Targets of Activity

To examine targets that are positively regulated by Wg in the wing, the zinc-finger protein Senseless (Sens) and the homeodomain protein Distal-less (Dll) were chosen. Sens is expressed in the proneural clusters on either side of the dorsoventral border, immediately adjacent to the Wg expression domain. Inhibition of Wg signaling with a dominant-negative TCF blocks Sens expression, demonstrating that it is a short-range target of Wg action. pygo-expressing cells outside the Wg expression domain completely lack Sens expression. The long-range target Dll is also always lost in clones overexpressing pygo. For reasons that are not clear, occasionally some expression persists just inside the clonal border (Parker, 2002).

pygo overexpression causes derepression of decapentaplegic (dpp) in leg imaginal discs. In the developing leg, wg and dpp are expressed in wedge-like domains just anterior to the posterior compartment, with wg highly enriched in the ventral half and dpp in the dorsal part. If Wg signaling is blocked, dpp expression becomes derepressed. If pygo is misexpressed using the patched-Gal4 driver, which is active in both the dpp and wg expression domains, then dpp expression (as judged by dpp-lacZ) is extended into the ventral compartment. This derepression of dpp expression is seen in the vast majority of leg discs examined and is again consistent with pygo overexpression antagonizing Wg signaling (Parker, 2002).

Protein Interactions

Wnt transduction is mediated by the association of ß-catenin with nuclear TCF DNA binding factors. The products of two newly identified Drosophila segment polarity genes, legless (lgs), and pygopus (pygo) are required for Wnt signal transduction at the level of nuclear ß-catenin. Lgs encodes the homolog of human BCL9; genetic and molecular evidence is provided that these proteins exert their function by physically linking Pygo to ß-catenin. These results suggest that the recruitment of Pygo permits ß-catenin to transcriptionally activate Wnt target genes and raise the possibility that a deregulation of these events may play a causal role in the development of B cell malignancies (Kramps, 2002).

The primary structure of Arm and its mammalian homolog ß-catenin consists of an N-terminal and a C-terminal tail flanking a central domain of ~500 residues composed of 12 armadillo repeats. These repeats pack against one another to form a superhelix that features a positively charged groove. The armadillo repeat domain mediates the binding of ß-catenin to cadherins, APC, Axin, and TCF. Despite their lack of significant sequence homologies, these proteins bind competitively to ß-catenin, presumably because they contact the same surface area of ß-catenin. If Lgs/BCL9 also binds to this surface, it would be expected to compete with Pan/TCF for the interaction with ß-catenin and could not be recruited to Wnt target genes. This issue was addressed by using peptide competition and coimmunoprecipitation experiments (Kramps, 2002).

Biotinylated peptides representing the N-terminal domains of hTCF4 (or Pan) were used to pull down labeled ß-catenin (or Arm protein) with avidin beads. This peptide-protein interaction was effectively disrupted by an excess of nonbiotinylated TCF or Pan peptides, but not by an excess of HD2 peptides. GST-BCL9 protein was used to pull down labeled hTCF4 in the presence of ß-catenin protein. hTCF4 is efficiently retained on BCL9-charged glutathion beads in the presence, but not absence, of ß-catenin, which apparently can function as a bridge between the two proteins. These results indicate that Lgs/BCL9 and Pan/TCF do not compete for their interaction with Arm/ß-catenin, but rather bind it simultaneously. This in turn suggests that Lgs/BCL9 is recruited to TCF binding sites of Wnt target genes (Kramps, 2002).

To address the role of Pygo in ß-catenin-mediated transcription, a TCF reporter gene (TOP-Flash) was used in immortalized human embryo kidney cells (HEK 293 cells). Low levels of a stable mutant form of ß-catenin (DeltaN-ß-catenin) were introduced into these cells to partially stimulate the pathway. The additional expression of hPYGO1 or hPYGO2 leads to a large increase in luciferase activity (30-fold). These levels are significantly higher than the sum of those produced by either treatment alone. This potentiation of ß-catenin activity by hPYGO1 and 2 appears to be mediated by the interaction of endogenous TCF protein with its DNA target sites, as it is only observed with TOP-Flash, which contains five optimal TCF binding sites, but not with the control reporter FOP-Flash, which contains five mutated sites (Kramps, 2002).

It can be concluded that Lgs and Pygo are required for the signaling activity of Arm, and that this function depends on the ability of Lgs to interact molecularly with Arm and on the ability of Pygo to molecularly interact with Lgs. Based on their subcellular localization and epistatic relationship with ArmS10, Lgs and Pygo are unlikely to exert their function by impeding proteasome-mediated degradation of Arm. They could play a role in permitting nuclear import or preventing nuclear export of Arm. However, this is thought to also be unlikely, because no difference is detected in subcellular localization of Arm in lgs mutant embryos. An alternative possibility is that Lgs and BCL9, respectively, function to tie Pygo to the ß-catenin-TCF complex, perhaps to allow Pygo to activate and sustain the expression of Wnt target genes. This hypothesis raises several predictions. (1) This model implies that the main task of Lgs/BCL9 is to serve as an adaptor to tether Pygo to Arm/ß-catenin. Thus, most of the Lgs/BCL9 protein should be dispensable, as long as Lgs HD1 is covalently linked to HD2, allowing the formation of a bridge between Arm/ß-catenin and Pygo. Lgs might even be entirely superfluous if Pygo is endowed with the ability to directly bind ß-catenin. (2) This model would require that Arm/ß-catenin is able to bind simultaneously, and stably, with both Pan/TCF and Lgs/BCL9. This in turn would necessitate separate binding sites on Arm/ß-catenin for Pan/TCF and Lgs/BCL9. (3) Pygo proteins would have to possess the ability to stimulate transcription when recruited to promoters of Wnt target genes (Kramps, 2002).

The Wnt signalling system controls many fundamental processes during animal development and its deregulation has been causally linked to colorectal cancer. Transduction of Wnt signals entails the association of ß-catenin with nuclear TCF DNA-binding factors and the subsequent activation of target genes. Using genetic assays in Drosophila, a presumptive adaptor protein, Legless (Lgs), has been identifed that binds to ß-catenin and mediates signalling activity by recruiting the transcriptional activator Pygopus (Pygo). This study characterizes the ß-catenin/Lgs interaction and shows that: (1) it is critically dependent on two acidic amino acid residues in the first Armadillo repeat of ß-catenin; (2) it is spatially and functionally separable from the binding sites for TCF factors, APC and E-cadherin; (3) it is required in endogenous as well as constitutively active forms of ß-catenin for Wingless signalling output in Drosophila, and (4) in its absence animals develop with the same phenotypic consequences as animals lacking Lgs altogether. Based on these findings, and because Lgs and Pygo have human homologues that can substitute for their Drosophila counterparts, it is inferred that the ß-catenin/Lgs binding site may thus serve as an attractive drug target for therapeutic intervention in ß-catenin-dependent cancer progression (Hoffmans, 2004).

This study is concerned with the question of how ß-catenin and Lgs interact molecularly with each other. The analysis addressed three issues: localization of the binding site on ß-catenin, specificity of this site vis-a-vis other partners of ß-catenin and in vivo significance of this interaction for Wg signal transduction. By means of site-directed mutagenesis the role of conspicuous ß-catenin residues in the binding to human LGS1 was examined. Two amino acids, D162 and D164, were identified that are both necessary for human LGS1 binding. Because substitutions of these residues with other amino acids did not affect the binding of several other proteins to ß-catenin, the role of these amino acids is interpreted as contact sites for human LGS1, rather than a structural function enhancing stability and/or three-dimensional conformation of ß-catenin. This conclusion, however, will need to be confirmed by determining the crystal structure of the ß-catenin/human LGS1 complex (Hoffmans, 2004).

Neither D162 nor D164 is required for binding to APC, E-cadherin or TCF4. Substitutions of these amino acids reduce binding to alpha-catenin twofold, but in vivo data suggest that this reduction does not prevent the assembly of adherens junctions. The specificity of the ß-catenin/human LGS1 interaction vis-a-vis that of ß-catenin and APC, E-cadherin or TCF4 is consistent with their respective locations on the surface of ß-catenin. While crystallographic studies show that APC, E-cadherin and TCF4 all bind to a common, extended surface within the groove of ß-catenin formed by Arm repeats 3-10, this analysis indicates that human LGS1 binds an acidic knob in Arm repeat 1. This knob is not only located more N terminally, it is also situated on the side of ß-catenin, which is opposite the groove. The spatial separation of these binding sites is in agreement with their separable functions observed in yeast binding assays, as well as with previous GST pull-down assays, in which simultaneous binding of TCF4 and human LGS1 to ß-catenin is observed (Kramps et al., 2002).

Thus, to assess the role of D162 and D164 in Wg transduction, mutant forms of Arm were subjected to various assays designed to reveal their in vivo function. Simple rescue and overexpression experiments have shown that transgenic Arm-D164A cannot substitute for endogenous Arm, and that the D164A mutation significantly reduces the constitutive signalling activity associated with N-terminal deletions of Arm. When tested in more advanced assays, it was found that D164 is required by wing disc cells to maintain Wg target gene expression and by developing embryos for segmentation. Together, these experiments support the conclusion that Arm signalling function relies on its capability to bind to Lgs throughout development (Hoffmans, 2004).

Although it is straightforward to interpret the results as a qualitative indication for the significance of the Arm/Lgs interaction, it is more difficult to assess their outcome in a quantitative manner. For example, the apparent residual expression of Dll in Arm-D164A cells may reflect perdurance of wild-type Arm or Dll proteins, but it could also indicate that a fraction of the Wg signal can be transmitted despite the D164A mutation. This latter scenario could in turn be attributed to some residual binding of Arm to Lgs, but it could also be explained by a partial redundancy of Lgs function. Lgs may be required for efficient Arm-mediated activation of Wg targets, but some activation may also occur in its absence. Consistent with this latter view, it was observed that animals lacking maternal and zygotic lgs product exhibit phenotypes equivalent to animals in which the sole source of Arm is the D164A transgene, yet neither of the two phenotypes are quite as severe as that of wg-null mutants (Hoffmans, 2004).

The Wnt pathway is highly conserved between Drosophila and vertebrates. The human homologues of Lgs (LGS1/BCL9) and Pygo (PYGO1 and PYGO2) can rescue lgs and pygo mutant flies, respectively. This suggests that these proteins have the same function in vertebrates and in Drosophila. It is possible therefore, that in vivo data can be extrapolated to Wnt signalling in mammals. Mutations in APC occur in more than 80% of inherited and sporadic colorectal cancers. These mutations lead to accumulation of free ß-catenin and as a result to overexpression of Wnt target genes. A chemical compound that interferes with the formation of the nuclear TCF/ß-catenin/Lgs/Pygo complex should in theory halt the progression of cancer. Such an anti-cancer drug must be highly specific though, since it should only disrupt the nuclear ß-catenin complex, but should not disrupt either the cytoplasmic ß-catenin/APC/Axin complex or the ß-catenin/E-cadherin complex at the cell membrane. APC, Axin and E-cadherin functions should not be compromised, since all three of them have tumour suppressor roles. This is not the case, however, for TCF and Lgs. Crystal structure data indicates that APC, Axin, E-cadherin and TCF4 partly use the same contact sites of ß-catenin for their binding. Therefore, designing an inhibitor that specifically disrupts the ß-catenin/TCF interaction is a difficult task. On the contrary, mapping and specificity results indicate that the ß-catenin/Lgs interaction site could be targeted without interfering with the binding of ß-catenin to APC and E-cadherin. Moreover, this analysis shows that genetic disruption of the Arm/Lgs interaction leads to severely reduced Wg signalling, suggesting that the protein-protein interaction between ß-catenin and Lgs may provide an attractive target for therapeutic intervention (Hoffmans, 2004).

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).

Pygopus residues required for its binding to Legless are critical for transcription and development

Pygopus and Legless/Bcl-9 are recently discovered core components of the Wnt signaling pathway that are required for the transcriptional activity of Armadillo/beta-catenin and T cell factors. It has been proposed that they are part of a tri-partite adaptor chain (Armadillo-->Legless-->Pygopus) that recruits transcriptional co-activator complexes to DNA-bound T cell factor. Four conserved residues at the putative PHD domain surface of Drosophila and mouse Pygopus have been identified that are required for their binding to Legless in vitro and in vivo. The same residues are also critical for the transactivation potential of DNA-tethered Pygopus in transfected mammalian cells and for rescue activity of pygopus mutant embryos. These residues at the Legless-->Pygopus interface thus define a specific molecular target for blocking Wnt signaling during development and cancer (Townsley, 2004a).

