can be increased several-fold without biological consequences. However, when the destruction machinery is inactivated either by Wg signal or mutation, Arm is synthesized but not destroyed, and thus levels of Arm rise. APC can bind Arm but in all probability, the APC is rapidly saturated, allowing accumulation of sufficient Arm to allow dTCF to effectively compete for binding. DE-cadherin, dAPC, dTCF and any other possible unknown partners together account for virtually all the Arm in a normal embryo; little if any free Arm is present. This model helps explain the differences in localization of the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus, added NLS or NES signals dramatically altered Arm’s intracellular distribution as expected, while in Drosophila the distribution of wild type Armadillo, Arm-NLS and Arm-NES are indistinguishable. It is proposed that this reflects differences in the level of expression. In flies, mutant Arm accumulates at near wild-type levels, so its binding partners can accommodate the additional protein. Arm bound to cadherin at the plasma membrane is unavailable for nuclear import; likewise Arm in a complex with dTCF is not available for export. Thus Arm-NLS and Arm-NES localization is primarily determined by their binding partners, resulting in a near normal localization. In contrast, Arm-NLS and Arm-NES expression levels in Xenopus likely exceed those of either endogenous beta-catenin or its binding partners. Free Arm is thus accessible to the nuclear import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in Wg signaling in normal cells? The fact that a subset of cells accumulate cytoplasmic but not nuclear Arm suggests that nuclear import may be regulated. In the simplest situation, addition of an NLS ought to promote Arm nuclear accumulation and trigger signaling, while addition of an NES should antagonize signaling. However, heterologous targeting signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does Arm-WT, while only a subset of the Arm-NLS lines are activated for signaling. In the case of Arm-NLS: in cells in which the destruction machinery is on, no free Arm is available for nuclear import or export. In cells with intermediate levels of Wg signaling, the destruction machinery may be slowed, allowing accumulation of cytoplasmic Arm in complex with APC, but not to sufficient levels to saturate APC and allow nuclear import. Only when signaling is fully activated would sufficient free Arm accumulate for nuclear import. Addition of an NLS would thus only alter the balance in cells near the signaling threshold. Further, if nuclear Arm is bound to dTCF, it may be inaccessible to the nuclear export machinery. The mechanisms by which Arm/beta-catenin enters nuclei remain unclear; dTCF-dependent and independent pathways may exist. The recent observation that beta Catenin may mediate its own nuclear transport, independent of importins, further complicates the issue. Additional levels of regulation may occur, beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).

The Wingless signaling pathway directs many developmental processes in Drosophila by regulating the expression of specific downstream target genes. The product of the trithorax group gene osa is required to repress such genes in the absence of the Wingless signal. The Wingless-regulated genes nubbin, Distal-less, and decapentaplegic and a minimal enhancer from the Ultrabithorax gene are misexpressed in osa mutants and repressed by ectopic Osa. Osa-mediated repression occurs downstream of the up-regulation of Armadillo but is sensitive both to the relative levels of activating Armadillo/Pangolin and repressing Groucho/Pangolin complexes that are present, and to the responsiveness of the promoter to Wingless. Osa functions as a component of the Brahma chromatin-remodeling complex; other components of this complex are likewise required to repress Wingless target genes. These results suggest that altering the conformation of chromatin is an important mechanism by which Wingless signaling activates gene expression (Collins, 2000).

In addition to transducing the wg signal in a complex with Arm, Pan is also required for the active repression of Wg target genes in the absence of the Wg signal. This repression requires the association of Pan with the corepressor Groucho (Gro). Gro functionally interacts with the histone deacetylase Rpd3, and this interaction is important for at least some of the repressive activity of Gro. Thus, both Osa-containing Brm complexes and Pan/Gro/Rpd3 complexes repress the expression of Wg target genes and probably mediate this repression by altering the local chromatin architecture at the promoters of these genes. Consistent with this, reduction of gro or rpd3 dosage reduces the ability of Osa to repress nub. The loss of nub expression caused by expression of UAS-Osa with ap-Gal4 is significantly rescued in wing discs homozygous for a hypomorphic allele of rpd3. Also, larvae transheterozygous for osaeld308 and groE48 often ectopically express nub in the wing disc, and 40% of transheterozygous adults have notum-to-wing transformations. These phenotypes are not seen when osa or gro single mutants are crossed to wild-type flies (Collins, 2000).

Interestingly, Gro has been shown to interact with the N-terminal tail of histone H3 and with the histone deacetylase Rpd3, and it has therefore been proposed that Gro mediates repression by altering chromatin structure. Consistent with this, a strong genetic interaction exists between osa and gro that suggests that their activities in repressing Wg target genes are closely related. Although it has not previously been reported that Rpd3 functions in the repression of wg target genes, reducing the function of rpd3 can partly rescue the loss of nub expression caused by the overexpression of Osa. Rpd3 is therefore important for the repression of Wg target genes; testing whether it is essential awaits the isolation of null alleles (Collins, 2000).

The loss of either osa or gro leads to ectopic expression of Wg target genes; thus, the activity of one is not sufficient to repress the expression of these genes without the activity of the other. Osa and Gro may, therefore, be mediating the same repressive event rather than acting in parallel. Interestingly, human SWI/SNF forms a repressor complex with Rb and the histone deacetylase HDAC. This complex interacts with the cyclin E promoter through the binding of Rb to E2F-1 and represses E2F-1 activation of cyclin E expression. This suggests the intriguing possibility that Osa and the Brm complex function in a larger repressor complex containing Gro and Rpd3 and that this complex is recruited to Wg target genes though the binding of Gro to Pan. However, Gro acts as a corepressor for a large number of transcription factors, and Osa cannot be required for all repression mediated by Gro because loss of osa does not result in neurogenic phenotypes like those caused by the loss of gro. Further research is needed to determine if Gro and/or Rpd3 can directly interact with components of the Brm complex and, if so, what determines the specificity of this interaction (Collins, 2000 and references therein).

A model for the regulation of gene expression by components of the Wg pathway is presented. The chromatin remodeling activity of the OsaBrm complex is required to maintain the chromatin at the promoters of wg-responsive genes in a repressive conformation. This would prohibit the association of other transcription factors with their binding sites and prevent the recruitment of components of the basal transcription machinery. Osa/Brm complexes may be recruited to Wg-responsive genes through an association with Pan/Gro/Rpd3 complexes. In response to the Wg signal, Arm is stabilized and accumulates in the cytosol. This accumulation of cytosolic Arm permits Arm to translocate to the nucleus and displace Gro from Pan and, in so doing, relieve the repression mediated by Gro, Rpd3, and Osa/Brm complexes. Arm may also promote a more open chromatin conformation by recruiting the HAT activity of dCBP, thus permitting the association of other transcription factors with their binding sites. Also, the stimulation of the DNA-bending activity of Pan by Arm may bring distantly spaced transcription factors into juxtaposition to promote the activation of gene expression. In the absence of osa, the chromatin is maintained in a more open and less repressive conformation. This would permit other transcription factors to interact with their binding sites at lower concentrations than would otherwise be possible. Under these conditions, the low levels of Arm that are always present in the cell may be sufficient to promote the activation of gene expression without the Wg signal (Collins, 2000).

Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. UbxVMB, a visceral mesoderm-specific enhancer of the Ubx gene, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf (Pangolin) binding sites in the UbxVMB enhancer. Thus, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on UbxVMB. A possible target of this activity is Pangolin. Since Pangolin has been shown to be capable of repressing Wingless targets, these results suggest that this repressive activity may be regulated by Notch. It is suggested that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of Pangolin and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with Pangolin (Lawrence, 2001).

These effects of loss of Notch function are not mediated by the Dpp responsive sites but require the integrity of at least one of two Pangolin binding sites on the enhancer, as do other activities of UbxVMB. This regulatory activity of Notch is likely to be different from that which mediates lateral inhibition since the activity of the enhancer is sensitive neither to Su(H) nor to forms of Notch, such as Nintra, that provide constitutive Notch signaling during lateral inhibition. Altogether, these results suggest the existence of an activity of Notch that antagonizes Wg signaling via Dsh. Thus, the removal of Notch function would lower the requirements for Dsh, as was observed. Similar situations have been described before in the development of muscle and peripheral nervous-system precursors in Drosophila and raise the possibility that, in addition to the Frizzled-mediated events, effective Wg signaling requires the downregulation of a Notch signaling event that might be independent of Su(H). These experiments suggest that a possible target of this event is the activity of Pangolin (Lawrence, 2001).

Members of the Pangolin family interact with Arm/ß-catenin to form complexes that can promote the transcription of Wnt/Wg targets in vitro and in vivo. However, with the exception of LEF-1, Tcf family members on their own do not promote the expression of Wnt target genes, and in some instances they can even repress the expression of these targets (Lawrence, 2001 and references therein).

The UbxVMB enhancer has provided a good model for the analysis of the role of Pangolin in Wg signaling. The optimal activity of this enhancer requires canonical Wg signaling via Dsh, Arm, and Pangolin, and for this reason it was surprising to observe that in the absence of Notch the activity of UbxVMB is independent of the canonical Wg pathway. The ability of the loss of Notch function to reverse the effects of the loss of function of Wingless signaling is most clearly demonstrated in the case of the UbxVMBM2 mutant enhancer. The activity of this enhancer is independent of Dpp but displays an absolute requirement for Wg signaling. However, while UbxVMBM2 is completely inactive in dsh mutants, it directs expression of a reporter in N;dsh double mutants (Lawrence, 2001).

The response of UbxVMB to the loss of Notch function, like that to Wg signaling, requires the integrity of at least one of the Pangolin sites in the context of the full enhancer and thus raises the possibility that these sites, and perhaps the activity of Pangolin itself, are the targets of Notch. It might be that the activity of UbxVMB is repressed by Pangolin in a Notch-dependent manner and that to signal efficiently, Wg must antagonize this repression. In the absence of Notch, this repression would not be implemented, which would lead to enhancer activity that is independent of Wg. This can account for the widespread and premature activity of the enhancer as well as the diminished requirements for Wg signaling that are observed in the absence of Notch (Lawrence, 2001).

Several observations indicate that Pangolin can act as a repressor, and recent results on the regulation of dpp expression in the VM of Drosophila support this possibility. The mutation of Pangolin binding sites in a Wg-dependent enhancer of the dpp gene results in spatially deregulated high levels of activity of the enhancer. This finding suggests that in this case Pangolin acts, primarily, as a repressor and that one function of Wg/Arm signaling might be to promote a nonrepressed state. Pangolin is also likely to act as a repressor at UbxVMB since the mutation of one Pangolin site, although lowering the overall levels of activity, expands the spatial domain of activity of the enhancer. These results provide further evidence for this repressive activity and suggest that Notch might be involved in it. However, in contrast to results with the dpp enhancer, the mutation of both Pangolin sites in UbxVMB abolishes enhancer activity. This finding indicates that at this enhancer Pangolin is also required as an activator, together with Arm (Lawrence, 2001).

In Drosophila, Pangolin can indeed behave as a repressor and an activator through interactions with different molecules. These results raise the possibility that its activity as a repressor, through interactions with transcriptional corepressors such as Groucho or CtBP, is modulated by a signaling event that depends on Notch. Wg signaling can thus lead to gene expression in two ways, by transcriptional activation through a Pangolin/Arm complex or by antagonizing the repressive activities of Pangolin. This dual activity of Wg signaling would explain the observations that in N;dsh double mutant embryos enhancer activity, although higher than in dsh mutants, is lower than that in N mutants. Thus, in N;dsh embryos the activity of the enhancer results from a derepression (inactivation of Pangolin repressor complexes) without the concomitant Arm activation mediated by Dsh (Lawrence, 2001).

The observations on UbxVMB parallel others in which Notch has been shown to antagonize Wg signaling independently of Su(H), and they raise the possibility that effective Wg signaling requires an antagonism of this repression. Interestingly, Wg and Dsh can bind to Notch, and therefore, the antagonism could be mediated by conformational changes in Notch induced at the cell surface through direct interactions between these molecules (Lawrence, 2001).

On the basis of these observations, it is suggest that in Drosophila, Wg signaling operates by regulating two molecular events: (1) repression of the expression of Wg targets implemented by Notch and (2) the Shaggy/GSK3-dependent degradation of Arm promoted by the Axin/APC complex. The regulation of both processes might be linked through the activity of Pangolin: the first event maintains its activity as a repressor, while the second one prevents its becoming an activator. Wg binding to Notch would modulate the repressive activity of Pangolin; then, through the activity of members of the Frizzled family of receptors, it would modulate the activity of Arm. It may be that Arm can only interact with a nonrepressor form of Pangolin and that, therefore, the 'effectiveness' of Wg signaling is determined by the amount of Notch signaling. A combination of antirepression and activation might help explain the observation that dominant-negative Frizzled and dominant-negative Pangolin have no effect on the activity of this enhancer, as they should have if activation was the only way to get expression. In addition, this would explain the observation that lowering Notch signaling increases the effectiveness of Arm signaling. It will be important to understand how Notch modulates Wg signaling (Lawrence, 2001).