To determine the residues within the PHD domain of Pygo that are required for its binding to Lgs, each conserved residue predicted to be at the surface and also the second of the putative zinc-coordinating cysteine residues (Cys-2; mutation C753A) were individually mutated. The wild-type and mutant domains fused to glutathione S-transferase were expressed in bacteria, and their binding to an in vitro-translated fragment of Lgs that spans both Pygo- and Armadillo-interacting domains (HD1 + 2) by pull-down assays. The wild-type PHD domain of Pygo binds efficiently to LgsHD1 + 2 in this assay, as does the ARD of Armadillo. Furthermore, the C753A mutation drastically reduces the binding of the PHD domain to Lgs, confirming the predicted structural importance of Cys-2. Of the other 15 point mutations, only 3 have a comparable effect on binding (namely L781A, T782A, and L789A) and one further mutation (A785V) reduces the binding significantly. The other mutations have little or no effect on the binding of Pygo to Lgs. These results identify Leu-781, Thr-782, Ala-785, and Leu-789 as four Pygo residues that are critical for the binding of Pygo to Lgs (Townsley, 2004a).

Interestingly, these residues are all located in the same loop (Loop 2) between the zinc-coordinating cysteines Cys-5 and Cys-6. Furthermore, if the PHD domain is projected onto the structurally related RING finger domain (from c-Cbl, a Glu-3 ubiquitin ligase), the four Loop2 residues from Pygo span a region that corresponds to an alpha-helical surface portion of c-Cbl that contacts its binding partner Rad18 (a Glu-3 ubiquitin ligase). Conversely, Loop1 residues (between Cys-2 and Cys-3) are predicted to be on the opposite surface of the PHD domain: Loop1 mutations do not substantially affect the binding between Pygo and Lgs. Thus, the Loop1 surface does not seem to be involved in binding to Lgs, whereas the above-mentioned four Loop2 residues of Pygo may all directly contact Lgs (Townsley, 2004a).

These Loop2 residues are either identical (Thr-782, Ala-785, Leu-789) or substituted by a similar amino acid (methionine instead of Leu-781) in mouse and human Pygo proteins. To test the function of the corresponding mammalian residues, two on each surface and also the structural Cys-2 residue in mPygo1 were mutated and their in vitro binding to Lgs HD1 + 2 (residues 232-555) was compared to that of wild-type mPygo1. This confirmed that Cys-2 and the two mutated Loop2 residues are critical for binding between mPygo1 and Lgs, whereas the Loop1 mutations do not substantially affect the binding. Thus, the binding between Lgs and Pygo proteins is highly comparable between Drosophila and mammals (Townsley, 2004a).

Next, a subset of the same mutations were introduced into full-length Pygo and wild-type and mutant Pygo were expressed together with Lgs HD1 + 2 in mammalian 293T cells. In the absence of Pygo, the Lgs fragment is largely cytoplasmic in these cells, although some nuclear staining is also observed. However, after co-transfection with Pygo, which itself is exclusively nuclear in these cells, HD1 + 2 is shifted efficiently into the nucleus, most likely because of its binding to Pygo. In support of this, HD1 + 2 is also shifted into the nucleus by the C-terminal fragment of Pygo that spans the PHD domain, whereas its nuclear-cytoplasmic distribution does not change if it is co-transfected with a Pygo deletion that lacks the PHD domain. The same is true for the Cys-2 mutation, although Cys-2 appears to aggregate somewhat in the nucleus (presumably because of misfolding), trapping the nuclear HD1 + 2 into these nuclear dots. Importantly, neither of the two Loop2 mutations tested (L789A and L781A) affects the nuclear-cytoplasmic distribution of HD1 + 2, whereas the two Loop1 mutants (N758A and D761A) are fully capable of shifting HD1 + 2 into the nucleus. Thus, the Loop2 residues are critical for the in vivo interaction between Pygo and Lgs, whereas the Loop1 residues are irrelevant for this interaction (Townsley, 2004a).

The same is essentially true for mPygo1. The full-length protein shifts HD1 + 2 into the nucleus as efficiently as Pygo, as do the two Loop1 mutants in mPygo1 (Q254A and N251A). In contrast, no change is seen with the Cys-2 mutant, nor with the Loop2 mutant L282A, confirming the importance of this loop. The other Loop2 mutant (T275A) still facilitates nuclear uptake of HD1 + 2 and is thus not severe enough to abolish the in vivo interaction between mPygo1 and Lgs, despite reducing their in vitro binding (Townsley, 2004a).

The conserved N-terminal residues of Pygo were also tested for their role in Pygo in vivo interaction with Lgs. These include a nuclear localization signal (NLS) and the N-box that spans a series of proline residues and an NPFXD motif. The latter can be an internalization signal and binds to a variety of endocytic proteins.Three sets of point mutations were generated in the N terminus and the mutants were tested in transfected 293T cells with and without HD1 + 2. Each of these mutants is fully competent in shifting Lgs to the nucleus, confirming that they are not required for the Lgs>Pygo interaction in the context of full-length Pygo. Notably, the Nnls mutation only weakly reduces the nuclear accumulation of Pygo, which is consistent with the observation that the C terminus of Pygo is nuclear despite not containing an NLS. Thus, the NLS and the C terminus of Pygo function redundantly to mediate its nuclear accumulation. In the case of the C terminus, this may be caused by a nuclear protein that binds to the PHD domain and anchors it in the nucleus (Townsley, 2004a).

It has been reported that overexpressed hPygo1 stimulates the transcription of a luciferase reporter linked to TCF-binding sites in transfected 293 cells ~30-fold if co-expressed with activated ß-catenin, suggesting that Pygo may act as a transcriptional co-activator. However, in experiments in transfected 293T cells, a very mild transcriptional stimulation (<2x) was observed with any of the mammalian Pygo proteins in the presence of varying amounts of activated ß-catenin. However, this stimulation seems to be insignificant because the same stimulation is seen with a Pygo protein whose PHD domain has been deleted. The same is true for Drosophila Pygo, which did not stimulate TCF-mediated transcription by itself, in the presence of Armadillo, and/or in the presence of dTCF. Notably, co-expression of increasing amounts of Lgs, Lgs HD1 + 2, or hBcl-9 does not improve the transcriptional stimulation. Therefore, it is concluded that overexpressed Pygo proteins cannot stimulate the transcription of TCF target genes in these cells, neither alone nor in the presence of co-expressed Lgs/Bcl-9 or TCF (Townsley, 2004a).

However, if tethered to DNA with the DNA-binding domain of GAL4 (GDB-Pygo), full-length Pygo efficiently stimulates transcription of a luciferase reporter linked to GAL4 upstream activating sequences. A similar stimulation has also been observed with a GDB fusion of Pygo that lacks its N terminus, suggesting that the latter is dispensable for transactivation. Consistent with this, the activity of GDB-Pygo is >10-fold reduced if its PHD domain is deleted. Likewise, the Cys-2 mutant is equally inactive in stimulating transcription. Thus, Pygo can stimulate transcription efficiently if tethered to DNA: this activity depends on its PHD domain (Townsley, 2004a).

Next, the mutants were tested as GDB-Pygo fusions. Individual point mutations only mildly reduce transactivation, maximally ~2.8x (C753A). Of the Loop2 mutations, the most severe one is L781A (<2x reduction). Interestingly, the most severe reduction (apart from C753A) is observed with the Loop1 mutation D761A. Other Loop1 and Loop2 mutations also show mild effects, and multiple mutations in the same loop (mLoop1, mLoop2) have more severe effects, whereas maximal effects are seen with simultaneous mutations in both loops. It is concluded that residues in both surface loops of the PHD domain contribute to the transcriptional activity of DNA-tethered Pygo. This conclusion implies that this activity not only depends on its binding to Lgs, but also on its ability to bind to another protein. The latter is unlikely to be Pygo itself, because no dimerization of Pygo nor of its C-terminal fragment has been detected in in vitro or in vivo binding assays. Therefore, an unknown Loop1-binding protein seems to synergize with Lgs in transcriptional activation (Townsley, 2004a).

The transcriptional activity of DNA-tethered Pygo measured in transfection assays is likely to depend on a variety of endogenous proteins, e.g., hPygo1 and hPygo2, which may mask the functional importance of specific residues under test. Thus, the GAL4 system was used to express three of the Pygo mutants in Drosophila embryos to see whether they could rescue mutants that lack endogenous Pygo. Wild-type Pygo expressed ubiquitously in the embryo rescues the 'denticle lawn' phenotype of pygo mutants to a considerable extent. Indeed, the rescued cuticles look essentially the same as those from paternally rescued pygo mutants (i.e., from embryos whose only source of wild-type Pygo is due to zygotic expression of the paternal allele). The same is true for the Nnpf mutant, suggesting that the NPFXD motif within the N-box is either not essential, or that it functions redundantly with other Pygo sequences. The mLoop1 mutant also rescues, so the conserved Loop1 residues are either not required in untethered Pygo, or they function redundantly with other Pygo sequences in flies. Also, the mLoop1 mutant may not be completely defective in binding to its cognate protein. In contrast, the mLoop2 mutant does not rescue pygo mutants at all (notably, this mutant is expressed at normal levels in embryos and produces similar phenotypes in the wing and eye as does wild-type Pygo). The same results were obtained in rescue assays of a hypomorphic pygo allele; in this case, wild-type Pygo, Nnpf, and mLoop1 fully restore the cuticles to wild-type. Thus, the four conserved Pygo Loop2 residues implicated in Lgs binding are critical for the function of Pygo during embryonic development (Townsley, 2004a).

Thus, four conserved residues have been identified in the PHD domain of Pygo required for its binding to Lgs that are critical for its function in transcription and embryonic development. These residues are predicted to be in a contiguous surface patch of the PHD domain and are thus likely to contact Lgs directly. Given the functional importance of these Loop2 residues, it is likely that blocking their binding to Lgs/Bcl-9 will reduce Wnt signaling activity in normal and malignant cells (Townsley, 2004a).

How Pygo and Lgs function molecularly in the transcription of Wnt target genes is still unclear. It has been suggested that Pygo is recruited to DNA-bound TCF through Lgs/Bcl-9 and Armadillo/ß-catenin and that the N terminus of Pygo recruits a co-activator complex or components of the general transcription machinery. Its PHD domain has been implicated in the former, and the current work demonstrates the importance of specific Loop2 residues in this recruitment step (Townsley, 2004a).

As yet, there is no direct evidence for the functional importance of the N terminus of Pygo. This portion of the protein is not required for transactivation by DNA-tethered Pygo. No function of the conserved NPFXD motif within the N-box was detected. However, it is possible that the mutations examined have not inactivated the N terminus sufficiently to reveal its function. Also, the N terminus may act redundantly with other Pygo sequences (Townsley, 2004a).

Perhaps the most puzzling result is the striking difference in the transactivation potential of untethered Pygo versus DNA-tethered Pygo: the latter is highly active in stimulating transcription, whereas the former is essentially inactive in transfected 293T cells. Similar results were obtained by other investigators who also found that overexpressed untethered Pygo proteins do not stimulate TCF directed transcription either in transfected 293 or 293T cells, even in the presence of co-expressed Bcl-9. Therefore, the suggestion that Pygo may act as a transcriptional co-activator may need revisiting. But how can the difference in activity between untethered and DNA-tethered Pygo be explained? Because the transcription assays measured the effects of overexpressed protein in conjunction with endogenous factors, a stimulatory effect could be observed only if none of these are limiting. Given that the direct linkage of Pygo to a DNA-binding domain confers on it high transactivation potential, this implies that the rate-limiting step in these assays is the recruitment of Pygo to DNA. Recall that this apparently involves the three proteins TCF-->ß-catenin>Lgs, but none of these are likely to limit the function of exogenous (untethered) Pygo; its transactivation potential cannot be revealed even if these factors are co-overexpressed. However, one possible explanation is that the direct tethering of Pygo to DNA by means of a DNA-binding domain is likely to be far more efficient than indirect recruitment by means of a three-protein adaptor chain. Bypassing this chain may allow detection of the intrinsic transactivation potential of Pygo (Townsley, 2004a).

Thus, transcription assays have identified conserved Loop1 residues that contribute to the transactivation potential of DNA-tethered Pygo. These Loop1 residues are predicted to be at a PHD domain surface opposite to the Pygo putative Lgs-interacting surface. They do not seem to be involved in intramolecular interactions nor in homo-dimerization, nor is this surface of the PHD domain predicted to be involved in the putative binding to phosphoinositides, so these residues are likely to mediate binding to an unknown ligand. The rescue assays in embryos have not mirrored these residue's functional importance in mammalian transcription assays for a number of possible reasons. The ultimate proof of these residue's functional significance will have to await the identification of their cognate Loop1-binding ligand (Townsley, 2004a).

Pygopus and Legless target Armadillo/beta-catenin to the nucleus to enable its transcriptional co-activator function

Wnt signalling controls the transcription of genes that function during normal and malignant development. Stimulation by canonical Wnt ligands activates beta-catenin (or Drosophila melanogaster Armadillo) by blocking its phosphorylation, resulting in its stabilization and translocation to the nucleus. Armadillo/beta-catenin binds to TCF/LEF transcription factors and recruits chromatin-modifying and -remodelling complexes to transcribe Wnt target genes. The transcriptional activity of Armadillo/beta-catenin depends on two conserved nuclear proteins recently discovered in Drosophila, Pygopus (Pygo) and Legless/BCL-9 (Lgs). Lgs functions as an adaptor between Pygo and Armadillo/beta-catenin, but how Armadillo/beta-catenin is controlled by Pygo and Lgs is not known. This study shows that the nuclear localization of Lgs entirely depends on Pygo, which itself is constitutively localized to the nucleus; thus, Pygo functions as a nuclear anchor. Pygo is also required for high nuclear Armadillo levels during Wingless signalling, and together with Lgs increases the transcriptional activity of beta-catenin in APC mutant cancer cells. Notably, linking Armadillo to a nuclear localization sequence rescues pygo and lgs mutant fly embryos. This indicates that Pygo and Lgs function in targeting Armadillo/beta-catenin to the nucleus, thus ensuring its availability to TCF during Wnt signalling (Townsley, 2004b).

Dissecting nuclear Wingless signalling: Recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins

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).

Pygopus and legless provide essential transcriptional coactivator functions to armadillo/β-catenin

Wnt signaling controls important aspects of animal development, and its deregulation has been causally linked to cancer. Transduction of Wnt signals entails the association of β-catenin with nuclear TCF DNA binding proteins and the subsequent activation of target genes. The transcriptional activity of Armadillo (Arm, the Drosophila β-catenin homolog) largely depends on two recently discovered components, Legless (Lgs) and Pygopus (Pygo). Lgs functions as an adaptor between Arm/β-catenin and Pygo, but different mechanisms have been proposed as to how Arm/β-catenin is controlled by Lgs and Pygo. Although Lgs and Pygo were originally thought to serve as nuclear cofactors for Arm/β-catenin to enhance its transactivation capacity, a recent analysis argued that they function instead to target Arm/β-catenin to the nucleus. This study used genetic assays in cultured cells and in vivo to discriminate between the two paradigms. Regardless of the measures taken to maintain the nuclear presence of Arm/β-catenin, a transcriptional-activation function of Pygo could not be bypassed. These findings therefore indicate that Arm/β-catenin depends on Lgs and Pygo primarily for its transcriptional output rather than for its nuclear import (Hoffmans, 2005).

Wingless signals are secreted glycoproteins controlling many fundamental processes during animal development. Whereas several responses to Wnt ligands appear to entail direct cytoplasmic responses organizing planar cell polarity and organ morphogenesis, a significant fraction of Wnt responses concern transcriptional changes in the nucleus. This latter aspect of Wnt-signal transduction is mediated by β-catenin and is often referred to as “canonical” or β-catenin-dependent Wnt signaling. The canonical Wnt pathway plays important roles in embryonic-cell-fate determination, and its constitutive activation is oncogenic in several adult mammalian tissues, most notably in the intestinal epithelium. Hence, it is of prime interest to understand how β-catenin activity can upregulate transcription of Wnt target genes. Although cytoplasmic β-catenin was originally discovered through its role in cell adhesion, a large body of evidence indicates that it is degraded in the absence of a Wnt signal but stabilized in its presence. As a consequence, β-catenin can sufficiently accumulate, translocate to the nucleus, and be directed to Wnt target genes by associating with DNA-binding TCF/LEF proteins. However, it is less clear how a cell-adhesion component, relocated to the nucleus, can promote and sustain the transcriptional activity of these targets (Hoffmans, 2005).

Using genetic assays in Drosophila, a presumptive adaptor protein, Legless (Lgs) has been identified, that binds to β-catenin and its Drosophila homolog, Armadillo (Arm), as well as to the nuclear protein Pygopus (Pygo). On the basis of biochemical and phenotypic analysis, it is proposed that nuclear β-catenin/Arm assembles a quaternary complex, consisting of TCF, β-catenin, Lgs, and Pygo, in which Pygo serves as a transcriptional activator to induce and/or maintain the transcription of Wnt/Wg target genes. Alternatively, however, the requirement for Lgs and Pygo in Wnt/Wg signaling could be attributed to a role in targeting and retaining β-catenin in the nucleus, increasing its net nuclear concentration and, hence, its activity. This latter view has recently gained recognition and experimental support by a cell-biological analysis of these components. This study set out to address the mechanistic role of Pygo by subjecting the two models to three different tests; each case comes to the conclusion, that Pygo functions mainly in the transcriptional output of β-catenin (Hoffmans, 2005).

In the first approach, the consequences of disrupting the molecular interaction between β-catenin and Lgs was examined. β-catenin/Arm amino acid residues required for Lgs binding have been identified and it was observed that mutant β-catenin forms lacking these residues are severely compromised in their signaling activity. This reduction in activity could be caused either by a failure of β-catenin/Arm to recruit the “transcriptional mediator” Pygo or by a reduced (as a result of diminished nuclear anchoring) nuclear-cytoplasmic ratio of β-catenin/Arm. An experiment was repeated in which N-terminally truncated and therefore constitutively active forms of Arm, ArmS10-wt and ArmS10-D164A (differing solely in one critical amino acid residue necessary for Lgs binding), were expressed in the embryonic epidermis of Drosophila. Expression of ArmS10-wt suppresses denticle formation—a read-out for a gain of Wg signaling activity—whereas the D164A mutation, which impairs binding to Lgs, efficiently abolished this gain-of-function activity. When subjected to an immunohistochemical analysis, however, the two genotypes visually differed neither in amount nor subcellular localization of the ArmS10 proteins. The D164A mutation was further used in a cellular assay in which a constitutively active form of β-catenin (S33Y) was tethered to the enhancer of a reporter gene by the DNA binding domain of Gal4. Whereas β-catenin caused strong transcriptional activation, the D164A form lost this activity almost completely. Importantly, however, both forms were expressed at equivalent levels in human cells and did not differ in their ability to localize in nuclei. Because Lgs mediates the binding of β-catenin to Pygo, these results are interpreted as evidence that a failure of Arm/β-catenin to recruit Pygo impedes the transcriptional activity of the former despite the fact that it is nuclearly localized (Hoffmans, 2005).

A second test was devised on the assumption that Lgs appears to function merely as an adaptor between Arm/β-catenin and Pygo, thereby linking Arm/β-catenin either to a transcriptional activator or a nuclear anchor. Such a passive role for Lgs can be inferred from the observations that Lgs is dependent on Pygo both for its signaling activity and for its nuclear localization. If the main role of Lgs would be to link Arm/β-catenin to the constitutively nuclear anchor Pygo, it should gain functional independence of Pygo when bestowed with a nuclear-localization signal (NLS). Lgs was therefore modified by replacing a C-terminal portion with sequences of a green fluorescent protein (LgsN-eGFP) and adding the NLS of SV40 large T-antigen N-terminally (NLS-LgsN-eGFP). These altered forms of Lgs were examined for their subcellular distribution and signaling function. The addition of a single NLS effectively conferred nuclear localization, as assessed in transfected cells. When tested for their signaling capacity in Drosophila S2 cells, LgsN and NLS-LgsN were found to be equally active in rescuing the RNAi-mediated knockdown of endogenous Lgs. However, these two forms of Lgs were equally inactive in rescuing the knockdown of endogenous Pygo. Consistent with this result, it was also found that the Lgs-rescuing activity of NLS-LgsN still depends on the HD1 domain, through which it binds Pygo. Together, these results indicate that constitutive nuclear targeting of Lgs does not bypass the requirement for Pygo in Wg signaling, suggesting that Pygo must provide a function beyond ensuring availability of Lgs and β-catenin in the nucleus of Wg-transducing cells (Hoffmans, 2005).

The third test aimed at assessing the role of the N-terminal homology domain (NHD) of Pygo. Drosophila Pygo and its two mammalian homologs, Pygo1 and Pygo2, share—in addition to their C-terminal plant homology domain (PHD) finger domain, through which they bind Lgs—a short N-terminally located sequence of amino acids. On the basis of the conservation of Pygo function and absence of further common domains, the NHD was proposed to serve as transactivation domain. It was first confirmed that the NHD core domain (amino acids 91 to 101) is not required for nuclear localization of Pygo because neither the deletion of the core nor the change of a conserved and functionally required amino acid (F99A) affected the nuclear localization of Pygo in cultured cells. Importantly, these alterations also had no discernible effect on the capacity of Pygo to bind Lgs. If Pygo and Lgs primarily function to target Arm/β-catenin to the nucleus, then NHD mutations should not seriously affect Wnt/Wg signaling. It was found, however, that Pygo-F99A—in contrast to wild-type Pygo—failed to rescue Pygo function in cultured cells and in vivo. The endogenous pygo gene was replaced with a genomic pygo-F99A transgene in vivo, and it was observed that both mutant and wild-type Pygo proteins are expressed at comparable levels without detectable differences in nuclear-cytoplasmic distribution. The most explicit argument for a role of the NHD in transactivation was obtained by analyzing mutant clones of imaginal cells in which either the pygo-wt or the pygo-F99A transgenes were the only source of full-length Pygo protein. Both transgenes rescued Lgs nuclear localization in the mutant clones to a similar extent; however, pygo-F99A—but not pygo-wt—showed severely reduced transcription of the Wg target gene senseless. Because Pygo protein bearing a mutant NHD retains the capacity to localize Lgs (and, by inference, Arm), it is inferred that the key function of the Pygo NHD is to confer transcriptional activity to Arm (Hoffmans, 2005).

In summary, the functions of Lgs and Pygo were tested in β-catenin-dependent Wnt/Wg signaling by devising experiments that separate a role in transcriptional activation of targets from a role in nuclear targeting or retention of Arm/β-catenin. In all three situations examined, the transcriptional output of Arm/β-catenin depended on Pygo activity despite measures to grant Arm/β-catenin such alleged nuclear retention. When Arm/β-catenin was tethered directly to DNA via the Gal4 DNA binding domain, or when Lgs was endowed with an NLS of its own, Arm/β-catenin activity was still dependent on the recruitment of Pygo. Likewise, in vivo, when the nuclear retention activity of Pygo was left intact, Arm was not able to transduce Wg and activate target genes without the Pygo NHD. Although it cannot be rule out that Lgs and Pygo function as a nuclear anchor for β-catenin, the results collectively argue that the primary requirement for the two Arm/β-catenin partners must be attributed to a transcriptional role that allows Arm/β-catenin to activate and/or sustain the expression of Wnt/Wg target genes. Although information is lacking on the biochemical nature of this transactivation activity, it is tempting to assume that it involves the NHD-mediated recruitment of a chromatin-modification complex or of factors mediating transcription initiation or elongation (Hoffmans, 2005).

Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo

The Wnt pathway controls cell fates, tissue homeostasis, and cancer. Its activation entails the association of β-catenin with nuclear TCF/LEF proteins and results in transcriptional activation of target genes. The mechanism by which nuclear β-catenin controls transcription is largely unknown. A novel Wnt/Wg pathway component has been genetically identify that mediates the transcriptional outputs of β-catenin/Armadillo. Drosophila Hyrax and its human ortholog, Parafibromin, components of the Polymerase-Associated Factor 1 (PAF1) complex, are required for nuclear transduction of the Wnt/Wg signal and bind directly to the C-terminal region of β-catenin/Armadillo. Moreover, the transactivation potential of Parafibromin/Hyrax depends on the recruitment of Pygopus to β-catenin/Armadillo. These results assign to the tumor suppressor Parafibromin an unexpected role in Wnt signaling and provide a molecular mechanism for Wnt target gene control, in which the nuclear Wnt signaling complex directly engages the PAF1 complex, thereby controlling transcriptional initiation and elongation by RNA Polymerase II (Mosimann, 2006).

Three lines of evidence argue for the notion that Hyx represents a component of the Wg pathway. (1) The initial observation that increased expression of hyx can overcome the dominant-negative effect of overexpressed lgs17E provides a first indication that Hyx positively influences Wg signaling outputs in vivo. lgs17E encodes an altered form of Lgs which contains a mutation in its Arm-interacting domain that severely decreases binding of Lgs to Arm and consequently the recruitment of Pygo to Arm. When provided in excess, Lgs17E protein likely impairs the function of nuclear Arm by outcompeting endogenous Lgs and thus disturbs the sensitive balance and/or sequence of factors normally recruited at Wg-responsive enhancers. Elevating the levels of a positively acting nuclear factor involved in Wg signaling, in this case Hyx, could readily explain the reversion of the Lgs17E phenotype in genetic assays. (2) The subsequent observation that genetic reduction of hyx function in imaginal discs as well as the RNAi-mediated knock-down of hyx expression in S2 cells caused a severe decrease in Wg pathway activity is a strong argument for a requirement of Hyx in Wg signaling. (3) Ultimate confirmation of the above genetic claims was the discovery of Hyx as a direct binding partner of Arm. Together these observations provide a solid basis for a model in which Hyx plays a key role in mediating the transcriptional output of Arm in response to Wg pathway activation. In contrast to the Arm partners Lgs and Pygo, Hyx is most likely not a component dedicated solely to the Wg pathway. The phenotypes associated with hyx loss-of-function mutations indicate that Hyx is involved in other developmental processes, possibly in the transcriptional output of some other signal transduction pathway(s) (Mosimann, 2006).