One difficulty with this model is the activity of the UbxVMB enhancer in the absence of both Notch and Dsh since, under these conditions, the levels of cytoplasmic Arm are low. It may be that the loss of Notch function alters some parameters of the interaction between Pangolin and Arm that allow very efficient functional association of Pangolin with these low levels of Arm. In this regard there is evidence that the phosphorylation of Tcf can regulate the activity of the Tcf/ ß-catenin complex. Further work should address these issues (Lawrence, 2001).

Drosophila Armadillo plays two distinct roles during development. It is a component of adherens junctions, and functions as a transcriptional activator in response to Wingless signaling. In the current model, Wingless signal causes stabilization of cytoplasmic Armadillo allowing it to enter the nucleus where it can activate transcription. However, the mechanism of nuclear import and export remains to be elucidated. Two gain-of-function alleles of Armadillo are shown to activate Wingless signaling by different mechanisms. The S10 allele localizes to the nucleus, where it activates transcription. In contrast, the DeltaArm allele localizes to the plasma membrane, and forces endogenous Arm into the nucleus. Therefore, DeltaArm is dependent on the presence of a functional endogenous allele of arm to activate transcription. DeltaArm may function by titrating Axin protein to the membrane, suggesting that Axin acts as a cytoplasmic anchor keeping Arm out of the nucleus. In axin mutants, Arm is localized to the nuclei. Nuclear retention is dependent on dTCF/Pangolin. This suggests that cellular distribution of Arm is controlled by an anchoring system, where various nuclear and cytoplasmic binding partners determine its localization (Tolwinski, 2001).

Since Arm import and export have been reported to be highly dynamic, a second mechanism must be in place to retain the imported Arm within the nucleus. One possibility is that dTCF/Pan anchors nuclear Arm to the DNA. By expressing a dominant negative form of TCF that interacts with DNA but no longer binds Arm, the nuclear accumulation observed following DeltaArm expression alone is blocked. Overexpressed dTCFDeltaN may occupy many of the DNA binding sites that Arm normally uses to stay in the nucleus, making it susceptible to export. Expression of dTCFDeltaN does not lead to complete exclusion of endogenous Arm from the nucleus, suggesting that there may be more relevant nuclear factors, possibly groucho. Overexpression of full-length dTCF does not lead to nuclear accumulation of endogenous Arm, suggesting that dTCF levels are not limiting. This is consistent with overexpression of dTCF having only a very subtle cuticle phenotype. However, overexpression of LEF-1 (a mammalian homolog of dTCF) in tissue culture cells, does lead to nuclear accumulation of ß-catenin. This was not observed in Drosophila embryos, suggesting that limiting levels of nuclear anchor may be a feature of specific cell types that have yet to be observed in Drosophila (Tolwinski, 2001).

A model is favored where the dynamic import and export of Arm is controlled by binding partners in the cytoplasm and the nucleus. Axin is involved in cytoplasmic anchoring, and dTCF/Pan is involved in nuclear retention. Arm retained in the cytoplasm is degraded unless it enters adherens junctions. In response to Wg, degradation stops, and Arm accumulates in the cytoplasm bound to Axin. Some Arm enters the nucleus where it binds dTCF/Pan. An equilibrium is reached as a result of active import and export, and inactive degradation. This is the situation in Arm stripes where diffuse staining throughout the cell is observed. However, the existence of anchoring offers a second level of signaling control that could induce a rapid and concentrated nuclear accumulation of Arm with no change in levels. Specific nuclear accumulation has been observed in Xenopus and sea urchin. Though levels were not measured, the striking lack of cytoplasmic ß-catenin is suggestive of a lack of cytoplasmic anchoring. Another response of this type may be what is observed in the epithelial to mesenchyme transition. Here, ILK is overexpressed in epithelial cells resulting in very high nuclear accumulation of ß-catenin without an increase in levels, suggesting the possibility of inhibition of cytoplasmic anchoring (Tolwinski, 2001).

Recently, two studies have suggested that APC is involved in the nuclear export of Arm/ß-catenin. APC contains a nuclear export signal (NES) which is required for efficient export of ß-catenin from the nucleus. Combining this result with the current data, it is proposed that there are at least two levels of control of Arm/ß-catenin localization involving cytoplasmic anchoring and active export. APC may play a role in preventing Arm/ß-catenin from accumulating in the nucleus due to dTCF binding. Both controls must be overcome to accumulate enough Arm/ß-catenin to activate transcription (Tolwinski, 2001).

Wnt-induced formation of nuclear Tcf-ß-catenin complexes promotes transcriptional activation of target genes involved in cell fate decisions. Inappropriate expression of Tcf target genes resulting from mutational activation of this pathway is also implicated in tumorigenesis. The C-terminus of ß-catenin is indispensable for the transactivation function, which probably reflects the presence of binding sites for essential transcriptional coactivators such as p300/CBP. However, the precise mechanism of transactivation remains unclear. An interaction between ß-catenin and Brg-1, a component of mammalian SWI/SNF and Rsc chromatin-remodeling complexes, is demonstrated. A functional consequence of reintroduction of Brg-1 into Brg-1-deficient cells is enhanced activity of a Tcf-responsive reporter gene. Consistent with this, stable expression of inactive forms of Brg-1 in colon carcinoma cell lines specifically inhibits expression of endogenous Tcf target genes. In addition, genetic interactions are observed between the Brg-1 and ß-catenin homologues in flies. It is concluded that ß-catenin recruits Brg-1 to Tcf target gene promoters, facilitating chromatin remodeling as a prerequisite for transcriptional activation (Barker, 2001).

Development in flies was selected as a model system to gain evidence for a functional interaction between Brg-1 and ß-catenin in vivo, assuming that this interaction would be conserved between mammals and Drosophila. It was asked whether reducing the gene dosage of brahma, the founder of the Brg-1 gene family, affects the mutant phenotypes caused by activation or depletion of Armadillo. First, a strain (GMR.Arm*) was used in which a constitutively activated form of Armadillo is overexpressed in the larval eye disc. The mutation in Arm* mimics the oncogenic point mutation S45F in the putative GSK3ß phosphorylation site of ß-catenin that renders the latter constitutively active. Oncogenic forms of ß-catenin such as this are potent transcriptional coactivators of Tcf. Flies bearing GMR.Arm* show rough and slightly glazed eyes whose size is reduced compared with the wild-type, due to late onset of apoptosis in the pupal disc caused by Arm* and dTcf. This phenotype is independent of armadillo gene dosage, but is reversed considerably towards wild-type in dTcf heterozygotes, whose gene dosage is reduced by half. This rough eye phenotype was reversed even further towards wild-type in brahma heterozygotes. Finally, a similar phenotypic suppression was observed in flies heterozygous for moira, a gene encoding another component of the Brahma complex. It is concluded that the mutant eye phenotype caused by activated Armadillo is as sensitive to the levels of Brahma complex components as it is to dTcf levels, indicating that the Brahma complex is required for the activity of Arm* (Barker, 2001).

It was also asked whether heterozygosity of Brahma complex genes would affect the mutant wing phenotype caused by Armadillo depletion in the wing disc. In the wing, armadillo is required for the integrity of the margin, and sequestration of Armadillo at the membrane by overexpression of the intracellular domain of cadherin (Armunder) in the posterior wing disc causes extensive notches in the posterior wing. This phenotype is worsened by heterozygosity for activating genes of the Wingless pathway, and suppressed by heterozygosity of antagonists of this pathway. In particular, in Armunder flies heterozygous for armadillo the posterior wing margin is completely absent, and the posterior wing area is much reduced. Likewise, dTcf heterozygotes showed on average slightly narrower wings, and less residual posterior margin than Armunder controls. This modifying effect of dTcf is much milder than that observed in the eye, perhaps reflecting a dual function of dTcf in the wing margin (activating as well as repressing) similar to that observed in the embryonic cuticle. Significantly, brahma heterozygotes show considerably narrower wings than the controls. Indeed, brahma heterozygosity enhances the wing margin phenotype as strongly as armadillo heterozygosity. Finally, a slight worsening of this phenotype is also observed in moira heterozygotes. These genetic experiments in flies indicate functional interactions between Brahma complex genes and Armadillo/dTcf. Consistent with this, it has been reported that embryos derived from near-sterile brm transheterozygous mothers show reduced expression of dTcf target genes such as Ultrabithorax and engrailed (Barker, 2001).

Taken together, these fly genetic data support the conclusions from experiments in mammalian cells that the Brg-1 complex contributes to the activity of the ß-catenin-Tcf transcription factor (Barker, 2001).

Groucho corepressor proteins, which repress Tcf target gene activity in the absence of Wnt signaling, are known to recruit histone deactylases and are likely to effect repression by altering chromatin structure. Additionally, a recent study has demonstrated a role for SWI/SNF-mediated chromatin remodeling of Tcf target gene promoters in ensuring effective repression of gene activity in the absence of ß-catenin during fly development. Potentially, Groucho proteins in complex with Tcf could recruit Brahma complexes to target gene promoters through an interaction mediated by the histone deacetylase rpd3. The data support a mechanism in which ß-catenin accumulation following Wnt signaling promotes the formation of ß-catenin-SWI/SNF (or -Rsc) complexes in the nucleus, in competition with Groucho repressor complexes. In cooperation with the histone-acetylating activity of ß-catenin-bound p300/CBP, Brg-1-associated complexes would then remodel the chromatin structure of target gene promoters into a conformation more accessible to the basal transcription machinery, enhancing transactivation of target genes and leading to cellular responses. The initially paradoxical observation that chromatin-remodeling complexes are required for both the activation and repression of perhaps the same set of target genes can be resolved by the finding that in vitro, SWI/SNF and Rsc can catalyse both forward and reverse nucleosome remodeling reactions (Barker, 2001).

Inappropriate activation of downstream target genes by the oncoprotein ß-catenin is implicated in development of numerous human cancers. ß-catenin and its fruitfly counterpart Armadillo act as coactivators in the canonical Wnt/Wingless pathway by binding to Tcf/Lef transcription factors. A conserved nuclear protein, named Chibby, has been identified in a screen for proteins that directly interact with the C-terminal region of ß-catenin. In mammalian cultured cells Chibby inhibits ß-catenin-mediated transcriptional activation by competing with Lef-1 to bind to ß-catenin. Inhibition of Drosophila Chibby by RNA interference results in segment polarity defects that mimick a wingless gain-of-function phenotype, and overexpression of the wingless target genes engrailed and Ultrabithorax. In addition, epistasis experiments indicate that chibby acts downstream of wingless and upstream of armadillo (Takemaru, 2003).

To investigate possible alternative mechanisms by which Cby inhibits ß-catenin-dependent gene activation, Myc-tagged ß-catenin and Flag-tagged Cby were co-transfected with increasing amounts of hemagglutinin (HA)-tagged Lef-1 (lymphoid-enhancer factor-1), then immunoprecipitated with an anti-Myc antibody. Because the amount of Cby co-precipitating with ß-catenin decreases as the amount of Lef-1 associating with ß-catenin increases, it is concluded that Cby competes with Lef-1 for binding to ß-catenin. In contrast, Cby has no apparent effect on the interaction between ß-catenin and APC or CBP. Additional data suggest that Cby negatively regulates ß-catenin-mediated transactivation by competing with Tcf/Lef transcription factors in mammalian cells (Takemaru, 2003).

A complex of Armadillo, Legless, and Pygopus coactivates dTCF to activate Wingless target genes

Upon receiving a Wnt signal, cells accumulate ß-catenin (Armadillo in Drosophila), which binds directly to TCF transcription factors, leading to the transcription of Wnt target genes. It is generally thought that ß-catenin/Armadillo is a transcriptional coactivator when bound to TCF in the nucleus and that this function is mediated by its C terminus. However, recent findings in Drosophila indicated that Armadillo may activate dTCF in the cytoplasm. This study reexamines the mechanism of Armadillo's signaling function in light of Legless and Pygopus, two nuclear factors recently discovered to be essential for this function. Armadillo, in order to activate dTCF, must enter the nucleus and form a complex with Legless and Pygopus. The ability of this complex to stimulate TCF-mediated transcription can be altered by linkage of a strong transcriptional activator or repressor to Armadillo. Furthermore, Armadillo is a strong transcriptional activator when fused to the yeast GAL4 DNA binding domain -- an activity that depends on regions of the Armadillo repeat domain that mediates binding to Legless and to chromatin modifying and remodeling factors. Finally, linkage of the N-terminal region of Pygopus, but not the C terminus of Armadillo, to dominant-negative dTCF can restore its signaling activity in transgenic flies. This evidence argues in favor of a revised coactivator factor model in which Armadillo's coactivator function depends on regions within its Armadillo repeat domain to which Legless/Pygopus and other transcriptional coactivators can bind. In contrast, the C terminus of Armadillo plays a less direct role in this function (Thompson, 2004).