The high degree of homology between Hyx and its single human ortholog suggested that Parafibromin serves the same function in Wnt signaling as Hyx in Wg signaling. Indeed, with the exception of genetic evidence for an in vivo requirement, equivalent lines of reasoning as those arrived at for Hyx argue for an important role of Parafibromin in human β-catenin signaling. What could this role be? It has recently been shown that Parafibromin/Hyx represents the Cdc73 subunit of a metazoan PAF1 complex (Rozenblatt-Rosen, 2005; Yart, 2005; Adelman, 2006). The yeast PAF1 complex has originally been found associated with initiating and elongating forms of RNAPII. Moreover, the PAF1 complex interacts genetically and physically with the histone H2B ubiquitination complex, the Set1 methylase-containing COMPASS complex, and Set2, thus conferring control over a number of distinct histone modifications on RNAPII. Together, these findings suggest important conserved functions of the PAF1 complex in coordinating histone modifications 'downstream' of chromatin preparation on target promoters to ensure proper initiation, elongation, and memory of transcription (Mosimann, 2006).

To date, Cdc73p has not been reported to interact directly or indirectly with a sequence-specific DNA binding transcription factor, and it is not clear how the PAF1 complex is recruited to its target genes. However, the metazoan homologs Parafibromin and Hyx share an extended N-terminal region, not present in Cdc73p, which was found to physically interact with the core Wnt/Wg component β-catenin/Arm. It is thus tempting to speculate that during metazoan evolution, Cdc73 homology proteins evolved in their N-terminal sequences interaction domains for certain signal transduction pathways, such as the Wnt/Wg pathway, while conserving C-terminal sequences for PAF1 complex and/or RNAPII association (Mosimann, 2006).

β-catenin/Arm has two “branches” of transcriptional output, an N-terminal and a C-terminal branch, which can be separated experimentally. The N-terminal activity maps to Arm repeat 1 and can be attributed to the recruitment of Lgs and Pygo. The current results suggest that Parafibromin/Hyx mediates an important aspect of the C-terminal output of β-catenin/Arm. The significance of any transcriptional activity mapping to C-terminal sequences of β-catenin/Arm is seemingly undermined by the finding that C-terminally truncated forms of Arm (such as the product of the allele armXM19) are able to drive Wg target gene expression under certain experimental conditions. However, the armXM19 allele exhibits robust signaling activity only when its product is 'forced' into the nucleus by overexpression of a membrane-tethered form of Arm and most likely uses the N-terminal Lgs/Pygo-dependent branch for this activity. Under physiological conditions, ArmXM19 is severely impaired for Wg signaling. ArmH8.6, which lacks only a distal portion of the CTD, retains residual transactivation potential at 18°C. This apparent correlation between signaling activity and the extent of C-terminal integrity of Arm might reflect the capacity of Arm to recruit Hyx, a view consistent with protein–protein interaction results (Mosimann, 2006).

Recent advances in the understanding of how transcriptional activators modulate gene transcription suggest a sequential recruitment of histone acetylases (such as CBP/p300) and chromatin-remodeling complexes (like SWI/SNF) to target genes before RNAPII is contacted to initiate transcription on the prepared chromatin. The β-catenin region encompassing Arm repeat 11 to the C terminus has been implicated in being necessary for chromatin remodeling using in vitro assays. Parafibromin/Hyx interacts with a region of β-catenin/Arm (repeat 12-C) that overlaps with the CBP/p300 binding site (repeat 10/11-C) and the Brg-1/Brm binding region (repeat 7-12). This raises the intriguing possibility of a concerted or sequential recruitment of chromatin remodeling factors during the control of Wnt/Wg-responsive genes to the C-terminal portion of β-catenin/Arm, as is being reported for other transcription factors. In such a scenario, CBP/p300 and Brg-1/Brm would, in sequential or arbitrary order, mediate chromatin remodeling steps at β-catenin/Arm-dependent target genes before the Parafibromin/Hyx-mediated recruitment of a PAF1-like complex orchestrates later transactivation steps involving the preparation of RNAPII with histone methylase complexes. In a final step, the PAF1 complex, including Parafibromin/Hyx, may be transferred from β-catenin/Arm to RNAPII to travel with it through the actively transcribed gene (Mosimann, 2006).

What role does the Wnt/Wg pathway component Pygo play in such a model? In several readouts it was found that the Parafibromin/Hyx-enhanced transactivation activity of β-catenin is dependent on Pygo. The Pygo-Parafibromin/Hyx dependence is interpreted as an indication for a more general cross talk between Pygo and proteins interacting with the C-terminal region of β-catenin/Arm. Thus, Pygo could act as a flexible recruitment module to facilitate the exchange or stabilization of transactivating complexes that sequentially bind to the β-catenin C terminus. It is therefore proposed that Wnt/Wg target gene activation might be a concerted, Pygo-guided process, which dynamically coordinates the sequential action of transcriptional modulators at the central scaffold protein β-catenin/Arm (Mosimann, 2006).

The yeast PAF1 complex shows cotranscriptional association with a wide range of genes and has therefore been considered a general transcription cofactor complex. However, deletion of individual components of this complex does not have a global effect on mRNA transcription but instead has a more selective impact on the transcription of only a subset of genes. Currently, aside from findings of an involvement in Wnt signaling, little is known about the target gene spectrum of metazoan PAF1-like complexes. Recently published data indicate that, as in yeast, the Drosophila PAF1-like complex is broadly associated with active genes but, functionally, Cdc73/Hyx seems only necessary for a subset of PAF1 complex targets. This would be consistent with a view that Parafibromin and Hyx provide an adaptor function only to certain transcription factors, such as, for example, β-catenin and Arm. Indeed, in vivo and in vitro assays indicate that in contrast to a cohort of other genes, whose expression is constitutive or controlled by other pathways, Wg targets are remarkably sensitive to reduction of Hyx levels. However, since the assays severely reduced but never abolished hyx expression, currently it is not possible to evaluate the extent to which Hyx activity is also required for the transcription of targets of other pathways, which potentially are more resilient to reductions in Hyx levels (Mosimann, 2006).

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).

BCL9-2 binds Arm/β-catenin in a Tyr142-independent manner and requires Pygopus for its function in Wg/Wnt signaling

The Wingless signal transduction pathway controls fundamental processes during animal development. Deregulation of the Wnt pathway has been causally linked to several forms of cancer, most notably to colorectal cancer. In response to Wnt signaling, Armadillo/β-catenin associates in the nucleus with DNA bound TCF and several co-factors, among them Legless/BCL9, which provides a link to Pygopus. Recently, the second vertebrate homologue of Legless, BCL9-2 (or B9L), was characterized and proposed to mediate Wnt signaling in a Pygopus-independent manner, by binding to a Tyrosine-142-phosphorylated form of β-catenin. This study examined the role of Tyrosine-142 phosphorylation in several assays and it was find not to be important for the recruitment of BCL9-2, nor for the transcriptional activity of β-catenin in cultured mammalian cells, nor is it important in Drosophila for Wg signaling activity in vivo. Furthermore, BCL9-2 can functionally replace Lgs both in cultured cells as well as in vivo and this rescue activity depends on the ability of BCL9-2 to bind Pygo. These results do not show a significant functional difference between BCL9-2 and BCL9 but rather suggest that the two proteins represent evolutionary duplicates of Legless, which have acquired distinct expression patterns while acting in a largely redundant manner (Hoffmans, 2007).

Previous studies have ascribed unique properties and functions to BCL9 and BCL9-2, namely that BCL9-2 functions independently of Pygo and depends on phosphorylation of tyrosine 142 of β-catenin to be able to interact with it. Initially attempts were made determine if residue D164 of β-catenin, crucial for BCL9 binding, was also important for BCL9-2 binding. This study was extended to compare the signaling properties of the two proteins in other available assays. Although the repertoire of these assays has some limitations (e.g. a deficit in vertebrate genetics), these experiments did not reveal any significant functional differences between the two human homologs. Instead, it appears that BCL9 and BCL9-2 represent evolutionary duplicates of Lgs that can function in a largely redundant manner (Hoffmans, 2007).

Evidence was first provided that binding of BCL9, BCL9-2 and Lgs to Arm/β-catenin does not depend on tyrosine residue 142 of Arm/β-catenin. Single amino acid substitutions of Y142 had no effect on the transcriptional activity of Arm/β-catenin, neither in vivo nor in tissue culture cells. It was next shown that BCL9-2 can functionally replace Lgs in vivo and in cultured cells, and that this activity of BCL9-2 depends on its ability to bind Pygo. Finally, it was found that BCL9 and BCL9-2 can interact with the same partners and form similar complexes in the nucleus of stimulated cells (Hoffmans, 2007).

It was previously reported that BCL9-2 interacts efficiently with β-catenin only if the latter is phosphorylated on tyrosine 142. In the absence of tyrosine kinase activity no interaction of the two proteins was detected in a special yeast two-hybrid system. Furthermore, a mutation of β-catenin's tyrosine 142 to alanine abrogated binding to BCL9-2. In contrast, the current binding studies indicate that the binding of Arm/β-catenin to BCL9-2 does not dependent on Y142 phosphorylation. In agreement with these results, BCL9-2 was also identified as a β-catenin binding partner in a standard yeast two-hybrid system that lacks protein tyrosine kinase activity. One explanation to account for the discrepancy between the two results may be attributed to the use of different lengths of β-catenin proteins in the binding assays. While full-length β-catenin was used in the present study, a previous study used a shortened protein containing only the Armadillo repeats, and another study used a form that in addition to the Arm repeats contained small parts of the N- and C-termini (Hoffmans, 2007).

The arm2a9 allele is the strongest arm allele available, has a frameshift mutation in Arm repeat 3 and fails to provide both adhesion function as well as Wg transduction activity. A transgene encoding Arm-Y142A was able to rescue arm2a9 males, but less efficiently as the control transgene encoding wild-type Arm. This difference was attributed to a slight deficit in adhesion function, a hypothesis supported by the observations that Arm-Y142A exhibited reduced α-catenin binding while it fully rescued a signaling-defective but adhesion-competent allele of arm (armS7X-2). Furthermore, no difference could be observed in Wg signaling activity when Arm-Y142A and Arm-wt were interrogated in a quantitative signaling assay in S2 cells. Together, these experiments indicate that the transduction of the Wg signal through Arm does not depend on residue Y142 (Hoffmans, 2007).

To compare the BCL9-2 and BCL9 proteins their ability to substitute for Lgs in Wg signaling was tested. Both human homologs are able to provide Lgs signaling function. Interestingly, previous studies reported that the signaling activity of BCL9-2 does not depend on Pygo binding, but on the C-terminus of BCL9-2. However, in the current replacement assays in Drosophila, found that both human proteins depended equally on their Pygo-binding domain (HD1). At the amino acid level BCL9 and BCL9-2 share three homologous regions of 20-30 amino acids in their C-termini. These short clusters are not found in Lgs; furthermore, it was previously found that the C-terminal half of Lgs is dispensable for Lgs function in Drosophila. Future studies will have to clarify a potential contribution of these C-termini to Wnt signaling in vertebrates. The conservation of these clusters in both BCL9 and BCL9-2 suggests that whatever the function of the C-terminus it is conserved between the two proteins. One approach that might shed light on this issue is to generate mice lacking BCL9 and BCL9-2 and see if the phenotypes of these animals can be rescued by a transgene ubiquitously expressing either only BCL9, BCL9-2 or Lgs. Less stringent signaling assays based on RNAi in cultured mammalian cells were uninformative, as the simultaneous addition of two siRNAs (against both bcl9 and bcl9-2) decreased their knock-down efficiency, complicating the interpretation of the results (Hoffmans, 2007).

Phylogenetic sequence comparisons indicate that a relatively recent event of gene duplication created the two BCL9 forms in vertebrates. The current results indicate that these proteins are essentially identical, at least regarding their interaction with β-catenin as well as regarding their capacity to transduce the Wg signal and to form higher order complexes containing β-catenin and Parafibromin. It is thus suggested that the BCL9 proteins function in a largely redundant manner, as adaptor proteins for the recruitment of Pygo to the β-catenin-TCF complex (Hoffmans, 2007).

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Wnt target gene transcription is mediated by nuclear translocation of stabilized β-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. The Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of β-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. It is proposed that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13 (Carrera, 2008).

The mediator complex was first defined in yeast as a large multisubunit complex required for transcription of RNA polymerase II (PolII)-dependent genes. Since then, its composition and function have been shown to be conserved in Drosophila, mouse, and human cells. The mediator complex can directly bind to Pol II and recruit it to target promoters, but it also appears to function at a step subsequent to Pol II assembly into the preinitiation complex. Several mediator subunits have been shown to act as adaptors for specific transcription factors, linking them to the mediator complex and allowing them to activate transcription (Carrera, 2008 and references therein).

Four subunits, Med12, Med13, Cdk8, and Cyclin C (CycC), form an accessory subcomplex known as the kinase module. Genetic and microarray analyses in yeast implicate the kinase module primarily in transcriptional repression. Many of its effects have been attributed to the Cdk8 kinase, which phosphorylates the C-terminal domain of Pol II, the Cyclin H component of the TFIIH general transcription factor, and other subunits of the mediator complex, as well as specific transcription factors. The large Med12 and Med13 proteins are required for specific developmental processes in Drosophila, zebrafish, and Caenorhabditis elegans, but their biochemical functions are not understood (Carrera, 2008 and references therein).