The model that Arm functions in the nucleus as a transcriptional activator of dTCF clearly predicts that exclusion of Arm from the nucleus by tethering to membranes should render it unable to signal. Two such nuclear-excluded, membrane-tethered forms of Arm have been examined in Drosophila: Sev-Arm, a fusion of the extracellular and transmembrane domains of Sevenless to Arm's N terminus, and Arm-CAAX, which features a CAAX-type palmitoylation sequence at its C terminus. The signaling activity of Arm transgenes can be measured by examining their ability to rescue Drosophila embryos that are maternally and zygotically mutant (henceforth: mutant) for arm. A severe impediment to this analysis is that arm null mutants (eg: armXP33 and arm4 also called armYD35) have adhesion defects in addition to defective Wingless signaling and, consequently, do not develop beyond oogenesis. Thus, mutant conditions that affect signaling, but not adhesion, must be used. The most commonly used signaling-mutant (but adhesion-competent) allele is armXM19, a truncation of the Arm C terminus that generates embryos with defective Wingless signaling (Thompson, 2004).

Surprisingly, both Sev-Arm and Arm-CAAX were reported to substantially rescue Wingless signaling in armXM19 mutants. The two possible interpretations of these results are (1) that these proteins signal independently of endogenous Arm and (2) that the ArmXM19 mutant protein can be induced to signal in the presence of these transgenes. Discrimination between these two possibilities requires examination of these transgenes in alternative arm mutant backgrounds. In the case of Arm-CAAX it was possible to use an effectively null mutant, armXP33 (which does not express detectable Arm protein), because Arm-CAAX is able to function in adhesion. Arm-CAAX was found to rescue the adhesion, but not the signaling defect of armXP33. In the case of Sev-Arm, analysis in a null mutant background is not possible because this transgene is not competent to rescue the adhesion defect. Attempts were made with arm043A01, an allele that produces both signaling and mild adhesion defects, but the results are unclear, because mutant embryos do not secrete a cuticle. Therefore, alternative mutant conditions were generated by expressing signaling-mutant (but adhesion-competent) Arm transgenes, ArmS6 and ArmS12, in an arm4 null-mutant background. These conditions (henceforth: ArmS6 and ArmS12 mutants) generated embryos whose cuticle phenotype was a lawn of denticles, indicating that Wingless signaling was inactive. Ubiquitous expression of Sev-Arm with the Gal4-UAS system was unable to rescue the Wingless-signaling defect of these embryos, whereas similar ubiquitous expression of Sev-Arm was able to rescue the cuticular phenotype of armXM19 mutants considerably. Similarly, as a control, an activated form of Armadillo, ArmS10, was able to rescue all three signaling-mutant conditions. It is concluded that Sev-Arm, like Arm-CAAX, is unable to signal in the absence of functional endogenous Arm and that the C-terminally truncated ArmXM19 protein retains significant signaling activity that is revealed by the expression of membrane-tethered forms of Arm (Thompson, 2004).

In addition to dTCF, two other ubiquitous factors, Legless (Lgs) and Pygopus (Pygo), are essential for Arm's signaling activity in Drosophila. In lgs or pygo mutants, Arm is unable to signal, even when it accumulates at unusually high levels throughout the cell. The localization of these proteins (either the endogenous protein or epitope-tagged versions expressed from a transgene) were examined in the embryonic epidermis where high levels of Wingless induce accumulation of Arm in stripes of cells. dTCF, Lgs, and Pygo are predominantly nuclear in all cells regardless of their state of signaling. Notably, no evidence was found for nuclear export of tagged, expressed dTCF in response to Wingless in the embryonic epidermis (Thompson, 2004).

Although genetic analysis of Lgs and Pygo has demonstrated that they are essential for Arm's signaling activity, it remains possible that these proteins simply provide an essential function for dTCF. No evidence was found that dTCF stability or localization are affected in pygo mutants. Note that both Lgs and Pygo function are compromised in pygo mutants, because Lgs depends on Pygo for its nuclear localization (F.M. Townsley, A. Cliffe, and M. Bienz, unpublished data, cited in Thompson, 2004). If Lgs and Pygo provide an essential function for Arm rather than dTCF, then providing dTCF with a strong transcriptional activator should bypass the requirement for Lgs and Pygo. A fusion protein of dTCF with the VP16 transcriptional activation domain (dTCF-VP16) that had been shown to rescue armXM19 mutants was therefore expressed in wild-type and pygo mutant embryos with the GAL4-UAS system. Unfortunately, expression of dTCF-VP16 arrests embryogenesis prior to cuticular differentiation. Therefore the expression of the engrailed gene, a target of Wingless signaling in the embryo that is downregulated in pygo mutants was examined. Expression of dTCF-VP16 is able to restore engrailed expression in these embryos. It is concluded that Lgs and Pygo are not required for dTCF's stability, localization, or DNA binding activity but, rather, for activation of dTCF by Arm (Thompson, 2004).

Consistent with this view, in vitro binding experiments have shown that the Lgs HD2 domain binds directly to the first four Armadillo repeats of Arm, while the Lgs HD1 domain binds to the PHD domain of Pygo. On this basis, it was proposed that Arm, Lgs, and Pygo may form a complex in vivo. To test this proposal, an HA-tagged version of Pygo was expressed in Drosophila embryos that also expressed Wingless to activate signaling in all cells. The tagged Pygo was immunoprecipitated with αHA antibodies. Both Arm and Lgs were found to be readily coimmunoprecipitated from embryos expressing HAPygo, but not from control embryos. It is concluded that Arm, Lgs, and Pygo form a nuclear complex in Wingless-stimulated cells in vivo. These findings strongly support the view that Arm activates dTCF in the nucleus, since Lgs and Pygo, two binding partners for Arm that are essential for this process, are nuclear proteins (Thompson, 2004).

The the Arm/dTCF transcription factor model of Wingless signal transduction need to be reconsidered in light of the discovery of Legless and Pygopus. This model was originally prompted by the findings that (1) activation of dTCF depends on a direct binding interaction with Arm; (2) TCF transcription factors are constitutively localized to the nucleus, whereas Arm enters the nucleus only upon signaling; and (3) the C terminus of Arm, which is absent in armXM19 mutants, can function as a transcriptional activator when tethered to DNA (Thompson, 2004).

The model for Arm function predicts that Arm must enter the nucleus in order to form an active transcription factor with dTCF on DNA. The results of this study show that membrane-tethered forms of Arm cannot directly activate dTCF, supporting the notion that Arm must enter the nucleus to do so. The ability of membrane-tethered Arm to signal in an armXM19 mutant background must therefore reflect that the armXM19 mutation is not a null and must retain some signaling activity that is enhanced by the presence of membrane-tethered Arm. A plausible explanation for this phenomenon is that membrane-tethered Arm recruits negative regulators of Arm, thereby stabilizing and/or promoting nuclear translocation of endogenous Arm. In support of this explanation, effects of this kind have, in fact, been observed with several different types of membrane-targeted Arm and β-catenin. Consideration of these results reveals a point of conflict with the original form of the Arm/dTCF transcription factor model, which proposes that the Arm C terminus is necessary and sufficient for Arm's coactivator function. The armXM19 mutation encodes an Arm protein that lacks its C terminus. If this truncated protein retains some signaling activity, then the C terminus cannot be the sole mediator of Arm's coactivator function. In support of this view, several different C-terminally truncated Arm and β-catenins appear to retain significant signaling activity under conditions of overexpression. Furthermore, Arm's C terminus can be substituted without loss of function by the C terminus of a different Armadillo repeat domain protein, Pendulin. Unlike the Arm C terminus, the Pendulin C terminus lacks transactivating activity when fused to the GAL4 DNA binding domain. It is concluded that the C terminus is not sufficient to mediate Arm's coactivator function but instead, is likely to be required in some way for the stability or activity of the Armadillo repeat domain. These findings undermine one block of evidence upon which the Arm coactivator model was originally founded (Thompson, 2004).

Evidence was therefore sought that Arm functions as a transcriptional activator. Arm's ability to activate TCF-mediated transcription, as measured in the Topflash assay, is enhanced by addition of a strong transcriptional activator and reduced by addition of a strong transcriptional repressor. Tethering of Arm to DNA with the GAL4 DNA binding domain reveals that Arm functions as a strong transcriptional activator. Furthermore, this activity of Arm was suppressed by mutations in the Armadillo repeat domain (S6 and S12) that prevent Arm from transducing Wingless signals in vivo. The results indicate that Arm indeed functions as a coactivator and that this function depends on regions in the Armadillo repeat domain that may recruit additional coactivating factors (Thompson, 2004).

Two candidates that may mediate Arm's coactivator function are Lgs and Pygo. Lgs and Pygo are constitutively nuclear proteins that bind to the Armadillo repeat domain upon signaling and are essential for Arm to activate dTCF. Furthermore, Lgs and Pygo appear to be present in the coactivator complex. The N terminus of Pygo (PygoΔPHD) is sufficient to mediate the function of Lgs and Pygo in Wingless signaling when targeted to Arm by fusion to the Lgs HD2 domain. The same region of Pygo has the capacity to function as a transcriptional activator and, when fused to dTCF, can partially bypass the requirement for Armadillo in Wingless signal transduction (Thompson, 2004).

The results argue that Lgs and Pygo directly contribute to transcriptional activation of the Arm/dTCF transcription factor. Although the Pygo N terminus has been defined as a transactivator, it is possible that other regions of Lgs and Pygo may also possess this activity. It is further possible that Lgs and Pygo may contribute indirectly to Arm's coactivator activity: for example, by facilitating nuclear import or retention of Arm (F.M. Townsley, A. Cliffe, and M. Bienz, unpublished data cited in Thompson, 2004).

In any case, it is unlikely that Lgs and Pygo are the sole mediators of Arm's coactivator activity. For example, while the Arm S6 mutation (in repeat 1) might be predicted to affect Lgs binding, the Arm S12 mutation affects the C-terminal repeats (repeats 10 and 11). It is inferred that an additional, essential coactivating factor(s) is recruited to the C-terminal Arm repeats. Two obvious candidate factors are the histone acetyltransferase CBP/p300 and the chromatin remodeling enzyme Brahma, both of which have been found to bind to C-terminal regions of the Armadillo repeat domain (Thompson, 2004 and references therein). The evidence presented in this study argues in favor of an extended Arm/dTCF transcription factor model in which Arm coactivates dTCF by recruiting Lgs, Pygo, and other factors to its Armadillo repeat domain. (Thompson, 2004).

Dissecting nuclear Wingless signalling: Recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins including Arm, Pan, and Legless

Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). This study analyses the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. Evidence is provided that together these four proteins form a chain of adaptors linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, these results suggest that the transcriptional output of Wg pathway activity largely relies on a chain of adaptors designed to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner (Stadeli, 2005).

The identification of β-catenin as the mediator of Wnt signals has spurred investigations and discussions of how a cell adhesion molecule (relocated to the nucleus) can function as a transcriptional activator. One step towards a molecular understanding of nuclear β-catenin function was the discovery of Lgs. Genetic and biochemical experiments indicated that both proteins assist β-catenin in its transactivator role and led to the hypothesis that Lgs functions to recruit Pygo to the β-catenin/TCF complex and hence to the regulatory regions of Wnt target genes. By a series of transcriptional assays in cultured Drosophila cells, this study shows that the four dedicated nuclear components of the Wg signalling pathway, Pan/TCF, Arm/β-catenin, Lgs, and Pygo, act together in a linear manner in order to activate target gene expression. The results have led to the formulation of a 'chain of adaptors' model for nuclear Wg signalling, assigning each of these four proteins a linker function connecting a proximal and a distal component, culminating in the recruitment of the NH2-terminal homology domain of Pygo (PygoNHD) to the promoter of Wg targets. In S2 cells, PygoNHD stimulates reporter gene expression when directly tethered to DNA, indicating that this domain is capable of bestowing transcriptional activity on Arm/β-catenin.

Arm/β-catenin is composed of three domains, each of which bears transcriptional activity when tethered to DNA: the NH2-terminal region. The activity of the NH2-terminus of Arm might not be relevant in the context of the full-length protein, since only an isolated NH2-terminal fragment of Arm[wt] shows transcriptional activity when tethered to DNA. In contrast, an NH2-terminal fragment of ArmS10 or ArmN-R4[D164A] does not show transcriptional activity in this assay. It is thus proposed that Arm's co-activator capacity maps primarily to the COOH-terminal domain and to the Lgs/Pygo recruiting region located in Arm repeats 1-4. While the COOH-terminus of Arm might activate gene expression by recruiting cofactors like the histone acetyltransferase CBP/p300 or the chromatin remodelling enzyme Brg-1/Brahma, the findings indicate that the activity of ArmR1-4 strictly relies on Lgs and Pygo (Stadeli, 2005).

The role of Lgs serving as an adaptor protein to link Pygo with Arm is widely accepted. In contrast, the role of Pygo is somewhat controversial. The finding that the replacement of PygoPHD by LgsHD2 or the fusion of LgsHD2 with PygoNHD results in chimeric proteins that can substitute for both Lgs and Pygo function implies that the LgsHD1 as well as the PygoPHD are dispensable if the PygoNHD is brought to Arm by more direct means. Interestingly, it has been shown in human 293T cells that the activity of DNA-tethered Pygo depends on its PHD. When similar assays were carried out using DNA-tethered constructs in Drosophila S2 cells, it was found that PygoΔPHD is about half as active as full-length Pygo. However, deletion of the NHD leads to a more pronounced decrease in activity, while the isolated NHD is almost as active as full-length Pygo, indicating that the NHD is the key activating domain of Pygo. Consistent with this view, the above-mentioned chimeric proteins lose their rescuing activity if the PygoNHD is mutated. Collectively, these results strongly suggest that Pygo, by means of its NHD, acts as a transcriptional co-activator in Wg signalling (Stadeli, 2005).

Analysis of DNA-tethered PygoNHD in S2 cells reveals that this domain does not act as a classical acidic activator domain. Rather, the conserved tripeptide NPF plays a crucial role in the activity of PygoNHD. Pygo is one out of 791 NPF motif-containing proteins in Drosophila. In yeast, a NPF motif has been found to serve as a recognition motif for proteins bearing Eps15 homology (EH) domains. Since EH domain-containing proteins might work as integrators of signals controlling cellular pathways as diverse as endocytosis, cell proliferation, or nucleocytosolic export, a possible functional link between such proteins and Pygo was examined. However, RNAi-mediated knock-down of all four Drosophila EH domain-containing proteins (CG1099, CG6148, CG6192, CG16932) in the UAS-luc/G4DBD-PygoNHD assay did not cause a decrease in reporter activity, suggesting that the PygoNHD, despite its NPF motif, does not rely on such EH domain-containing proteins for its capacity to activate transcription (Stadeli, 2005).

As an extension of this 'chain of adaptors' model, it is tempting to assume that Pygo, through its NHD, might recruit factors with enzymatic activities, such as histone acetyl transferases (HAT) or chromatin remodelling proteins. Such interactions may depend on an intact Pygo NPF motif. It is important to mention that mutations in this motif, although almost abolishing activity of the isolated DNA-tethered PygoNHD, retain some activity in the context of the full-length protein. It is possible that these mutations do not completely prevent presumptive interactions with positive regulators of transcription; some other part of Pygo may act in a partially redundant manner together with the NHD. Confirmation of such explanations will have to await the identification of PygoNHD interacting proteins (Stadeli, 2005).

Wingless-independent association of Pygopus with dTCF target genes

The Wnt signaling pathway controls numerous cell fates during animal development. Its inappropriate activity can lead to cancer in many human tissues. A key effector of the canonical Wnt pathway is β-catenin (or Drosophila Armadillo), a highly unstable phosphorylated protein that shuttles rapidly between nucleus and cytoplasm. Wnt signaling inhibits its phosphorylation and degradation; this allows it to associate with TCF/LEF factors bound to Wnt target genes and to stimulate their transcription by recruiting chromatin modifying and remodeling factors. The transcriptional activity of Armadillo/β-catenin also depends on Pygopus (Pygo), a nuclear protein with which it associates through the Legless/BCL9 adaptor. It has been proposed that Pygo associates with TCF target genes during Wnt signaling through Armadillo and Legless to recruit a transcriptional coactivator through its Nbox motif. This study reports that Pygo is associated constitutively with dTCF target genes in Drosophila salivary glands and tissue-culture cells. The evidence indicates that this association depends on dTCF and on the Nbox motif of Pygo, but not on Legless. An alternative model is proposed according to which Pygo functions at the onset of Wnt signaling, or at low signaling levels, to capture Armadillo at dTCF target genes, thus enabling the interaction between Armadillo and dTCF and, consequently, the Armadillo-mediated recruitment of transcriptional coactivators (de la Roche, 2007).

Pygo could act as an Armadillo-loading factor whose function might be essential at limiting levels of activated Armadillo, either at low Wingless signaling levels or during the early phase of a Wingless response. Thus, Pygo could target even low levels of nuclear Armadillo to dTCF loci, thereby facilitating the efficient interaction between DNA-bound dTCF and Armadillo and enabling the subsequent recruitment of transcriptional cofactors. It is conceivable that the adaptor chain would rearrange after the capture of Armadillo, which might enable a putative transactivation function of the Nbox binding factor, consistent with a dual role of Pygo. In essence, this model envisages that Pygo predisposes dTCF target genes for efficient activation in response to Wingless. It explains why Pygo is required for efficient nuclear accumulation of Legless and Armadillo and why this requirement is bypassed by high levels of nuclear Armadillo. Note that some dTCF target genes in Drosophila or mammals may not rely on this predisposing function of Pygo, and some modes of Wnt-induced transcription may proceed without it. The ultimate test of this model will depend on the identification of the Nbox binding factor and its proposed role in predisposing TCF target genes to Wnt-induced transcription (de la Roche, 2007).

Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains

A key effector of the canonical Wnt pathway is β-catenin, which binds to TCF/LEF factors to promote the transcription of Wnt target genes. In the absence of Wnt stimulation, β-catenin is phosphorylated constitutively, and modified with K48-linked ubiquitin for subsequent proteasomal degradation. Trabid is a positive regulator of Wnt signaling in mammalian and Drosophila cells. Trabid shows a remarkable preference for binding to K63-linked ubiquitin chains with its three tandem NZF fingers (Npl4 zinc finger), and it cleaves these chains with its OTU (ovarian tumor) domain. These activities of Trabid are required for efficient TCF-mediated transcription in cells with high Wnt pathway activity, including colorectal cancer cell lines. Trabid can bind to and deubiquitylate the APC tumor suppressor protein, a negative regulator of Wnt-mediated transcription. Epistasis experiments indicate that Trabid acts below the stabilization of β-catenin, and that it may affect the association or activity of the TCF-β-catenin transcription complex. These results indicate a role of K63-linked ubiquitin chains during Wnt-induced transcription (Tran, 2008).

In the absence of Wnt stimulation, an active mechanism operates to down-regulate β-catenin, the key effector of the canonical Wnt pathway. This involves the constitutive phosphorylation of β-catenin by the Axin complex that also includes APC, glycogen synthase kinase 3β (GSK3β), and casein kinase I. Phosphorylated β-catenin is recognized by β-TrCP, an F-box-containing adaptor, which binds to an SCF ubiquitin ligase. The latter conjugates a K48-linked ubiquitin chain to phosphorylated β-catenin, which earmarks it for subsequent proteasomal degradation. Binding of Wnt ligand to its transmembrane receptor leads to Dishevelled-mediated inhibition of the Axin complex; as a consequence, unphosphorylated β-catenin accumulates and associates with TCF/LEF factors in the nucleus, to stimulate the transcription of Wnt target genes. This involves the recruitment of a wide range of transcriptional cofactors to the C terminus of β-catenin, including the chromatin modifier and remodeling factors CBP, Brg-1, and SET1; TATA-binding and associated factors; and the transcriptional elongation factor Parafibromin. Through a more N-terminal segment, β-catenin binds to the BCL9-Pygopus complex, to recruit an unknown transcriptional coactivator and/or to facilitate efficient recruitment of β-catenin to TCF target genes. Thus, the key output of canonical Wnt signaling is a switch in transcription (Tran, 2008 and references therein).

The same SCF ubiquitin ligase complex that regulates β-catenin also controls the NF-kappaB pathway: Following TNF-α stimulation, this complex conjugates a K48-linked ubiquitin chain to phosphorylated IkappaB, an inhibitor of NF-kappaB. The subsequent proteasomal degradation of ubiquitylated IkappaB allows NF-kappaB to accumulate in the nucleus and to stimulate the transcription of NF-kappaB target genes. Notably, several components of the NF-kappaB pathway are also regulated by K63-linked polyubiquitylation. The TNF receptor-associated factors TRAF2, TRAF6, RIP, and the NEMO subunit of the IkappaB kinase are modified by K63-linked ubiquitin chains, which are thought to mediate the recruitment and activation of their effectors. For example, the binding of TAB2 to K63-polyubiquitylated TRAF6, NEMO, and RIP appears to be required for activation of NFkappaB. TAB2 binds to its polyubiquitylated substrates via its NZF motif (Npl4 zinc finger), one of many different ubiquitin-binding domains that bind to the commonly recognized hydrophobic "Ile44 patch" of the ubiquitin surface (Tran, 2008 and references therein).

The deubiquitylating (DUB) enzyme A20 antagonizes the NF-kappaB pathway, by removing the K63-linked polyubiquitylation of RIP, which appears to be necessary for its ability to down-regulate TNF-alpha-induced NF-kappaB activity. A20 belongs to the OTU (ovarian tumor) family of DUBs, which includes the largely uncharacterized proteins Cezanne and Trabid. The OTU domain is a conserved cysteine protease that possesses DUB activity (Balakirev, 2003). Overexpressed Cezanne can also down-regulate NF-kappaB signaling, whereas overexpressed Trabid had negligible effects on this pathway (Tran, 2008).

This study reports the discovery of Trabid as a new APC-interacting protein and a positive regulator of Wnt responses in mammalian cells and Drosophila. Its variant OTU domain exhibits DUB activity, and it deubiquitylates APC. Its remarkable preference for binding to, and cleaving, K63-linked ubiquitin chains are critical for Trabid's function in TCF-mediated transcription. Epistasis analysis suggests that the TCF-β-catenin complex is the functional target of Trabid (Tran, 2008).

A yeast two-hybrid (Y2H) screen was conducted with the armadillo repeat domain (ARD) of mouse Apc, and 13 independent positives were counterscreened with a mutant ARD bearing a point mutation in its putative surface that, in Drosophila, causes inactivation of E-APC. This revealed two isolates that were sensitive to this mutation. One of these encodes an internal fragment of mouse Cezanne overlapping its OTU domain (residues 252-403), which shows high sequence similarity to Trabid, and also to dTrbd, the Trabid ortholog in Drosophila and only representative of the A20 OTU family in flies. The Cezanne Y2H fragment, and a similar OTU-spanning fragment from human Trabid, both coimmunoprecipitate preferentially with wild type rather than mutant ARD in cotransfected 293 cells. The same selective association is also seen with full-length Flag-dTrbd. Furthermore, Flag-Trabid coimmunoprecipitates with coexpressed GFP-APC(1-1447), an ARD-spanning APC fragment similar to APC type 1 truncations typically found in colorectal cancer cells, but not with coexpressed GFP-APC(918-1698) that lacks the ARD. These sequence-specific interactions in yeast and mammalian cells indicate that Trabid, Cezanne, and dTrbd may be bona fide direct ARD ligands (Tran, 2008).

To test the function of the single Trabid ortholog of Drosophila in its Wingless response, the trbd locus (CG9448) was deleted by homologous recombination. Surprisingly, trbd-null mutants were viable and fertile, suggesting that trbd may be redundant with another gene. trbd function was tested in a more sensitive assay by asking whether lowering the dose of Trabid by half (in trbd heterozygotes) would affect the rough eye phenotype due to ectopic Wingless in the eye imaginal disc. Indeed, trbd heterozygosity suppresses this phenotype, similarly to dTCF heterozygosity. trbd heterozygosity also suppressed the rough eye phenotype due to overexpressed Armadillo (the β-catenin of Drosophila) to some extent. In contrast, no genetic interactions was observed between trbd and components of EGF receptor signaling, a pathway that controls differentiation in the developing fly eye: The rough eye phenotype due to activation or inhibition of this pathway remained unchanged in trbd heterozygotes. Similarly, there was no interaction between trbd and the Notch pathway, whose stimulation also causes rough eyes. These results suggest that dTrbd is required for an efficient Wingless response, but not for other signaling responses in the eye, consistent with results in human cell lines (Tran, 2008).

To date, the only known function of ubiquitylation in the Wnt pathway relates to protein turnover, especially that of its key effector β-catenin, typically conferred by K48-linked ubiquitin chains. The discovery of Trabid, a DUB enzyme that exhibits a remarkable preference for K63-linked ubiquitin, and its functional link to APC and the Wnt response of mammalian and Drosophila cells implies a distinct role of ubiquitylation in this pathway, namely, one mediated by K63-linked ubiquitin chains. These chains are likely to affect the activity rather than the stability of proteins, and Trabid may affect the transcriptional response to Wnt signaling by antagonizing the ubiquitylation of APC (Tran, 2008).

This study showed that Trabid is a bona fide DUB and that it cleaves preferentially K63-linked ubiquitin. This activity resides in its OTU domain, which possesses intrinsic DUB activity, as has been shown previously for other members of the OTU family (Tran, 2008).