Secreted proteins of the Wnt family play important roles in both development and oncogenesis. Transcription of Wnt target genes is mediated by nuclear translocation of stabilized Armadillo (Arm)/β-catenin and its binding to the HMG box transcription factor TCF. The adaptor protein Legless (Lgl)/Bcl-9 links Armβ-catenin to Pygopus (Pygo); the N-terminal homology domain (NHD) of Pygo is essential for Wnt-regulated transcriptional activation and is thought to interact with unknown general transcriptional regulators. This study shows that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo (kto) and skuld (skd) (Treisman, 2001), are essential for the transcription of Wingless (Wg) target genes in vivo and a Wg-responsive reporter in cultured cells. skd and kto act downstream of Arm stabilization and are required for the function of the NHD of Pygo when fused to an exogenous DNA-binding domain. Skd and Kto interact with Pygo in vivo through the NHD. It is suggested that this interaction recruits the mediator complex to allow for the transcription of Wg target genes (Carrera, 2008).

Two domains of Arm/α-catenin are important for the activation of Wnt target genes: (1) Arm repeats 1-4, which act by binding Lgs and thus recruiting Pygo, and (2) a C-terminal transcriptional activation domain. The C-terminal domain has been shown to bind to the histone acetyltransferases p300 and CBP, Hyrax/Parafibromin, which recruits histone modification complexes, and directly to the Med12 mediator complex subunit. However, this domain is insufficient for target gene activation in vivo, which requires Lgs, Pygo, and an amino acid in Arm that is critical for Lgs binding. In addition, although the C-terminal domain is a strong activator in cell culture, it is not sufficient to replace the function of Arm in vivo when fused to dTCF, whereas the activation domain of Pygo is. It has been proposed that Pygo interacts with unidentified general transcriptional regulators through its NHD. The current results suggest that the Pygo NHD recruits the mediator complex through the Kto/Med12 and Skd/Med13 subunits and that these subunits are essential for its activation function (Carrera, 2008).

An alternative view of the role of Pygo is that it acts as a nuclear anchor for Lgs and Arm. This model has been further refined by recent data showing that Pygo is constitutively localized to Wg target genes in a manner dependent on its NHD and on TCF, and it might function there to capture Arm. However, the finding that PygoDeltaPHD-GAL4 is sufficient to activate UAS-GFP expression in all cells in vivo strongly supports an additional activation function for Pygo. It is suggested that this function reflects its ability to recruit the mediator complex. Interestingly, the C. elegans Med12 and Med13 homologues have been implicated in the transcriptional repression of Wnt target genes although these effects have not been shown to be direct (Yoda, 2005; Zhang, 2000). Their dispensability for Wnt target gene activation may reflect the absence of pygo homologues in the worm genome (Carrera, 2008).

The kinase module of the mediator complex is commonly thought to have a repressive function; it has been shown to sterically hinder recruitment of Pol II, and Ras signaling promotes transcriptional elongation by inducing loss of this module from the mediator complex bound to C/EBP-regulated promoters. However, recent results suggest that the kinase module can play a role in transcriptional activation as well as repression. An exclusively repressive function would be difficult to reconcile with the observation that the genome-wide occupancy profiles of Cdk8 and Med13 characterized by ChIP match that of the core mediator complex. The current results support an essential and direct function for the Med12 and Med13 subunits in the activation of Wg target genes. The transcriptional and phenotypic profiles of mutants in the four subunits of the yeast kinase module are very similar. However, Drosophila cdk8 and cycC are required for only a subset of the functions of skd and kto (Loncle, 2007) that does not include Wg target gene activation. Therefore, Med12 and Med13 may have gained additional functions during the evolution of higher eukaryotes. The identical defects of the two mutants may reflect the requirement for Skd to stabilize the Kto protein. Similarly, Med24 stabilizes Med16 and Med23 and promotes their incorporation into the mediator complex (Carrera, 2008 and references therein).

Several mediator complex subunits act as adaptors that link specific transcription factors to the mediator complex. For example, Med1 interacts with nuclear receptors; Med23 interacts with phosphorylated Elk-1, the adenovirus E1A protein, and Heat shock factor; Med16 interacts with differentiation-inducing factor; and Med15 interacts with Smad2/3 and Sterol regulatory element-binding protein. The current results show that, despite their location in a module that is not part of the core mediator complex, Med12 and Med13 act as adaptors for Pygo. These subunits also are likely to act as adaptors for additional transcription factors because mutations in Drosophila and other organisms have other phenotypes that cannot be explained by loss of Wg signaling. Indeed, Med12 has been shown to interact with both Sox9 and Gli3. The yeast Med13 homologue is a target for Ras-regulated PKA phosphorylation, suggesting the interesting possibility that Wg or other signals might directly regulate the activity of Med12 or Med13. Finally, because skd and kto are not essential for normal cell proliferation or survival, they may provide targets for the treatment of Wnt-driven cancers (Carrera, 2008).

The PHD domain is required to link Drosophila Pygopus to Legless/β-catenin and not to histone H3

In Drosophila Pygopus (Pygo) and Legless (Lgs)/BCL9 are integral components of the nuclear Wnt/Wg signaling machine. Despite intense research, ideas that account for their mode of action remain speculative. One proposition, based on a recently discovered function of PHD fingers, is that Pygo, through its PHD, may decipher the histone code. This study found that human, but not Drosophila, Pygo robustly interacts with a histone-H3 peptide methylated at lysine-4. The different binding behavior is due to a single amino acid change that appears unique to Drosophilidae Pygo proteins. Rescue experiments with predicted histone binding mutants showed that in Drosophila the ability to bind histones is not essential. Further experiments with Pygo-Lgs fusions instead demonstrated that the crucial role of the PHD is to provide an interaction motif to bind Lgs. The results reveal an interesting evolutionary dichotomy in Pygo structure-function, as well as evidence underpinning the chain of adaptors model (Kessler, 2009).

This study has carefully tested the idea that histone binding contributes to the function of Pygo. The results demonstrate that in Drosophila the ability to bind H3K4me is not required for Pygo function. Instead the results suggest that the sole crucial function of the dPygoPHD finger is as a link in the chain of adaptors, which recruits the transcriptional activator potential of the NHD to Wg target genes (Kessler, 2009).

The findings do not exclude that in other species histone binding by the PygoPHD has a functional importance. Indeed it is tempting to speculate that the different behavior of mammalian and Drosophila Pygo with respect to histone binding accounts for their different requirement in vivo. In mice, where histone binding appears convincing, Pygo loss of function has a 'mild' phenotype. In Drosophila, where histone binding is apparently absent, Pygo has a positive role that is reflected in a strong loss of function phenotype. Since the effect of Pygo loss of function in other organisms has not been documented, it remains unclear if mice represent the rule and Drosophila the exception, or vice versa. If the conservation of the W in the K4 cage is an indication then Drosophila is the exception. One paradigm that would account for the observations is that Pygo has both positive and negative modes of action; histone binding providing the negative role. A possible mechanism for this model was suggested by Mosimann (2009): Pygo binds H3K4me via its PHD finger. By using BCL9 as adaptor it can retain β-catenin in the proximity of a Wingless response independently of TCF. Pygo-dependent tethering of β-catenin to methylated histones frees TCF to recruit co-repressors (e.g. Groucho and CtBP), which then counteract the β-catenin-nucleated activating chromatin remodeling processes. However, this mechanism relies on the existence of a Histone/Pygo/BCL9 complex, which the results suggest may not form (Kessler, 2009).

Whatever the case, the demonstration that dPygo’s role in Wg signaling can be reduced to the N-terminal Homology Domain (NHD) of Pygopus suggests that its primary positive function lies in this domain. The list of transcriptional complexes which interact with the NHD is small but growing, a trend which should continue. Via the interaction of the PHD with HD1 of Lgs this polygamy of interactions is targeted to Wnt target genes. In this way Pygo may act co-operatively with the C-terminus of Arm/β-catenin -- recruiting complexes required for Wg/Wnt target gene activation (Kessler, 2009).

Evolutionary adaptation of the fly Pygo PHD finger toward recognizing histone H3 tail methylated at Arginine 2

Pygo proteins promote Armadillo- and beta-catenin-dependent transcription, by relieving Groucho-dependent repression of Wnt targets. Their PHD fingers bind histone H3 tail methylated at lysine 4, and to the HD1 domain of their Legless/BCL9 cofactors, linking Pygo to Armadillo/beta-catenin. Intriguingly, fly Pygo orthologs exhibit a tryptophan > phenylalanine substitution in their histone pocket-divider which reduces their affinity for histones. This study used X-ray crystallography and NMR to discover a conspicuous groove bordering this phenylalanine in the Drosophila PHD-HD1 complex-a semi-aromatic cage recognizing asymmetrically methylated arginine 2 (R2me2a), a chromatin mark of silenced genes. A structural model of the ternary complex reveals a distinct mode of dimethylarginine recognition, involving a polar interaction between R2me2a and its groove, the structural integrity of which is crucial for normal tissue patterning. Notably, humanized fly Pygo derepresses Notch targets, implying an inherent Notch-related function of classical Pygo orthologs, disabled in fly Pygo, which thus appears dedicated to Wnt signaling (Miller, 2013).

An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP

TCF/LEF factors (see Drosophila Pangolin) are ancient context-dependent enhancer-binding proteins that are activated by β-catenin (see Drosophila Armadillo) following Wnt signaling. They control embryonic development and adult stem cell compartments, and their dysregulation often causes cancer. β-catenin-dependent transcription relies on the NPF motif of Pygo proteins. This study used a proteomics approach to discover the Chip/LDB-SSDP (ChiLS) complex as the ligand specifically binding to NPF. ChiLS also recognizes NPF motifs in other nuclear factors including Runt/RUNX2 and Drosophila ARID1, and binds to Groucho/TLE. Studies of Wnt-responsive dTCF enhancers in the Drosophila embryonic midgut indicate how these factors interact to form the Wnt enhanceosome, primed for Wnt responses by Pygo. Together with previous evidence, this study indicates that ChiLS confers context-dependence on TCF/LEF by integrating multiple inputs from lineage and signal-responsive factors, including enhanceosome switch-off by Notch. Its pivotal function in embryos and stem cells explain why its integrity is crucial in the avoidance of cancer (Fiedler, 2015).

TCF/LEF factors (TCFs) were discovered as context-dependent architectural factors without intrinsic transactivation potential that bind to the T cell receptor α (TCRα) enhancer via their high mobility group (HMG) domain. They facilitate complex assemblies with other nearby enhancer-binding proteins, including the signal-responsive CRE-binding factor (CREB) and the lineage-specific RUNX1 (also called Acute Myeloid Leukemia 1, AML1). Their activity further depends on β-catenin, a transcriptional co-factor activated by Wnt signaling, an ancient signaling pathway that controls animal development and stem cell compartments, and whose dysregulation often causes cancer. The context-dependence of TCFs is also apparent in other systems, for example in the embryonic midgut of Drosophila where dTCF integrates multiple signaling inputs with lineage-specific cues during endoderm induction. The molecular basis for this context-dependence remains unexplained (Fiedler, 2015).

In the absence of signaling, T cell factors (TCFs) are bound by the Groucho/Transducin-like Enhancer-of-split (Groucho/TLE) proteins, a family of co-repressors that silence TCF enhancers by recruiting histone deacetylases (HDACs) and by 'blanketing' them with inactive chromatin. TLEs are displaced from TCFs by β-catenin following Wnt signaling, however this is not achieved by competitive binding but depends on other factors. One of these is Pygopus (Pygo), a conserved nuclear Wnt signaling factor that recruits Armadillo (Drosophila β-catenin) via the Legless/BCL9 adaptor to promote TCF-dependent transcription. Intriguingly, Pygo is largely dispensable in the absence of Groucho, which implicates this protein in alleviating Groucho-dependent repression of Wg targets (Fiedler, 2015).

Pygo has a PHD and an N-terminal asparagine proline phenylalanine (NPF) motif, each essential for development and tissue patterning. Much is known about the PHD finger, which binds to Legless/BCL9 and to histone H3 tail methylated at lysine 4 via opposite surfaces that are connected by allosteric communication. By contrast, the NPF ligand is unknown, but two contrasting models have been proposed for its function (1">Figure 1) (Fiedler, 2015).