Trabid exhibits an unprecedented selectivity for binding to K63- versus K48-linked ubiquitin chains, which is conferred by a minimal module of at least two linked NZF motifs. In contrast, an individual NZF motif (e.g., that of TAB3) binds indiscriminately to K48- and K63-linked ubiquitin chains, consistent with structural analysis that revealed the commonly used surface of ubiquitin for NZF binding. Notably, the three specificity-conferring NZF motifs of Trabid are not only a defining feature of Trabid orthologs, but also a fairly unique one: Although NZF domains with ubiquitin-binding signatures are found in a number of proteins, arrays of such NZF fingers are rare. It is conceivable that tandem NZF arrays confer specificity for K63-linked ubiquitin by topological restriction: K48-linked chains are known to form relatively compact structures, whereas K63-linked chains are likely to be extended (Tran, 2008).

Evidence suggests that the NZF motifs of Trabid contribute to its function in TCF-mediated transcription, although they are not required for its intrinsic DUB activity in vitro. These motifs may have a targeting function in vivo, similarly to the ubiquitin binding by the TAB adaptors, which is thought to facilitate the recruitment of ubiquitylated substrates to the TAB-associated TAK1 kinase (Tran, 2008).

Epistasis analysis indicated that Trabid acts below the stabilization of β-catenin. Consistent with this, only minor effects of Trabid depletion were detected on the ubiquitylation of β-catenin (by K48-linked ubiquitin) and on its steady-state levels. These results argue against a direct function of Trabid in proteasomal degradation (Tran, 2008).

In contrast, it was discovered that APC becomes hyperubiquitylated in Trabid-depleted cells, indicating that APC is not only a binding partner of Trabid, but also a substrate of its DUB activity. Thus, at least one function of Trabid is to antagonize the ubiquitylation of APC. This is likely to involve K63-linked ubiquitin chains, given Trabid’s preference for cleaving these chains. Also, the hyperubiquitylation of APC in Trabid-depleted cells, despite being more pronounced after MG132 treatment, neither seems to promote the degradation of APC itself, nor that of its key target, β-catenin. It thus appears that the deubiquitylation of APC by Trabid has a regulatory output (rather than antagonizing the proteasomal turnover of APC mediated by Axin in the absence of Wnt stimulation. For example, it might regulate the activity of APC in transcription. Further work will be required to clarify the molecular nature of these APC ubiquitylations, and their effects on APC’s stability and/or activity (Tran, 2008).

The results from the epistasis analysis and LEF1 chimeras suggest a role of Trabid in TCF-mediated transcription. Consistent with this, preliminary results from chromatin immunoprecipitation experiments indicate that Trabid is associated with the c-myc promoter and upstream sequences. Trabid's prime physiologically relevant substrate could be APC itself, which is recruited (together with β-TrCP) to TCF target genes to repress their transcription during sustained Wnt signaling. If so, this would imply that Trabid inactivates APC; hyperubiquitylation of APC in Trabid-depleted cells would result in its hyperactivation, possibly leading to hyperrepression (or precocious repression) of Wnt target genes, which would explain why the Wnt response is attenuated in these cells. Alternatively, the prime functional target of Trabid might be an unknown nuclear protein, a negative regulator like APC whose deubiquitylation results in its inactivation or a positive regulator of Wnt-induced transcription that becomes activated as a result of Trabid's DUB action; in the latter case, hyperubiquitylation would be expected to result in inactivation (Tran, 2008).

How does Trabid affect the TCF-mediated transcription? No change was detected in the steady-state association between β-catenin and TCF in Trabid-depleted cells. Nevertheless, the results from the LEF chimeras suggest that Trabid may affect the dynamic association or dissociation of the TCF-β-catenin complex: These chimeras might bypass the requirement for Trabid because of their direct linkage to TADs, which allows them to recruit (or retain) coactivators efficiently -- possibly more efficiently than normal TCF factors whose transcriptional activity during Wnt signaling relies on transient, and perhaps labile, association with limited amounts of nuclear β-catenin. It is therefore conceivable that Trabid increases, for example, the rate of TCF-β-catenin complex formation or the lifetime of its activity (Tran, 2008).

Ubiquitylation is beginning to emerge as a versatile controlling factor in transcriptional switches, including regulatory ubiquitylation events such as monoubiquitylation of histones. There are as yet no known regulatory transcription events that rely on K63-linked ubiquitin, although DNA repair can be regulated by K63-linked ubiquitylation (of PCNA). The turnover of regulatory ubiquitylations via DUBs may be as important as the ubiquitylations themselves, one example being the negative regulation of H2B monoubiquitylation by Ubp8, a DUB associated with the yeast histone acetylation SAGA complex. Interestingly, in vitro transcription from a β-catenin-dependent chromatin template was attenuated by a DUB inhibitor (Tran, 2008).

Given its requirement for efficient TCF-mediated transcription in colorectal cancer cell lines, Trabid has potential as a molecular target for inhibitory drugs in colorectal cancer. Obviously, more work is required to validate this target, but Trabid appears attractive because of (1) its apparent specificity for Wnt responses, (2) its enzymatic activity as a protease, and (3) its catalytic pocket that is likely to be unique among OTU domain proteases (Tran, 2008).

Wnt signaling has a key role in maintaining intestinal stem cell compartment. Furthermore, a recent compilation of gene expression data sets has identified relatively few genes (<40) commonly up-regulated in a variety of stem cell types (including skin bulge, hematopoietic, embryonic, and neural stem cells), which include TCF3 and TCF4 as well as Trabid. Notably, TCF3 plays a role in maintaining skin stem cells in an undifferentiated state, and TCF4 is required for maintaining crypts in the intestinal epithelium. Given the high Trabid levels in these stem cell compartments and Trabid's role in TCF-mediated transcription, it is conceivable that Trabid may have a function in stem cell self-renewal or differentiation (Tran, 2008).

Homeodomain-interacting protein kinases (Hipks) promote Wnt/Wg signaling through stabilization of β-catenin/Arm and stimulation of target gene expression

The Wnt/Wingless (Wg) pathway represents a conserved signaling cascade involved in diverse biological processes. Misregulation of Wnt/Wg signal transduction has profound effects on development. Homeodomain-interacting protein kinases (Hipks) represent a novel family of serine/threonine kinases. Members of this group (in particular Hipk2) are implicated as important factors in transcriptional regulation to control cell growth, apoptosis and development. This study provides genetic and phenotypic evidence that the sole Drosophila member of this family, Hipk, functions as a positive regulator in the Wg pathway. Expression of hipk in the wing rescues loss of the Wg signal, whereas loss of hipk can enhance decreased wg signaling phenotypes. Furthermore, loss of hipk leads to diminished Arm protein levels, whereas overexpression of hipk promotes the Wg signal by stabilizing Arm, resulting in activation of Wg responsive targets. In Wg transcriptional assays, Hipk enhances Tcf/Arm-mediated gene expression in a kinase-dependent manner. In addition, Hipk can bind to Arm and Drosophila Tcf, and phosphorylate Arm. Using both in vitro and in vivo assays, Hipk was found to promote the stabilization of Arm. Similar molecular interactions were observed between Lef1/β-catenin and vertebrate Hipk2, suggesting a direct and conserved role for Hipk proteins in promoting Wnt signaling (Lee, 2009).

Metazoan development is a highly dynamic and complex process that requires the action of several key signal transduction pathways. Their activity must be tightly regulated to ensure the proper patterning and growth of tissues. Regulation of a signaling pathway can occur at any level within the pathway, from perturbation of ligand-receptor interactions to regulation of the activity of transcription factors in the nucleus. Some regulators affect the on or off state of pathways, whereas others are involved in fine-tuning, an essential aspect that ensures that accurate and physiologically necessary levels of signaling are achieved without excessive signaling, which can have deleterious effects (Lee, 2009).

The ability of the Wnt pathway to control different developmental events in a temporally and spatially specific manner requires coordination between numerous regulators. Canonical Wnt signaling controls cell fate by regulating transcription of target genes. Wingless (Wg), a secreted glycoprotein, is the best characterized of the seven Drosophila Wnt ligands, and initiates the canonical pathway by binding to the Frizzled2 (Fz2) and LRP5/6/Arrow co-receptors. This leads to the activation of Dishevelled, which then inhibits the activity of the destruction complex composed of Axin, glycogen synthase kinase 3β (GSK-3β)/Zw3 and adenomatous polyposis coli (APC). As a result, cytosolic Drosophila β-catenin called Armadillo (Arm) accumulates and enters the nucleus to interact with a Tcf/Lef (Drosophila Tcf) family transcription factor to promote target gene expression. In the absence of Wg signaling, the Axin/GSK-3β/APC complex promotes the proteolytic degradation of Arm, whereas transcriptional co-repressors bind to Tcf and repress transcription (Lee, 2009).

The Nemo-like kinase family (Nlk) of protein kinases can regulate activation of Tcf/Lef target genes. In Drosophila, Nemo (Nmo) inhibits Drosophila Tcf activity, and is itself a transcriptional target of the Wg pathway. Recently Homeodomain-interacting protein kinase 2 (Hipk2) was proposed to participate in a kinase cascade to activate Nlk during the regulation of the Myb transcription factor. Therefore, attempts were made in this study to identify whether this regulation was perhaps more general and whether Drosophila Hipk played a role in regulating Nmo, and thus also Wg signaling. It was rapidly learned that Hipk exerts a positive effect on Wg signaling, distinct from Nmo, which has been more fully characterized using the developing wing as a model system (Lee, 2009).

The patterning of the adult wing blade is a tightly regulated process involving numerous essential signaling pathways, including Wg, Notch, EGFR and TGFβ, making it an excellent tissue in which to examine regulatory and epistatic relationships between many genes involved in patterning. The adult wing blade possesses five longitudinal veins (LI-LV) that extend proximally to distally. These are connected by the anterior cross vein (ACV) and posterior crossvein (PCV). The Wg pathway acts at several stages of wing patterning and growth. Wg is expressed along the dorsal/ventral boundary, which in imaginal discs is a stripe bisecting the wing imaginal disc, and in adult wings gives rise to the wing margin and bristles that surround the edge of the wing blade. Loss of wg can lead to loss of the entire wing, to wing to notum transformations, to wing notching or to loss of bristles along the entire wing margin. Wg also promotes proliferation in the wing disc and ectopic Wg can induce outgrowths from the ventral surface of the wing (Lee, 2009).

Hipk2 is a member of a conserved family of serine/threonine kinases. Vertebrate species possess four Hipk proteins (Hipk1-4) that have evolved distinct functions. Singly mutant Hipk1 and Hipk2 mice are viable, whereas double mutant mice die before birth. Drosophila possesses a single Hipk ortholog (which has been referred to as both Hipk and Hipk2) that shares extensive sequence homology within the kinase domain with members of the vertebrate family (Lee, 2009).

Vertebrate Hipk2 has been the most extensively studied member of the family. Biochemical studies have identified a growing list of Hipk2 interactors, including proteins involved in transcriptional regulation, chromatin remodeling and key components of evolutionarily conserved signaling pathways. However, the biological investigation of these interactions in multicellular organisms has been minimal. In vivo examination of murine Hipk2 protein function has thus far revealed a role in neurogenesis and homeotic transformation of the skeleton. Studies in Drosophila using transgenic flies expressing Hipk transgenes have uncovered a role for Hipk in regulating the global corepressor Groucho. Using loss-of-function mutant analyses, a role has been identified for Hipk in promoting Notch signaling during Drosophila eye development (Lee, 2009 and references therein).

This study presents an analysis of the function of Hipk in Drosophila canonical Wg signaling. Genetic studies show that ectopic hipk can rescue phenotypes owing to loss-of-function wg alleles or inhibition of the pathway with a dominant-negative Fz2 receptor. Immunohistochemical studies show that hipk positively regulates expression of Wg targets, and that Hipk can act to stabilize cellular levels of the Arm protein in wing discs. Wnt reporter assays show that both Drosophila Hipk and mouse Hipk2 can promote the Wnt-responsive Topflash reporter. In addition, Hipk/Hipk2 can promote the stabilization of Arm/β-catenin in cell culture and in vivo. These results suggest that Hipk is a positive regulator of the Wg pathway that refines Wg activity during wing development. The findings suggest that these roles may be conserved across species (Lee, 2009).

Loss of zygotic hipk results in pupal or larval lethality. Whole-mount in situ hybridization reveals that hipk is expressed broadly in a non-uniform pattern in multiple stages of development, including all imaginal discs. Removal of the maternal contribution caused embryonic lethality characterized by twisted embryos and head holes, showing that hipk is an essential gene for Drosophila development. These analyses have focused on the most severe allele, hipk4, which causes early larval lethality, or pupal lethality in trans to hipk3. Given the embryonic lethality caused by loss of maternal Hipk, it is speculated that maternally contributed Hipk perdures and obscures its requirement at later stages and impacts the severity of mutant phenotypes. The FLP/FRT technique was used to generate mutant somatic clones to examine the requirements for Hipk in patterning adult structures (Lee, 2009).

This study focused on the role of hipk in the development of the wing. Clones of cells mutant for hipk4 show ectopic veins in the anterior region of the wing blade along LII, loss of the PCV and occasional notches in the wing margin. Reducing hipk function by expression of two independent Gal4-responsive hipk-RNAi constructs in the wing pouch with sd-gal4 or vg-Gal4 also caused a wing notching phenotype reminiscent of those seen upon decreased Wg signaling (Lee, 2009).

Next, the effects of ectopic expression of hipk was studied using the Gal4-UAS system. Phenotypes were observed that suggested that hipk is involved in promoting Wg signaling. Expression of one copy of hipk in the central domain of the imaginal wing discs with omb-Gal4 induced the formation of an additional wing margin and outgrowths emanating from the distal most tip of the ventral surface of the wing. Expression of two copies of UAS-hipk enhanced this phenotype and caused the outgrowths to extend further distally from the ventral plane of the wing. These effects phenocopied the effects observed in ectopic wg-expressing clones. Similarly, expression of UAS-hipk in the wing phenocopies the ectopic venation pattern seen both upon ectopic expression of activated Arm (ArmS10) with bs-Gal4 and in nmoDB24/nmoadk2 mutants. Although control of wing vein patterning is not generally attributed to Wg signaling, ectopic venation has been observed upon elevated Wg signaling. For example, ectopic expression of constitutively active Arm by en-Gal4 in the posterior region of the wing, or ubiquitously with 69B-Gal4 or MS1096-Gal4, leads to disturbed and ectopic venation. Moreover, loss-of-function clones of sgg/zw3 (encoding the fly homolog of GSK3β, a component of the destruction complex) induce the formation of ectopic veins. The results of these phenotypic analyses of hipk are surprising because they demonstrated that Nmo and Hipk did not act in concert to inhibit Wg signaling. Rather, hipk mutant and gain-of-function phenotypes suggest a role in promoting the Wg pathway (Lee, 2009).

This study has revealed that Hipk possesses an intrinsic ability to promote Wg pathway activity and this regulatory function for Hipk is conserved in both Drosophila and mammalian cells. Through a combination of genetic and biochemical analyses, the data reveal that Hipk proteins promote Tcf/Lef1-mediated transcription. Additionally, Hipks enhances the stabilization of Arm/β-catenin in several cell lines and hipk mutant clones in the wing disc have diminished Arm protein levels. Overexpression of Hipk induces a broader domain of stabilized Arm, suggesting Hipk is required to maintain the signaling pool of cytosolic Arm. A model is proposed in which Hipk promotes the Wnt/Wg signal via its regulation of Arm stabilization (Lee, 2009).

Nlk is a conserved antagonist of the Lef1/β-catenin transcriptional complex. This research has shown that Nmo is also an inducible antagonist of the Wg signal in the developing Drosophila wing. It was previously reported that Wnt1 induces the activation of a putative Tak1-Hipk2-Nlk kinase cascade to promote the degradation of the Myb transcription factor. The current study sought to delineate the physiological relevance of this potential kinase cascade; specifically, it was determined whether these interactions played a role in the regulation of the Wg pathway. The data reveal that in Drosophila Nmo and Hipk do not form a kinase cascade in this context, rather they exert opposing effects on the same pathway, probably through distinct mechanisms (Lee, 2009).

The Wg morphogen can bring forth a spectrum of biological processes. Sustaining maximal levels of signaling could be accomplished through the amplification and enhancement of the signal in the wing margin. In support of this model, it was found that overexpression of Hipk expands the expression of Wg targets such as Dll, Sens and Ac. Transcriptional assays reveal that both Drosophila Hipk and mouse Hipk2 enhance the transcriptional activity of Tcf and Lef1, respectively, in a kinase-dependent manner. These findings strongly suggest that Hipk and Hipk2 function to enhance the activity of the transcriptional complex to promote the Wg/Wnt signal (Lee, 2009).

Accumulation of stabilized Arm is paramount to effective Wg signaling. Failure to escape the destruction complex results in Arm degradation and inhibition of Tcf-mediated gene activation. Thus understanding the regulation of Arm is central to the global understanding of how the Wg signal is modulated. In these studies, a role was revealed for Hipk in Arm stabilization. This feature is highlighted by the loss of stabilized Arm in hipk mutant clones. Additionally, in hipk mutant discs, overexpressed wild-type Arm fails to accumulate, despite its expression in domains of high Wg signaling. These findings demonstrate that Hipk plays an important role in Arm stabilization. Hipk may reduce the ability of Arm to interact with destruction complex components or may increase the nuclear retention of Arm. In the absence of Hipk, either of these scenarios would give the destruction complex more access to Arm. In agreement with such a model, it was found that increasing Hipk activity in the wing surpasses the inhibitory effects of the degradation machinery and expands the perimeter of stabilized Arm. Furthermore, it was found that the presence of Hipk or Hipk2 in cell culture stabilizes Arm/β-catenin. Thus, the enhanced transcriptional activity is probably due to the elevated availability of Arm protein (Lee, 2009).

It is crucial for normal development to maintain the proper amounts of β-catenin, since elevated levels of β-catenin can lead to cancer. Elaborate regulatory networks in the cytoplasm and nucleus are vital to maintaining appropriate levels of β-catenin. It is well documented that phosphorylation in the N terminus of β-catenin is crucial for its negative regulation. A chain of phosphorylation events begins when Casein Kinase I (CKI) primes β-catenin for successive modifications by GSK-3β. Central to this event is Axin, which provides a scaffold for APC, CK1, GSK-3β and β-catenin. N-terminally phosphorylated β-catenin is ubiquitinated by βTrCP ubiquitin ligase and targeted for degradation via the proteasome (Lee, 2009).

Wnt signaling promotes the accumulation of β-catenin; however, some of the mechanisms governing this process remain enigmatic. Although overexpression of wild-type β-catenin/Arm is unable to overcome the effects of the degradation machinery, Wnt-stimulated β-catenin can resist the activity of the destruction complex. Although achieving stabilized pools of β-catenin represents the core goal of the Wnt pathway, high levels of β-catenin are not always coupled with elevated transcription. For example, in Xenopus, alanine substitution of one of the GSK3 target residues leads to elevated β-catenin levels, without causing an increase in Tcf-mediated transcription. Thus, further posttranslational modifications of β-catenin are necessary to potentiate its signaling activity (Lee, 2009).

Furthermore, phosphorylation can affect β-catenin stability by affecting protein-protein interactions that regulate protein turnover and activity. Phosphorylation of β-catenin by Cdk5 inhibits APC binding to β-catenin, whereas phosphorylation by CK2 promotes β-catenin stability and transcriptional activity (Lee, 2009).

Recent advances have begun to unravel the molecular complexity that controls β-catenin-mediated transcription within the nucleus. Upon pathway activation, Tcf recruits Arm to the enhancers of Wg-responsive genes where Arm forms multiple transcriptional complexes along its length. Formation of these transcriptional units is needed for the transmission of the Wg/Wnt signal. Recent studies have shown that phosphorylation (distinct from the N-terminal phosphorylation that triggers β-catenin destruction) may modulate its ability to recruit these co-factors. This study found that the Hipk-dependent stabilized form of Arm is transcriptionally active and induces the expression of Wg targets, suggesting modification by Hipk may promote protein interactions (Lee, 2009).

APC and the cell-adhesion molecule E-cadherin compete with Tcf for overlapping binding sites on β-catenin. Competition between proteins may play an important role in the regulation of the Wnt signaling pathway. It is proposed that Hipks may promote the stability of Arm/β-catenin by excluding further interactions with other proteins, including those that antagonize Arm/β-catenin. Given that Hipks can also bind to Tcf/Lef1, it is predicted that these proteins may act synergistically to displace the inhibitory partners of β-catenin. In agreement with such a role, it is observed that Lef1 enhances the interaction between Hipk2 and β-catenin, and these interactions may insulate β-catenin from components of the degradation machinery (Lee, 2009).

Although Hipk phosphorylates Arm, the functional significance of this modification has yet to be determined. Hipk might facilitate the interactions between Arm and its transcriptional co-factors, as Hipk2 phosphorylation has been shown to affect gene regulation by modifying the composition of various transcriptional complexes. Hipk may also enhance the formation of the β-catenin/Tcf transcriptional complex by inducing a conformational change and/or reducing the affinity of possible inhibitors for β-catenin through the phosphorylation of β-catenin. Recently, it has been reported that Hipk2 could antagonize β-catenin/Tcf-mediated transcription in a kinase-independent manner. Although these data appear in conflict with the current findings, it was observed that the effect of Hipk2 on transcription is very cell type- and target gene-dependent, suggesting Hipk2 function is affected by its cellular context, most probably owing to the availability of targets and co-factors (Lee, 2009).

The dynamic localization of Hipk2 in the nucleus, nucleoplasm and in cytosolic speckles suggests that the protein may carry out distinct roles in each site. Given the growing list of interacting proteins, it is tempting to speculate that specific Hipk function is determined in part through its particular localization. It is also possible that Hipk/Hipk2 may act as a scaffolding protein, bringing together multiple binding partners. Ongoing biochemical studies will further uncover the molecular significance of these interactions. Hipk proteins are emerging as important components of multiple signaling networks. The current study describes the roles of Hipk and Hipk2 as Wnt/Wg regulators and sheds light on the regulatory mechanisms governing this conserved pathway (Lee, 2009).

Erect Wing facilitates context-dependent Wnt/Wingless signaling by recruiting the cell-specific Armadillo-TCF adaptor Earthbound to chromatin

During metazoan development, the Wnt/Wingless signal transduction pathway is activated repetitively to direct cell proliferation, fate specification, differentiation and apoptosis. Distinct outcomes are elicited by Wnt stimulation in different cellular contexts; however, mechanisms that confer context specificity to Wnt signaling responses remain largely unknown. Starting with an unbiased forward genetic screen in Drosophila, a novel mechanism was recently uncovered by which the cell-specific co-factor Earthbound 1 (Ebd1), and its human homolog jerky, promote interaction between the Wnt pathway transcriptional co-activators β-catenin/Armadillo and TCF to facilitate context-dependent Wnt signaling responses (Benchabane, 2011). In the same genetic screen an unanticipated requirement was found for Erect Wing (Ewg), the fly homolog of the human sequence-specific DNA-binding transcriptional activator nuclear respiratory factor 1 (NRF1), in promoting contextual regulation of Wingless signaling. Ewg and Ebd1 functionally interact with the Armadillo-TCF complex and mediate the same context-dependent Wingless signaling responses. In addition, Ewg and Ebd1 have similar cell-specific expression profiles, bind to each other directly and also associate with chromatin at shared genomic sites. Furthermore, recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. These findings provide in vivo evidence that recruitment of a cell-specific co-factor complex to specific chromatin sites, coupled with its ability to facilitate Armadillo-TCF interaction and transcriptional activity, promotes contextual regulation of Wnt/Wingless signaling responses (Xin, 2011).

Genome-wide expression profiling has revealed that the vast majority of Wnt target genes are context specific, and, by contrast, globally activated target genes are quite rare. Factors that confer context-dependent target gene activation in response to activation of this widely used signal transduction pathway are of fundamental clinical importance, as they provide putative targets for therapeutic intervention; however, the identity of these factors, and their mechanism of action remain poorly understood, and have posed a daunting challenge for the Wnt signaling field (Xin, 2011).

An unbiased forward genetic approach was undertaken to identify genes that promote Wingless signal transduction. The design of the genetic screen allowed identification of any gene that impacts Wingless signaling downstream, or at the level of the Apc/Axin destruction complex, which in principle could encompass a wide range of functions. Surprisingly, however, among the relatively small number of genetic complementation groups revealed in the screen, two cell-specific chromatin-associated co-factors were identified that had not been implicated in Wingless signaling previously, but directly associate with each other and probably mediate the same context-specific Wingless-dependent developmental process. The combined activities of the two factors in this complex not only result in its recruitment to discrete chromatin sites, but also promote activity of the Armadillo-TCF transcription complex. Together, these unanticipated findings uncover a novel mechanism that facilitates context-specific regulation of Wingless signaling (Xin, 2011).

It was recently discovered that the novel Drosophila CENPB domain protein Ebd1 and its human homolog Jerky contribute to Wnt/Wingless target gene activation by enhancing β-catenin-TCF complex formation and β-catenin recruitment to chromatin (Benchabane, 2011). Since Ebd1 is expressed in a cell-restricted pattern, these findings provided evidence that context-dependent responses to Wnt/Wingless stimulation are facilitated by cell-specific co-factors that interact with both β-catenin/Armadillo and TCF to enhance complex formation and activity. However, whether Ebd1 conferred context specificity solely by functioning as a cell-specific Armadillo-TCF adaptor or also by associating with distinct chromatin sites was not known (Xin, 2011).