This study used a proteomics approach to discover that the NPF ligand is an ancient protein complex composed of Chip/LDB (LIM-domain-binding protein) and single-stranded DNA-binding protein (SSDP), also called SSBP. This complex controls remote Wnt- and Notch-responsive enhancers of homeobox genes in flies (Bronstein, 2011), and remote enhancers of globin and other erythroid genes in mammals, integrating lineage-specific inputs from LIM-homeobox (LHX) proteins and other enhancer-binding proteins. Using nuclear magnetic resonance (NMR) spectroscopy, this study demonstrated that Chip/LDB-SSDP (ChiLS) binds directly and specifically to Pygo NPFs, and also to NPF motifs in Runt-related transcription factors (RUNX) proteins and Osa (Drosophila ARID1), whose relevance is shown by functional analysis of Drosophila midgut enhancers. Furthermore, Groucho was identified as another new ligand of ChiLS by mass spectroscopy. This study thus define the core components of a Wnt enhanceosome assembled at TCF enhancers via Groucho/TLE and RUNX, primed for timely Wnt responses by ChiLS-associated Pygo. The pivotal role of ChiLS in integrating the Wnt enhanceosome provides a molecular explanation for the context-dependence of TCFs (Fiedler, 2015).

The discovery of ChiLS as the NPF ligand of Pygo proteins led to the definition of the core components of a multi-protein complex tethered to TCF enhancers via Groucho/TLE and RUNX, and slated for subsequent Wnt responses by Pygo (see Model of the Wnt enhanceosome). ChiLS also contacts additional enhancer-binding proteins via its LID, including lineage-specific and other signal-responsive factors, thereby integrating multiple position-specific inputs into TCF enhancers, which provides a molecular explanation for the context-dependence of TCF/LEF. This complex will be referred to as the Wnt enhanceosome since it shares fundamental features with the paradigmatic interferon β-responsive enhanceosome (Panne, 2007). Its components are conserved in placozoa, arguably the most primitive animals without axis and tissues with only a handful of signaling pathways including Wnt, Notch and TGF-β/SMAD, suggesting that the Wnt enhanceosome emerged as the ur-module integrating signal-responses (Fiedler, 2015).

Other proteins have been reported to interact with the Pygo N-terminus, but none of these recognize NPF. It is noted that this N-terminus is composed of low-complexity (intrinsically disordered) sequences that are prone to non-specific binding (Fiedler, 2015).

NPF is a versatile endocytosis motif that binds to structurally distinct domains, including eps15 homology (EH) domains in epsin15 homology domain (EHD) protein. Indeed, EHDs were consistently identified in lysate-based pull-downs with triple-NPF baits. EHDs are predominantly cytoplasmic, and do not interact with nuclear Pygo upon co-expression, nor are any of the Drosophila EHDs required for Wg signaling in S2 cells. ChiLS is the first nuclear NPF-binding factor (Fiedler, 2015).

NPF binding to ChiLS appears to depend on the same residues as NPF binding to EHD domains, that is, on the aromatic residue at +2, the invariant P at +1, N (or G) at 0 and NPF-adjacent residues, including negative charges at +3 and +4 (whereby a positive charge at +3 abolishes binding to EHD). Indeed, an intramolecular interaction between the +3 side-chain and that of N predisposes NPF to adopt a type 1 β-turn conformation, which increases its affinity to the EHD pocket, while the -1 residue undergoes an intermolecular interaction with this pocket. ChiLS also shows a preference for small residues at -1 and -2, similarly to N-terminal EHDs although RUNX seems to differ at -1 and -2 from Pygo and MACC1 (F/L A/E/D vs S A, respectively) (Fiedler, 2015).

Groucho/TLE is recruited to TCF via its Q domain, which tetramerizes. Intriguingly, the short segment that links two Q domain dimers into a tetramer is deleted in a dTCF-specific groucho allele that abolishes dTCF binding and Wg responses, suggesting that TCF may normally bind to a Groucho/TLE tetramer (Fiedler, 2015).

Groucho/TLE uses its second domain, the WD40 propeller, to bind to other enhancer-binding proteins on Wnt-responsive enhancers, most notably to the C-terminal WRPY motif of RUNX proteins (common partners of TCFs in Wnt-responsive enhancers). This interaction can occur simultaneously with the WD40-dependent binding to ChiLS, given the tetramer structure of Groucho/TLE. In turn, RUNX uses its DNA-binding Runt domain to interact with HMG domains of TCFs, and to recruit ChiLS. RUNX thus appears to be the keystone of the Wnt enhanceosome since it binds to the enhancer directly while undergoing simultaneous interactions with Groucho/TLE (through its C-terminal WRPY motif), TCF and ChiLS (though its Runt domain) (Fiedler, 2015).

In line with this, Runt has pioneering functions in the early Drosophila embryo, shortly after the onset of zygotic transcription, and in the naïve endoderm as soon as this germlayer is formed, in each case prior to the first Wg signaling events. RUNX paralogs also have pioneer-like functions in specifying cell lineages, that is, definitive hematopoiesis in flies and mammals (Fiedler, 2015).

The model predicts that ChiLS, once tethered to the enhanceosome core complex, recruits Pygo via NPF to prime the enhancer for Wnt responses (see Model of the Wnt enhanceosome). Given the dimer-tetramer architecture of ChiLS, its binding to Pygo can occur simultaneously to its NPF-dependent binding to RUNX. In turn, tethering Pygo to the Wnt enhanceosome may require Pygo's binding to methylated histone H3 tail, similarly to Groucho/TLE whose tethering to enhancers depends on binding to hypoacetylated histone H3 and H4 tails. Interestingly, Pygo's histone binding requires at least one methyl group at K4-the hallmark of poised enhancers. Indeed, Drosophila Pygo is highly unorthodox due to an architectural change in its histone-binding surface that allows it to recognize asymmetrically di-methylated arginine 2-a hallmark of silent chromatin. Thus, the rare unorthodox Pygo proteins may recognize silent enhancers even earlier, long before their activation, consistent with the early embryonic function of Pygo, prior to Wg signaling (Fiedler, 2015).

Overcoming the OFF state imposed on the enhancer by Groucho/TLE involves Pygo-dependent capturing of β-catenin/Armadillo, which recruits various transcriptional co-activators to its C-terminus. Although these include CREB-binding protein (CBP), a histone acetyl transferase, its tethering to TCF enhancers is likely to co-depend on CRE-binding factors (CREB, c-Fos) and SMAD which synergize with Armadillo to activate these enhancers-similarly to the interferon-β enhanceosome where CBP recruitment also co-depends on multiple enhancer-binding proteins (Panne, 2007). The ensuing acetylation of the Wnt enhancer chromatin could promote the eviction of Groucho/TLE whose chromatin anchoring is blocked by acetylation of histone H3 and H4 tails, thus initiating the ON state (Fiedler, 2015).

Osa antagonizes Wg responses throughout development, and represses UbxB through its CRE, which also mediates repression in response to high Wg signaling. Osa could therefore terminate enhancer activity, by displacing HAT-recruiting enhancer-binding proteins such as CREB and c-Fos from CREs and by cooperating with repressive enhancer-binding proteins such as Brinker (a Groucho-recruiting repressor that displaces SMAD from UbxB) to re-recruit Groucho/TLE to the enhancer, thereby re-establishing its OFF state. Notably, Osa binds Chip, to repress various Wg and ChiLS targets including achaete-scute and dLMO (Fiedler, 2015).

Therefore, ChiLS is not only a coincidence detector of multiple enhancer-binding proteins and NPF proteins, but also a switch module that exchanges positively- and negatively-acting enhancer-binding proteins (through LID) and NPF factors, to confer signal-induced activation, or re-repression. Its stoichiometry and modularity renders it ideally suited to both tasks. It is noted that the interferon-β enhanceosome does not contain a similar integrating module, perhaps because it is dedicated to a single signaling input (Fiedler, 2015).

ChiLS is essential for activation of master-regulatory genes in the early embryo, similarly to DNA-binding pioneer factors such as Zelda (in the Drosophila embryo) or FoxA (in the mammalian endoderm) which render enhancers accessible to enhancer-binding proteins. Moreover, ChiLS maintains HOX gene expression throughout development, enabling Wg to sustain HOX autoregulation, a mechanism commonly observed to ensure coordinate expression of HOX genes in groups of cells (Fiedler, 2015).

Another hallmark of pioneer factors is that they initiate communication with the basal transcription machinery associated with the promoter. Chip is thought to facilitate enhancer-promoter communication, possibly by bridging enhancers and promoters through self-association. Indeed, Ldb1 occupies both remote enhancers and transcription start sites (e.g., of globin genes and c-Myb), likely looping enhancers to the basal transcription machinery at promoters which requires self-association, but possibly also other factors (such as cohesin, or mediator) (Fiedler, 2015).

It is noted that the chromatin association of Ldb1 has typically been studied in erythroid progenitors or differentiated erythroid cells, following activation of erythoid-specific genes. It would be interesting (if technically challenging) to examine primitive cells, to determine whether ChiLS is associated exclusively with poised enhancers prior to cell specification or signal responses (Fiedler, 2015).

Previous genetic analysis in Drosophila has linked chip predominantly to Notch-regulated processes. Likewise, groucho was initially thought to be dedicated to repression downstream of Notch, before its role in antagonizing TCF and Wnt responses emerged. Moreover, Lozenge facilitates Notch responses in the developing eye, and in hematocytes. Indeed, the first links between Groucho/TLE, RUNX and nuclear Wnt components came from physical interactions, as in the case of ChiLS. The current work indicates that these nuclear Notch signaling components constitute the Wnt enhanceosome. Although the most compelling evidence for this notion is based on physical interactions, the genetic evidence from Drosophila is consistent with a role of ChiLS in Wg responses (Bronstein, 2010). Indeed, mouse Ldb1 has been implicated in Wnt-related processes, based on phenotypic analysis of Ldb1 knock-out embryos and tissues. Notably, Ldb1 has wide-spread roles in various murine stem cell compartments that are controlled by Wnt signaling (Fiedler, 2015).

An interesting corollary is that the Wnt enhanceosome may be switchable to Notch-responsive, by NPF factor exchange and/or LMO-mediated enhancer-binding protein exchange at ChiLS. Hairy/Enhancer-of-split (HES) repressors could be pivotal for this switch: these bHLH factors are universally induced by Notch signaling, and they bind to ChiLS enhancers to re-recruit Groucho/TLE via their WRPW motifs. HES repressors may thus be capable of re-establishing the OFF state on Wnt enhancers in response to Notch (Fiedler, 2015).

Notably, restoring a high histone-binding affinity in Drosophila Pygo by reversing the architectural change in its histone-binding surface towards human renders it hyperactive towards both Wg and Notch targets even though pygo is not normally required for Notch responses in flies. Humanized Pygo may thus resist the Notch-mediated shut-down of the Wnt enhanceosome, owing to its elevated histone affinity that boosts its enhancer tethering, which could delay its eviction from the enhanceosome by repressive NPF factors. The apparent Notch-responsiveness of the Wnt enhanceosome supports the notion that orthodox Pygo proteins (as found in most animals and humans) confer both Wnt and Notch responses (Fiedler, 2015).

Previous genetic studies have shown that the components of the Wnt enhanceosome (e.g., TCF, RUNX, ChiLS and LHX) have pivotal roles in stem cell compartments, as already mentioned, suggesting a universal function of this enhanceosome in stem cells. It is therefore hardly surprising that its dysregulation, that is, by hyperactive β-catenin, is a root cause of cancer, most notably colorectal cancer but also other epithelial cancers. Indeed, genetic evidence implicates almost every one of its components (as inferred from the fly counterparts) in cancer: AML1 and RUNX3 are tumour suppressors whose inactivation is prevalent in myeloid and lymphocytic leukemias, and in a wide range of solid tumors including colorectal cancer, respectively. Likewise, ARID1A is a wide-spread tumor suppressor frequently inactivated in various epithelial cancers. Furthermore, many T-cell acute leukemias can be attributed to inappropriate expression of LMO2. Intriguingly, AML1 and ARID1A behave as haplo-insufficient tumor suppressors, consistent with the notion that these factors compete with activating NPF factors such as Pygo2, RUNX2 and possibly MACC1 (predictive of metastatic colorectal cancer) for binding to ChiLS, which will be interesting to test in future. The case is compelling that the functional integrity of the Wnt enhanceosome is crucial for the avoidance of cancer (Fiedler, 2015).

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).


The expression profile of pygo in embryos was examined using in situ hybridization. pygo is expressed at relatively high levels in pre-blastoderm embryos and this staining is absent in germline clones of pygo , indicating that it is maternal in origin. pygo expression drops rapidly after this early high level, and low levels of signal are observed throughout the embryos for the rest of embryogenesis. For example, at full germband extension, pygo expression is at such low levels that visualization of the message requires overstaining, as judged by significant signal in embryos lacking maternal and zygotic pygo. It is believed that the allele used (pygo10) is a molecular null, although it cannot be ruled out that some small amount of aberrant mRNA is produced. In either case, the data indicate a high degree of maternally provided message, followed by a low level of ubiquitous zygotic expression. This continues into larval development, where pygo appears to be expressed at low levels in no distinctive pattern (e.g. the wing imaginal disc) (Parker, 2002).