This study provides multiple lines of evidence consistent with the model that the putative sequence-specific DNA-binding transcriptional activator Ewg is a physical and functional partner of Ebd1 in regulating context-specific Wingless signaling, and is required for chromatin recruitment of the Ewg-Ebd1 complex. Together, these studies suggest that Ebd1 and Ewg act together in the same context-specific Wingless-dependent developmental process, interact functionally with Armadillo-TCF, are expressed in similar cell-restricted patterns, associate physically with each other and are recruited to similar chromatin sites throughout the genome. Furthermore, all recruitment of Ebd1 to chromatin is abolished in the absence of Ewg. Based on these findings, a model is proposed in which context specificity in Wingless signaling is achieved in part through association of Ewg with distinct enhancer sites, and recruitment of Ebd1 to these enhancers. In addition, previous work suggested that by binding both Armadillo and TCF, Ebd1 stabilizes the Armadillo-TCF complex (Benchabane, 2011). Together, these findings support the model that Ebd1 promotes recruitment of the Armadillo-TCF complex to enhancers containing Ewg-binding sites, thereby facilitating context specificity in Wingless signal transduction. Alternatively, Ebd1 might also function in an Ewg-independent manner by solely providing an Armadillo-TCF adaptor function that stabilizes the Armadillo-TCF complex and thereby facilitates transcriptional activation of a subset of Wingless target genes requiring relatively high Armadillo-TCF levels (Xin, 2011).

The ability to visualize endogenous Ebd1 and Ewg on larval salivary gland polytene chromosomes reveals that Ewg is essential for association of Ebd1 with chromatin, and that nearly all, if not all, chromatin sites with which Ebd1 associates are also bound by Ewg. By contrast, the association of Ewg with chromatin is not dependent on Ebd1; furthermore, not all Ewg-associated chromatin sites are bound by Ebd1, which probably reflects the broader role for Ewg in muscle and neuronal development. Together, these findings suggest that Ewg is necessary but not sufficient for recruitment of Ebd1 to chromatin, and either additional factors or specific DNA sequences are important for association of Ebd1 with chromatin. In particular, the presence of CENPB-type DNA-binding domains in Ebd1 indicates that Ebd1 may bind DNA directly, and therefore the association of Ebd1 with chromatin may require not only Ewg, but also sequence-specific Ebd1 DNA-binding sites. If so, Ewg might bind and remodel specific chromatin sites, thereby allowing Ebd1 to access nearby cognate binding sites or, alternatively, physical association with Ewg may result in a conformational change in Ebd1 that enables DNA binding. Similarly, association of Ebd1 with the Armadillo-TCF complex may facilitate association of Ebd1 and/or Armadillo-TCF with distinct chromatin sites; currently, this hypothesis could not be tested in vivo, since fixation and immunostaining conditions do not detect the indirect association of endogenous Armadillo with polytene chromosomes. Nonetheless, the extensive physical and genetic interactions between Ewg, Ebd1 and Wingless pathway transcriptional components, coupled with association of Ewg-Ebd1 with distinct chromatin sites support the model that context-specific activation of Wingless target genes is facilitated by interaction of Armadillo-TCF with cell-specific co-activator complexes at specific sites in chromatin (Xin, 2011).

The findings also suggest that Ebd1-Ewg complex function is required for only a context-specific subset of the Wingless-dependent processes that direct indirect flight muscles (IFM) development. For example, Wingless secreted from larval wing imaginal disc epithelia specifies the fate of myoblasts associated with the wing disc that are important for IFM development. In addition, Wingless signaling regulates the specification of tendon cells and the position of flight muscle attachment sites. Further evidence for Wingless signaling in IFM development was revealed by hypomorphic wingless mutants, some of which display a morphologically normal external appearance, but are nonetheless flightless. Indeed, analysis of these hypomorphic wingless mutants reveals defects in late steps of muscle differentiation that result in partial loss of IFMs (Benchabane, 2011). In addition, strong genetic interactions was found between Ewg and the Armadillo-TCF complex. Together with the genetic and physical interactions between Ewg and Ebd1, as well as previous findings that Ebd1 facilitates Wingless signaling in adult muscle development (Benchabane, 2011), the genetic interaction between Ewg and TCF provides further evidence supporting the model that Ewg and Ebd1 facilitate Wingless-dependent adult flight muscle development. However, no Ewg expression is detected in IFM precursors until pupation, suggesting that Ewg does not participate in early steps of Wingless-dependent specification of IFM myoblasts or tendon cells, but instead promotes later steps in IFM growth and differentiation. Previous studies revealed that context specificity in Wnt signaling responses is facilitated by either crosstalk between signal transduction pathways or replacement of TCF with tissue-specific DNA-binding transcription co-factors that associate with β-catenin. Thus, these findings suggest a novel mechanism in which a sequence-specific DNA-binding transcription factor recruits a cell-specific Armadillo-TCF adaptor to modulate context-specific Wingless signaling responses (Xin, 2011).

Ewg activity in neuronal differentiation can be functionally replaced by its human homolog nuclear respiratory factor 1 (NRF1) (Haussmann, 2008), a sequence-specific DNA-binding transcription factor important for activation of genes required in mitochondrial biogenesis and respiration, and for regulation of histone gene expression. Functional analysis of vertebrate NRF1 during development has been hampered by early lethality, as mouse Nrf1-null embryos die near the time of uterine implantation. Fortuitously, however, inactivation of the zebrafish NRF1 homolog, not really finished (nrf), has improved understanding of NRF1 function during vertebrate development; fish nrf-null mutants survive up to 14 days of development with the primary developmental defect restricted to markedly smaller brains and retinas. The tissue-restricted nature of the fish nrf-null mutant phenotype was unexpected, given the broader expression of the fish nrf gene and the global roles for NRF1 in mitochondrial and histone function revealed by in vitro studies. However, these in vivo analyses revealed that fish Nrf, like fly Ewg, acts in specific tissue-restricted developmental processes. Of note, Wnt signaling is required for patterning of the mouse central nervous system. Similarly, Wnt signaling is also crucial for brain and eye development in zebrafish, thus raising the possibility that, like Ewg, vertebrate Nrf may also facilitate a context-specific subset of Wnt-dependent developmental processes (Xin, 2011).

Jerky/Earthbound facilitates cell-specific Wnt/Wingless signalling by modulating beta-catenin-TCF activity

Wnt/Wingless signal transduction directs fundamental developmental processes, and upon hyperactivation triggers colorectal adenoma/carcinoma formation. Responses to Wnt stimulation are cell specific and diverse; yet, how cell context modulates Wnt signalling outcome remains obscure. In a Drosophila genetic screen for components that promote Wingless signalling, Earthbound 1 (Ebd1), a novel member in a protein family containing Centromere Binding Protein B (CENPB)-type DNA binding domains, was identified. Ebd1 is expressed in only a subset of Wingless responsive cell types, and is required for only a limited number of Wingless-dependent processes. In addition, Ebd1 shares sequence similarity and can be functionally replaced with the human CENPB domain protein Jerky, previously implicated in juvenile myoclonic epilepsy development. Both Jerky and Ebd1 interact directly with the Wnt/Wingless pathway transcriptional co-activators β-catenin/Armadillo and T-cell factor (TCF). In colon carcinoma cells, Jerky facilitates Wnt signalling by promoting association of β-catenin with TCF and recruitment of β-catenin to chromatin. These findings indicate that tissue-restricted transcriptional co-activators facilitate cell-specific Wnt/Wingless signalling responses by modulating β-catenin-TCF activity (Benchabane, 2011).

How does cell identity influence transcriptional activation of Wnt/Wingless target genes? Starting with an unbiased genetic screen for components that facilitate Wingless signalling, an unanticipated role was discovered for the novel Drosophila CENPB domain protein Ebd1 in promoting cell-specific responses to Wingless signalling. Ebd1 is required for Wingless transduction in a restricted developmental context, and is expressed in only a subset of cells in which Wingless transduction is active, indicating that Ebd1 functions as a context-specific facilitator, and not as a general component in the Wingless pathway. Indeed, the absence of Ebd1 expression in many tissues is likely critical for proper development, as attempts to ectopically express Ebd1 using several tissue-specific promoters resulted in cell and/or organismal lethality. Further, within Ebd1-expressing cells, some Wingless-dependent developmental processes proceed normally upon Ebd loss, suggesting that only a subset of Wingless target genes is dependent on Ebd. Specifically, flight muscle loss resulting from Wingless pathway inactivation is more severe than that resulting from Ebd loss, even upon simultaneous elimination of all five Drosophila CENPB proteins. Together, these data provide genetic evidence that cell-specific responses to Wingless signalling are mediated in part by tissue-specific co-factors that modulate activity of the Armadillo/TCF transcription complex (Benchabane, 2011).

In vivo epistasis experiments support the model that Ebd1 functions at the level of the Armadillo-TCF transcriptional complex, and in vitro data indicate that Jerky and Ebd1 contribute to Wnt/Wingless target genes activation by enhancing β-catenin-TCF complex formation and β-catenin recruitment to chromatin. Thus, Jerky and Ebd likely serve an adaptor function similar to that previously documented for human transducin β-like protein 1 (TBL-1) (Li, 2008). However, by contrast with TBL-1, which promotes activation of all Wnt target genes analysed, only certain Wingless-dependent developmental events (and by extension, associated target genes) require Ebd. How does an Arm-TCF adaptor protein such as Ebd1 promote the transcription of only a subset of Wingless target genes? The data are consistent with two distinct models. Ebd may act solely as an Arm-TCF adaptor, such that Wingless target genes requiring relatively high Arm-TCF complex levels may be dependent on Ebd for activation, whereas activation of target genes requiring lower Arm-TCF levels may be Ebd independent. Alternatively, given the presence of its CENPB DNA binding domains, Ebd1 may provide two functions -- to act as an Armadillo-TCF adaptor and to also associate with cognate sites in enhancer elements. Thus, Ebd1 DNA-binding specificity may also contribute directly to Wingless target gene selection. The existence of cell-specific factors such as Ebd that modulate Arm-TCF activity was presaged by previous studies, which indicated that binding of DNA by TCF is a regulated process; not all TCF-binding site clusters bind TCF in vivo, and TCF binding at a particular locus varies between cell types, suggesting that optimal TCF binding to cognate sites likely requires recruitment by cell-specific transcription co-factors (Benchabane, 2011).

Taken together with previous studies, these findings reveal that modulation of Wnt/Wingless responses is facilitated by several distinct modes of transcriptional regulation. For example, recruitment of β-catenin to chromatin can occur in a TCF-independent manner, through association of β-catenin with sequence-specific DNA-binding co-activators that extend the range of Wnt/β-catenin target genes. In addition, cooperative signalling between the Wnt/Wingless pathway and other signal transduction pathways also modulates target gene specificity. For instance, molecular cross-talk at the transcriptional level between the mammalian Wnt pathway and the TGFβ or JNK pathway is mediated by interaction of Lef-1 with Smads and TCF-4 with c-Jun, respectively. Further, some modulators of Wingless pathway activity, such as Lines and Split ends/Spenito, do not associate with Armadillo or TCF directly, but instead act downstream or in parallel with Armadillo-TCF to specify cell fate. The current findings reveal that context-dependent responses to Wnt/Wingless stimulation are also facilitated by cell-specific co-factors that interact with both β-catenin and TCF to enhance their activity (Benchabane, 2011).

The human CENPB domain protein Jerky shares functional homology with Ebd1. Mouse Jerky was originally identified as a neuronally enriched protein in a murine epilepsy model; heterozygous inactivation of jerky results in recurrent generalized seizures, whereas homozygous jerky inactivation also results in growth and fertility defects. The subsequent mapping of human Jerky (JH8/JRK) to chromosome 8p24 revealed linkage to a susceptibility locus for juvenile myoclonic epilepsy and childhood absence epilepsy. Recently, a genome-wide RNA interference screen identified human Jerky as an activator of a TCF-dependent reporter as well as endogenous Wnt/β-catenin target genes in colon carcinoma cells. Jerky was found to facilitate the activation of a subset, but not all endogenous Wnt/β-catenin target genes examined, paralleling the in vivo analysis of Ebd function. The current findings indicate that Jerky facilitates Wnt pathway-dependent transcription by stabilizing the β-catenin-TCF complex and promoting recruitment of β-catenin to chromatin. These findings also raise the possibility that attenuation of Wnt transduction underlies generalized seizures, as well as growth and fertility defects in mice with reduced Jerky activity, and may contribute to development of certain types of childhood epilepsy (Benchabane, 2011).

Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9

Wnt/β-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing β-catenin via the Legless/BCL9 adaptor. This study used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of β-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these β-catenin co-factors may coordinate Wnt and nuclear hormone responses (van Tienen, 2017).

The Wnt/β-catenin signaling cascade is an ancient cell communication pathway that operates context-dependent transcriptional switches to control animal development and tissue homeostasis. Deregulation of the pathway in adult tissues can lead to many different cancers, most notably colorectal cancer. Wnt-induced transcription is mediated by T cell factors (TCF1/3/4, LEF1) bound to Wnt-responsive enhancers, but their activity depends on the co-activator β-catenin (Armadillo in Drosophila), which is rapidly degraded in unstimulated cells. Absence of β-catenin thus defines the OFF state of these enhancers, which are silenced by Groucho/TLE co-repressors bound to TCF via their Q domain. This domain tetramerizes to promote transcriptional repression (Chodaparambil, 2014), which leads to chromatin compaction apparently assisted by the interaction between Groucho/TLE and histone deacetylases (HDACs) (van Tienen, 2017).

Wnt signaling relieves this repression by blocking the degradation of β-catenin, which thus accumulates and binds to TCF, converting the Wnt-responsive enhancers into the ON state. This involves the binding of β-catenin to various transcriptional co-activators via its C-terminus, most notably to the CREB-binding protein (CBP) histone acetyltransferase or its p300 paralog, resulting in the transcription of the linked Wnt target genes. Subsequent reversion to the OFF state (for example, by negative feedback from high Wnt signaling levels near Wnt-producing cells, or upon cessation of signaling) involves Groucho/TLE-dependent silencing, but also requires the Osa/ARID1 subunit of the BAF (also known as SWI/SNF) chromatin remodeling complex which binds to β-catenin through its BRG/BRM subunit. Cancer genome sequencing has uncovered a widespread tumor suppressor role of the BAF complex, which guards against numerous cancers including colorectal cancer, with >20% of all cancers exhibiting at least one inactivating mutation in one of its subunits, most notably in ARID1A. Thus, it appears that failure of Wnt-inducible enhancers to respond to negative feedback imposed by the BAF complex strongly predisposes to cancer (van Tienen, 2017).

How β-catenin overcomes Groucho/TLE-dependent repression remains unclear, especially since β-catenin and TLE bind to TCF simultaneously (Chodaparambil, 2014). Therefore, the simplest model envisaging competition between β-catenin and TLE cannot explain this switch, which implies that co-factors are required. One of these is Pygo, a chromatin reader binding to histone H3 tail methylated at lysine 4 (H3K4m) via its C-terminal PHD finger (Fiedler, 2008). In Drosophila where Pygo was discovered as an essential co-factor for activated Armadillo, its main function appears to be to assist Armadillo in overcoming Groucho-dependent repression. It has been discovered recently that Pygo associates with TCF enhancers via its highly conserved N-terminal NPF motif that binds directly to the ChiLS complex, composed of a dimer of Chip/LDB (LIM domain-binding protein) and a tetramer of SSDP (single-stranded DNA-binding protein, also known as SSBP). Notably, ChiLS also binds to other enhancer-bound NPF factors such as Osa/ARID1 and RUNX, and to the C-terminal WD40 domain of Groucho/TLE, and thus forms the core module of a multiprotein complex termed 'Wnt enhanceosome' (Fiedler, 2015). This study proposed that Pygo renders this complex Wnt-responsive by capturing Armadillo/β-catenin through the Legless adaptor (whose orthologs in humans are BCL9 and B9L, also known as BCL9-2). The salient feature of this model is that the Wnt enhanceosome keeps TCF target genes repressed prior to Wnt signaling while at the same time priming them for subsequent Wnt induction, and for timely shut-down via negative feedback depending on Osa/ARID1 (Fiedler, 2015; van Tienen, 2017 and references therein).

This study assessed the function of Legless and BCL9/B9L within the Wnt enhanceosome. Using a proximity-labeling proteomics approach (called BioID) in human embryonic kidney (HEK293) cells, a compelling association was uncovered between BCL9/B9L and the core Wnt enhanceosome components, regardless of Wnt signaling. Co-immunoprecipitation (coIP) and in vitro binding assays based on Nuclear Magnetic Resonance (NMR) revealed that BCL9 and B9L associate with TLE3 through their C-termini, and that they bind directly to Chip/LDB-SSDP via their evolutionary conserved homology domain 3 (HD3). These elements are outside the sequences mediating the adaptor function between Pygo and Armadillo/β-catenin, but they are similarly important for Wnt responses during Drosophila development and in human cells, as is shown by CRISPR/Cas9-based genome editing. The results consolidate and refine the Wnt enhanceosome model, indicating a constitutive scaffolding function of BCL9/B9L within this complex. The evidence further suggests that BCL9/B9L but not Pygo undergoes a β-catenin-dependent rearrangement within the enhanceosome upon Wnt signaling (see Model of the Wnt enhanceosome), gaining proximity to TCF, which might trigger enhanceosome switching (van Tienen, 2017).

This study has uncovered genetic and physical interactions between two constitutive core components of the Wnt enhanceosome and the C-terminus of Legless/BCL9. The first of these is ChiLS, the core module of the Wnt enhanceosome (Fiedler, 2015): ChiLS is a direct and specific ligand of the α-helical HD3 element of B9L and, likely, of other Legless/BCL9 orthologs, given the strong sequence conservation of this α-helix. The physiological relevance of this interaction with ChiLS is underscored by genetic analysis in flies. The evidence thus implicates HD3 as an evolutionary conserved contact point between Legless/BCL9 and ChiLS, although the primary link between these two proteins appears to be provided by Pygo (van Tienen, 2017).

A second link between the Legless/BCL9 C-terminus and the Wnt enhanceosome is mediated by the WD40 domain of TLE/Groucho. Given evidence from RIME, this link is also likely to be direct although, for technical reasons, it has not been possible to prove this. The function of the C-terminus of Legless/BCL9 for transducing Wnt signals was revealed by the wg-like phenotypes in Drosophila larvae and flies and by their defective transcriptional Wg responses, and by the loss of transcriptional Wnt responses in BCL9/B9L-deleted human cells. The evidence indicates that Legless/BCL9 undergoes three separate functionally relevant interactions with distinct components of the Wnt enhanceosomewith Pygo, ChiLS and Groucho/TLE. Importantly, BioID revealed that these interactions are constitutive, preceding Wnt signaling, and that they hardly change upon Wnt stimulation. Taken together with its multivalent interactions with the Wnt enhanceosome, this is consistent with Legless/BCL9 being a core component of this complex, providing a scaffolding function that facilitates its assembly and/or maintains its cohesion (van Tienen, 2017).

Following Wnt stimulation, Legless/BCL9 undergoes an additional physiologically relevant interaction, by binding to (stabilized) Armadillo/β-catenin via HD2. Legless/BCL9 thus confers Wnt-responsiveness on the Wnt enhanceosome through its ability to capture Armadillo/β-catenin. In other words, in addition to scaffolding the enhanceosome, Legless/BCL9 also earmarks this complex for Wnt responses. Intriguingly, the BioID data indicated that the capture of β-catenin by Legless/BCL9 triggers its rearrangement within the complex, apposing its C-terminus to TCF. This apparent β-catenin-dependent apposition is consistent with structural data showing that BCL9/B9L HD2 is closely apposed to TCF when in a ternary complex with β-catenin. The evidence supports the notion of Legless/BCL9 acting as an Armadillo loading factor, facilitating access of Armadillo/β-catenin to TCF, but argues against the original co-activator hypothesis which posited that Legless/BCL9 is recruited to TCF by Armadillo/β-catenin exclusively in Wnt-stimulated cells. Whatever the case, the β-catenin-dependent apposition of the Legless/BCL9 C-terminus to TCF is likely to trigger Wnt enhanceosome switching from OFF to ON, resulting in the relief of Groucho/TLE-dependent repression and culminating in the Wnt-dependent transcriptional activation of linked target genes (van Tienen, 2017).

This transition of the Wnt enhanceosome from OFF to ON is accompanied by a proximity gain between Legless/BCL9 and CBP/p300, likely to reflect at least in part its de novo binding to Armadillo/β-catenin. However, the evidence indicates that CBP/p300 is associated with the Wnt enhanceosome prior to Wnt signaling, possibly via direct binding to B9L as suggested by RIME, and that the docking of Armadillo/β-catenin to the Wnt enhanceosome strengthens its association with CBP/p300, and/or directs the histone acetyltransferase activity of CBP/p300 towards its substrates, primarily the histone tails. By acetylating these tails, CBP/p300 appears to promote Wnt-dependent transcription in flies and human cells. Indeed, CBP-dependent histone acetylation has been observed at Wg target enhancers in Drosophila although, interestingly, this preceded transcriptional activation. This is consistent with BioID data, indicating constitutive association of CBP/p300 with the Wnt enhanceosome (van Tienen, 2017).

It seems plausible that histone acetylation at Wnt target enhancers is instrumental in antagonizing the compaction of their chromatin imposed by Groucho/TLE, which depends on its tetramerization via its Q domain as well as its binding to HDACs. Indeed, HDACs were found near the bottom of the BioID lists, and one of the top hits identified by B9L was GSE1, a subunit of the BRAF-HDAC complex. However, CBP/p300 also has non-histone substrates within the Wnt enhanceosome, including dTCF in Drosophila whose Armadillo-binding site can be acetylated by dCBP, which thus blocks the binding between the two proteins and antagonizes Wg responses. It thus regulates Wnt-dependent transcription positively as well as negatively, similarly to Groucho/TLE which not only silences Wnt target genes but also earmarks them for Wnt inducibility, as a core component of the Wnt enhanceosome. It is intriguing that both bimodal regulators are associated constitutively with this complex. A corollary is that the docking of Armadillo/β-catenin to the Wnt enhanceosome changes their substrate specificities and/or activities (van Tienen, 2017).

An important refinement of the initial enhanceosome model is with regard to the BAF complex, which appears to be a constitutive component of the Wnt enhanceosome, as indicated by BioID data. This complex is highly conserved from yeast to humans, and it contains 15 subunits in human cells (Kadoch, 2015), including the DNA-binding Osa/ARID1 subunit. A wealth of evidence from studies in flies and mammals indicates that this complex primarily antagonizes Polycomb-mediated silencing of genes, most notably of the INK4A locus which encodes an anti-proliferative factor, which could explain why the BAF complex functions as a tumor suppressor in many tissues. However, recall that this complex also specifically antagonizes Armadillo/β-catenin-mediated transcription, likely via its BRG/BRM subunit which directly binds to β-catenin. Evidence from studies in Drosophila of Wg-responsive enhancers suggests that this complex mediates a negative feedback from high Wg signaling levels near Wg-producing cells which results in re-repression, imposed by the Brinker homeodomain repressor and its Armadillo-binding Teashirt co-repressor. The same factors may also install silencing on Wnt-responsive enhancers upon cessation of Wnt signaling. Notably, mammals do not have a Brinker ortholog, which could explain some of the apparent functional differences between flies and mammals with regard to the BAF complex (Kadoch, 2015). However, the closest mammalian relatives of Teashirt are the Homothorax/MEIS proteins, a family of homeodomain proteins whose expression can be Wnt-inducible. They are thus candidates for Wnt-induced repressors that confer BAF-dependent feedback inhibition (van Tienen, 2017).

Notably, none of BioID lists contained RUNX proteins. Based on functional evidence from Drosophila midgut enhancers, it is proposed that these proteins (which bind to both enhancers and Groucho/TLE) are pivotal for initial assembly of the Wnt enhanceosome at Wnt-responsive enhancers during early embryonic development, or in uncommitted progenitor cells of specific cell lineages (Fiedler, 2015). However, HEK293 cells are epithelial cells and may thus not express any RUNX factors. In any case, the negative BioID results suggest that RUNX factors function in a hit-and-run fashion. Evidently, the Wnt enhanceosome complex, once assembled at Wnt-responsive enhancers, can switch between ON and OFF states without RUNX (van Tienen, 2017).

In summary, this study has uncovered a fundamental role to Legless/BCL9 as a scaffold of the Wnt enhanceosome, far beyond its role in linking Armadillo/β-catenin to Pygo. Indeed, the function of Legless/BCL9 may extend beyond transcriptional Wnt responses, as indicated by the unexpected discovery of its strong association with nuclear co-receptor complexes. Potentially, these associations underlie the observed cross-talk between Wnt/β-catenin and nuclear hormone receptor signaling, documented extensively in the literature, including evidence for direct activation of the androgen receptor by β-catenin. Furthermore, a strong association between TLE1 and the estrogen receptor has been discovered in breast cancer cells, where TLE1 assists the estrogen receptor in its interaction with chromatin and its proliferation-promoting function. This is reminiscent of the role of Groucho/TLE as a cornerstone of the Wnt enhanceosome, proposed to earmark TCF enhancers for subsequent β-catenin docking and transcriptional Wnt responses (Fiedler, 2015). It will be interesting to test experimentally the putative roles of BCL9/B9L and Pygo in enabling cross-talk between β-catenin and nuclear hormone receptor signaling, both during normal development and in cancer (van Tienen, 2017).

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

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