Effects of Mutation or Deletion

A previously uncharacterized gene, gammy legs (gam/pygopus) was identified in a screen for genes that inhibit Wg signaling when overexpressed, taking advantage of the EP strategy and the BDGP collection of 2300 EP insertions. The starting point for the screen was a severely reduced eye created by P[GMR-GAL4] and P[UAS-wg] (GMR/wg). Each EP stock was crossed to GMR/wg flies and the F1 progeny were scored for suppression of the small eye phenotype. The ORF of gam has no known motifs except for a PHD domain at its carboxyl terminus. The function of this domain is not understood, though it is found in several transcription factors and chromatin remodeling proteins. In addition to suppressing the GMR/wg phenotype, overexpression of gam can block Wg inhibition of morphogenetic furrow progression, and can inhibit specification of the wing margin by endogenous wg activity. Epitasis experiments indicate that gam acts downstream of dishevelled and armadillo (arm) in the Wg signaling pathway. In addition to the dominant Gal4-dependent phenotypes, flies homozygous for the EP insertion in the gam locus have a recessive lethal phenotype. Homozygotes die as pharates, exhibiting reduced antenna and malformed legs. In gam mutants, dpp-lacZ is expressed at high levels on both dorsal and ventral sides of legs and antenna, indicating dorsalization of the legs and ventralization of the antenna. This is consistent with gam mutants having a reduction in Wg signaling. Therefore gam is a gene that inhibits Wg signaling when overexpressed and when reduced in activity. One explanation for this is that Gam may act in a trimeric complex (A/Gam/B). Loss of Gam prevents the complex from forming and overexpression favors the formation of A/Gam and Gam/B heterodimers over the trimeric complex. Future directions includes testing for binding of Gam to Arm or DTcf, as well as generating null allele, to determine how important this gene is for Wg signaling (Cadigan, 2001).

Overexpression of pygo clearly results in phenotypes consistent with a block in Wg signaling. However, a more physiologically relevant test of the importance of pygo for Wg function is the analysis of pygo mutants. Deletions of the pygo locus were created via imprecise excision of the EP(3)1076 transposon. Several deficiencies were generated, the most useful of which is pygo10. This deletion removes the splice acceptor of the first intron and the first 295 residues of the Pygo ORF. Therefore, it is believed that pygo10 is null for pygo activity. It fully complements a null allele of rod (rodX2) (Scaerou, 1999), in contrast to pygo9, which removes the transcription start site of rod. Mutants in gamma-cop, the gene on the other side of pygo, do not exist: whether pygo10 compromises its activity could not be tested. However, since the 3'UTR of gamma-cop and the intergenic region between it and pygo are unaffected in pygo10, this is considered unlikely. Thus, pygo10 is a deletion specific for pygo (Parker, 2002).

pygo10 homozygotes (zygotic mutants) have an early pupal lethal phenotype, as do pygo10/pygo9 transheterozygotes. However, embryos lacking maternal pygo fail to hatch, even when zygotic pygo is provided from wild-type males. pygo mutant embryos were subjected to cuticle analysis. Wild-type embryos have a distinctive patterning of denticles on their ventral cuticle, with each denticle belt arranged in a trapezoidal pattern with intermittent naked cuticle. A wg mutant does not form naked cuticle and has a characteristic denticle lawn phenotype. When mothers producing pygo10 mutant eggs are crossed with pygo10 heterozygotes, two classes of mutant phenotype are observed. Approximately half the cuticles exhibit a denticle lawn extremely similar to wg mutants. The other half have a reduction in the number of denticle belts, with some denticle fusions. This phenotypic class was also observed when the fathers were wild type for pygo, indicating that they are pygo maternal mutants. Thus, embryos lacking both maternal and zygotic pygo have a cuticle phenotype indicating a loss of Wg signaling (Parker, 2002).

To confirm that loss of pygo activity is responsible for the phenotypes described above, pygo mutant phenotypes were examined in the presence of P[UAS-pygo] and P[Daughterless-Gal4] (P[Da-Gal4]), which is ubiquitously active during embryogenesis. When pygo maternal mutants contain P[Da-Gal4] but not P[UAS-pygo], 99% of the embryos have a reduction of abdominal denticle belts. The mutant cuticles have six denticle belts; no embryos had all eight. When pygo maternal mutants contain P[Da-Gal4] and P[UAS-pygo], there is a considerable shift of the phenotypic range to the right. Some cuticles had seven abdominal belts, and 4% of the progeny had all eight. Keeping in mind that only half of progeny contain P[Da-Gal4], the data suggest that ubiquitous expression of pygo can significantly rescue the reduction of denticle belts in maternal pygo mutants (Parker, 2002).

P[Da-Gal4]/P[UAS-pygo] can also significantly rescue pygo maternal/zygotic mutants. In control crosses where P[UAS-pygo] is omitted, approximately half (52%) of the embryos have a denticle lawn. When the pygo transgene is included, only a quarter (25%) of the progeny have a lawn of denticles, which is the predicted result since only half the embryos have zygotic P[Da-Gal4]. In fact, the ratio of full to lesser lawns is even better than expected, which could be due to some maternal expression of P[Da-Gal4]. There is also an increase in the number of progeny with seven or eight denticle belts (22% versus 2%), which are presumably rescued pygo maternal mutants. These results indicate that pygo is the gene responsible for the embryonic phenotypes observed in pygo10 mutant embryos (Parker, 2002).

The pygo embryonic phenotype was further characterized using molecular markers. The Engrailed (En) protein is normally expressed in epidermal stripes of single segment periodicity. In wg mutants, the En stripes are initiated normally but fade from the epidermis by full germband extension. In embryos lacking maternal and zygotic pygo, the En pattern begins normally but alternating stripes become slightly irregular during germband extension. By full germband extension, the En stripes are largely absent but some expression does remain. Wg signaling positively regulates its own striped expression at the same stage, indirectly through maintenance of Hedgehog expression and by a more direct Wg autoregulatory loop. The Wg stripes are normal at early stages but fade at full germband extension in a pygo mutant. In addition, the dorsal derepression of Wg expression seen in wg mutants is also observed in the pygo mutants. Both the En and Wg expression patterns in pygo maternal/zygotic mutants are consistent with Wg signaling being severely compromised in the absence of pygo (Parker, 2002).

In the mesoderm, Wg is needed for expression of Even skipped (Eve) in a subset of pericardial cells. In pygo null mutants, the pericardial Eve expression is completely absent. Eve is still expressed in the CNS in both wg and pygo mutants, though the pattern is more severely disrupted in pygo. The Eve-positive RP2 neurons, which are absent in wg mutants, are also missing in pygo mutants, once again consistent with pygo being required for Wg signaling (Parker, 2002).

pygo10 homozygotes and pygo10/pygo9 transheterozygotes die around mid-pupation, indicating that maternal pygo expression can provide enough activity for viability until this stage. However, the imaginal discs of third instar pygo10 homozygotes are severely reduced in size and display abnormal morphology. Therefore, the role of pygo was examined in these tissues using mosaic analysis (Parker, 2002).

In the wing imaginal disc, Wg signaling at the dorsoventral boundary of the presumptive wing blade is required for formation of an adult structure known as the wing margin. Wings from flies containing clones of pygo10 frequently contain notches, caused by a loss of wing margin. To confirm that these notches are due to a loss of Wg signaling, molecular markers were examined at third larval instar. Loss of pygo causes derepression of Wg adjacent to the stripe and does not affect Wg expression in the stripe. pygo clones in the wing disc also show a cell autonomous loss of expression of the Wg targets Sens and Dll. The Sens result was observed with 100% penetrance. In the case of Dll, all clones had reduced expression, with Dll completely absent 28% of the time, a large reduction with 57% frequency, and a modest reduction in 15% of the clones. Thus, as in the embryo, loss of pygo results in a dramatic reduction in several Wg-dependent readouts, though the results with En and Dll suggest that Wg signaling may still occur at a modest level without pygo (Parker, 2002).

In the developing eye, misexpression of wg at low levels with the sevenless promoter (P[sev-wg]) results in a morphologically normal eye, except that the interommatidial bristles are absent. Expression at higher levels with GMR-wg represses the bristles and causes a severe reduction in eye size. Clones of pygo10 in either misexpression background completely suppress the effects of Wg (Parker, 2002).

Finally, reduction of pygo in the leg disc gives phenotypes consistent with loss of Wg signaling. Wg signaling is required for ventral leg identity, at least in part by repressing the dorsally expressed gene dpp. To examine the role of pygo in the leg, a hypomorphic allele was used. In addition to the Gal4-dependent phenotypes observed with EP(3)1076, Gal4-independent recessive phenotypes were found. To avoid confusion, the EP(3)1076 allele is referred to as pygoEP in this context. pygoEP homozygotes are late pupal lethal, and exhibit several defects in their exoskeleton, including malformed legs. The sex comb, a stout row of bristles seen on the ventral side of the first leg in Drosophila males is missing in the pygoEP legs. At the molecular level, the dpp-lacZ reporter, which is normally expressed primarily dorsally, becomes derepressed ventrally in pygoEP legs. Once again, loss of pygo results in a phenotype consistent with a loss of Wg signaling. The fact that every Wg readout examined is pygo-dependent suggests that it is a core component of the pathway (Parker, 2002).

The Wingless (Wg)/Wnt signal transduction pathway regulates many developmental processes through a complex of Armadillo(Arm)/ß-catenin and the HMG-box transcription factors of the Tcf family. A new component, Pygopus (Pygo), plays an essential role in the Wg/Wnt signal transduction pathway. Wg signaling is diminished during embryogenesis and imaginal disc development in the absence of pygo activity. Pygo acts downstream or in parallel with Arm to regulate the nuclear function of Arm protein. pygo encodes a novel and evolutionarily conserved nuclear protein bearing a PHD finger that is essential for its activity. Pygo can form a complex with Arm in vivo and possesses a transcription activation domain(s). A Xenopus homolog of pygo (Xpygo) has been isolated. Depletion of maternal Xpygo by antisense deoxyoligonucleotides leads to ventralized embryonic defects and a reduction of the expression of Wnt target genes. Together, these findings demonstrate that Pygo is an essential component in the Wg/Wnt signal transduction pathway and is likely to act as a transcription co-activator required for the nuclear function of Arm/ß-catenin (Belenkaya, 2002).

Detailed functional analyses of the pygo mutant strongly argue that Pygo is an essential component in the Wg signal transduction pathway and is likely to be required universally for all the Wg signaling events in embryogenesis and imaginal disc development. Two lines of evidence support this conclusion: (1) Wg signaling in pygo mutants is defective in all the embryonic developmental processes examined, including ventral cuticle patterning, midgut constriction, embryonic central nervous system and specification of cardiac precursor cells; (2) Pygo is required for cells to respond to Wg input, for both positive and negative gene regulation in imaginal disc development. This is in contrast to other genes such as teashirt, which is specifically required for a subset of late Wg-dependent functions in the embryonic trunk segments where the teashirt gene is expressed. So far, no tissue is known in which Wg transduces its signaling in the absence of Pygo activity (Belenkaya, 2002).

The Wg/Wnt signal transduction pathway is conserved in both vertebrate and invertebrate. Loss-of-function studies in Xenopus provide strong evidence that Pygo is also required for Wnt signaling in vertebrate development. The Xenopus homolog of Pygo, XPygo, shares a significant degree of homology with Drosophila Pygo, particularly in the PHD finger domain at the C-terminal region. Depletion of maternal Xpygo mRNA by antisense oligos leads to ventralized embryonic defects and a reduction in the expression of various Wnt target genes. These results are consistent with the role of Pygo in Drosophila, suggesting that XPygo is crucial for Wnt signaling in embryonic development in Xenopus. Consistent with these results, Thompson (2002) has shown that a disruption of human PYGO1 and PYGO2 by double-stranded (ds) RNA interference (RNAi) leads to a reduction of the expression of ß-catenin/TCF target gene expression in colorectal cancer cells. Transfection of PYGO1 can enhance the TCF-mediated transcription in transient transfection assays. Together, these results strongly suggest that Pygo is essential for Wnt signaling in vertebrates as well (Belenkaya, 2002).

Genetic analyses provide strong evidence that the PHD finger in Pygo plays a crucial and specific role in Wg signaling. Wg signaling is defective in both embryogenesis and imaginal disc development in the pygoF107 mutant. pygoF107 contains a point mutation that converts amino acid 802 cysteine into tyrosine, which is the last conserved cysteine in the PHD finger. Both structural determination and mutational analysis suggest this is a critical residue for the function of the PHD finger domain. Interestingly, while both pygoF15 and pygoF66, two null alleles of pygo, have defects in addition to those in Wg signaling in embryogenesis (they exhibit pair-rule like phenotypes that have denticle deletions), pygoF107 exhibits only defects specifically associated with Wg signaling. Thus, these results suggest that the PHD finger domain in Pygo may provide a specific motif that is dedicated to Wg signaling, possibly involving the formation of a Pygo-Arm multi-protein complex(es). Consistent with these studies, Kramps (2002) has shown that Pygo interacts with Arm via Legless. The PHD domain in Pygo is required for the interaction between Pygo and Legless. The pygo130 allele used in their work is a specific deletion in PHD domain. Additional embryonic defects have been observed that are associated with both pygoF15 and pygoF66 null alleles. Similar results have also been observed by Parker (2002), suggesting that the remaining portion of Pygo has an additional role in embryonic development. The role of Pygo in regulating pair-rule gene expression in embryonic development is currently being investigated (Belenkaya, 2002).

To understand the molecular mechanism(s) by which Pygo participates in Wg signaling, detailed genetic epistasis analysis and molecular studies were carried out. These results support a model in which Pygo acts as a transcription co-activator required for activation of Wg/Wnt target genes. The following evidence supports this conclusion. (1) Genetic epistasis analysis in both embryos and wing disc places Pygo downstream of Axin. Further experiments have demonstrated that Pygo is not involved in the post-translation control and subcellular localization of Arm protein. These results thus provide strong evidence that Pygo acts downstream or in parallel with Arm to regulate the nuclear function of Arm activity. (2) Consistent with genetic epistasis analysis, it was found that Pygo contains a nuclear localization signal and is localized in the nuclei when Pygo-GFP fusion protein is expressed in 293T cells. The co-immunoprecipitation experiment provided molecular evidence that Arm and Pygo proteins are present in vivo in a multi-protein complex. (3) Like many other co-activators that can activate transcription when fused to a DNA binding domain(s), it has also been observed that Pygo has an intrinsic activation function when examined as a GAL4 fusion protein. The results in this report are in agreement with a model in which Pygo is linked to Arm protein via Legless and acts as a transcription co-activator required for the activity of Arm/ß-catenin-Tcf complex. It remains to be determined whether Pygo recruits the Arm/ß-catenin-Tcf complex to the basal transcriptional machinery or to chromatin remodeling complexes (Belenkaya, 2002).

The role of Pygopus in the differentiation of intracardiac valves in Drosophila

Cardiac valves serve an important function; they support unidirectional blood flow and prevent blood regurgitation. Wnt signaling plays an important role in the formation of mouse cardiac valves and cardiac valve proliferation in Zebrafish, but identification of the specific signaling components involved has not been addressed systematically. Of the components involved in Wnt signal transduction, pygopus (pygo), first identified as a core component of Wnt signaling in Drosophila, has not been investigated with respect to valve development and differentiation. This study took advantage of the Drosophila heart model to study the role of pygo in formation of valves between the cardiac chambers. Cardiac-specific pygo knockdown in the Drosophila heart was found to cause dilation in the region of these cardiac valves, and their characteristic dense mesh of myofibrils does not form and resembles that of neighboring cardiomyocytes. In contrast, heart-specific knockdown of the transcription factors, arm/beta-Cat, lgs/BCL9, or pan/TCF, which mediate canonical Wnt signal transduction, shows a much weaker valve differentiation defect. Double-heterozygous combinations of mutants for pygo and the Wnt-signaling components have no additional effect on heart function compared with pygo heterozygotes alone. These results are consistent with the idea that pygo functions independently of canonical Wnt signaling in the differentiation of the adult interchamber cardiac valves (Tang, 2014).


Adelman, K., et al. (2006). Drosophila paf1 modulates chromatin structure at actively transcribed genes. Mol. Cell. Biol. 26: 250-260. Medline abstract: 16354696

Andrews, P. G., Lake, B. B., Popadiuk, C. and Kao, K. R. (2007). Requirement of Pygopus 2 in breast cancer. Int. J. Oncol. 30(2): 357-63. Medline abstract: 17203217

Basto, R., Gomes, R. and Karess, R. E. (2000). Rough Deal and Zw10 are required for the metaphase checkpoint in Drosophila. Nature Cell Biol. 2: 939-943. 11146659

Bauer, A., Chauvet, S., Huber, O., Usseglio, F., Rothbacher, U., Aragnol, D., Kemler, R. and Pradel, J. (2000). Pontin52 and reptin52 function as antagonistic regulators of beta-catenin signalling activity. EMBO J. 19: 6121-6130. 9843967

Belenkaya, T. Y., et al. (2002). pygopus encodes a nuclear protein essential for Wingless/Wnt signaling. Development 129: 4089-4101. 12163411

Bronstein, R. and Segal, D. (2011). Modularity of CHIP/LDB transcription complexes regulates cell differentiation. Fly (Austin) 5: 200-205. PubMed ID: 21406967

Bronstein, R., Levkovitz, L., Yosef, N., Yanku, M., Ruppin, E., Sharan, R., Westphal, H., Oliver, B. and Segal, D. (2010). Transcriptional regulation by CHIP/LDB complexes. PLoS Genet 6: e1001063. PubMed ID: 20730086

Cadigan, K. M., Parker, D. S., Jemison, J., Klinedienst, S. and Kohen, R. (2001). The role of the gammy legs gene in Wingless signaling. Annual Dros. Res. Conf. 42: 144. FlyBase abstract

Carrera, I., Janody, F., Leeds, N., Duveau, F. and Treisman, J. E. (2008). Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13. Proc. Natl. Acad. Sci. 105(18): 6644-9. PubMed Citation: 18451032

Chodaparambil, J. V., Pate, K. T., Hepler, M. R., Tsai, B. P., Muthurajan, U. M., Luger, K., Waterman, M. L. and Weis, W. I. (2014). Molecular functions of the TLE tetramerization domain in Wnt target gene repression. EMBO J 33(7): 719-731. PubMed ID: 24596249

de la Roche, M. and Bienz, M. (2007). Wingless-independent association of Pygopus with dTCF target genes. Curr. Biol. 17(6): 556-61. Medline abstract: 17320388

Fiedler, M., Sanchez-Barrena, M. J., Nekrasov, M., Mieszczanek, J., Rybin, V., Muller, J., Evans, P. and Bienz, M. (2008). Decoding of methylated histone H3 tail by the Pygo-BCL9 Wnt signaling complex. Mol Cell 30(4): 507-518. PubMed ID: 18498752

Fiedler, M., Graeb, M., Mieszczanek, J., Rutherford, T. J., Johnson, C. M. and Bienz, M. (2015). An ancient Pygo-dependent Wnt enhanceosome integrated by Chip/LDB-SSDP. Elife 4:e09073. PubMed ID: 26312500

Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F. and Kemler, R. (2000). The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. EMBO J. 19: 1839-1850. 10775268

Hoffmans, R. and Basler, K. (2004). Identification and in vivo role of the Armadillo-Legless interaction. Development 131: 4393-4400. 15294866

Hoffmans, R., Stadeli, R. and Basler, K. (2005). Pygopus and legless provide essential transcriptional coactivator functions to armadillo/β-catenin. Curr. Biol. 15(13): 1207-11. 16005293

Hoffmans, R. and Basler, K. (2007). BCL9-2 binds Arm/β-catenin in a Tyr142-independent manner and requires Pygopus for its function in Wg/Wnt signaling. Mech. Dev. 124(1): 59-67. 17113272

Kadoch, C. and Crabtree, G. R. (2015). Mammalian SWI/SNF chromatin remodeling complexes and cancer: Mechanistic insights gained from human genomics. Sci Adv 1(5): e1500447. PubMed ID: 26601204

Kessler, R., Hausmann, G. and Basler, K. (2009). The PHD domain is required to link Drosophila Pygopus to Legless/β-catenin and not to histone H3. Mech. Dev. 126(8-9): 752-9. PubMed Citation: 19493659

Kramps, T. (2002). Wnt/Wingless signaling requires BCL9/Legless-mediated recruitment of Pygopus to the nuclear ß-Catenin-TCF complex Cell 109: 47-60. 11955446

Krieghoff, E., Behrens, J. and Mayr, B. (2006). Nucleo-cytoplasmic distribution of β-catenin is regulated by retention. J. Cell Sci. 119(Pt 7): 1453-63. 16554443

Lake, B. B. and Kao, K. R. (2003). Pygopus is required for embryonic brain patterning in Xenopus. Dev. Biol. 261: 132-148. 12941625

Li, B., Mackay, D. R., Ma, J. and Dai, X. (2004). Cloning and developmental expression of mouse pygopus 2, a putative Wnt signaling component. Genomics 84(2): 398-405. 15234002

Li, B., et al. (2007). Developmental phenotypes and reduced Wnt signaling in mice deficient for pygopus 2. Genesis 45(5): 318-25. Medline abstract: 17458864

Loncle N., et al. (2007). Distinct roles for Mediator Cdk8 module subunits in Drosophila development. EMBO J. 26(4): 1045-54. PubMed Citation: 17290221

Miller, T. C., Mieszczanek, J., Sanchez-Barrena, M. J., Rutherford, T. J., Fiedler, M. and Bienz, M. (2013). Evolutionary adaptation of the fly Pygo PHD finger toward recognizing histone H3 tail methylated at Arginine 2. Structure 21: 2208-2220. PubMed ID: 24183574

Mosimann, C., Hausmann, G. and Basler, K. (2006). Parafibromin/Hyrax activates Wnt/Wg target gene transcription by direct association with β-catenin/Armadillo. Cell 125(2): 327-41. Medline abstract: 16630820

Mosimann, C., Hausmann, G. and Basler, K. (2009). Beta-catenin hits chromatin: regulation of Wnt target gene activation. Nat. Rev. Mol. Cell Biol. 10: 276-286. PubMed Citation: 19305417

Nakamura, Y., et al. (2007). Crystal structure analysis of the PHD domain of the transcription co-activator Pygopus. J. Mol. Biol. 370(1): 80-92. Medline abstract: 17499269

Panne, D., Maniatis, T. and Harrison, S. C. (2007). An atomic model of the interferon-beta enhanceosome. Cell 129: 1111-1123. PubMed ID: 17574024

Parker, D. S., Jemison, J. and Cadigan, K. M. (2002). Pygopus, a nuclear PHD-finger protein required for Wingless signaling in Drosophila. Development 129: 2565-2576. 12015286

Popadiuk, C. M., et al. (2006). Antisense suppression of pygopus2 results in growth arrest of epithelial ovarian cancer. Clin. Cancer Res. 12(7 Pt 1): 2216-23. Medline abstract: 16609037

Rozenblatt-Rosen, O., et al. (2005). The parafibromin tumor suppressor protein is part of a human Paf1 complex. Mol. Cell. Biol. 25; 612-620. Medline abstract: 15632063

Scaerou, F., Aguilera, I., Saunders, R., Kane, N., Blottiere, L. and Karess, R. (1999). The rough deal protein is a new kinetochore component required for accurate chromosome segregation in Drosophila. J. Cell Sci. 112: 3757-3768. 10523511

Schwab, K. R., et al. (2007). Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development. BMC Biol. 5: 15. Medline abstract: 17425782

Sierra, J., Yoshida, T., Joazeiro, C. A. and Jones, K. A. (2006). The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes. Genes Dev. 20(5): 586-600. 16510874

Song, N., et al. (2007). pygopus 2 has a crucial, Wnt pathway-independent function in lens induction. Development 134(10): 1873-85. Medline abstract: 17428831

Stadeli, R. and Basler, K. (2005). Dissecting nuclear Wingless signalling: Recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins. Mech. Dev. 122(11): 1171-82. 16169192

Tang, M., Yuan, W., Bodmer, R., Wu, X. and Ocorr, K. (2014). The role of Pygopus in the differentiation of intracardiac valves in Drosophila. Genesis 52: 19-28. PubMed ID: 24265259

Thompson, B., Townsley, F., Rosin-Arbesfeld, R., Musisi, H. and Bienz, M. (2002). A new nuclear component of the Wnt signalling pathway. Nat. Cell Biol. 4: 367-373. 11988739

Thompson, B. J., et al. (2004). A complex of Armadillo, Legless, and Pygopus coactivates dTCF to activate Wingless target genes. Curr. Biol. 14: 458-466. 15043810

Townsley, F. M., Thompson, B., and Bienz, M. (2004a). Pygopus residues required for its binding to Legless are critical for transcription and development. J. Biol. Chem. 279: 5177-5183. 14612447

Townsley, F. M., Cliffe, A. and Bienz, M. (2004b). Pygopus and Legless target Armadillo/beta-catenin to the nucleus to enable its transcriptional co-activator function. Nat. Cell Biol. 6: 626-633. 15208637

Treisman, J. (2001). Drosophila homologues of the transcriptional coactivation complex subunits TRAP240 and TRAP230 are required for identical processes in eye-antennal disc development. Development 128: 603-615. PubMed Citation: 11171343

van Tienen, L. M., Mieszczanek, J., Fiedler, M., Rutherford, T. J. and Bienz, M. (2017). Constitutive scaffolding of multiple Wnt enhanceosome components by Legless/BCL9. Elife 6: e20882. PubMed ID: 28296634

Yang, L., Lin, C., Jin, C., Yang, J. C., Tanasa, B., Li, W., Merkurjev, D., Ohgi, K. A., Meng, D., Zhang, J., Evans, C. P. and Rosenfeld, M. G. (2013). lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs. Nature 500: 598-602. PubMed ID: 23945587

Yart, A., et al. (2005). The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol. Cell. Biol. 25: 5052-5060. Medline abstract: 15923622

pygopus: Biological Overview | Evolutionary homolog | Regulation | Developmental Biology | Effects of Mutation

date revised: 30 April 2017

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