BIOLOGICAL OVERVIEW

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the DNA-binding protein TCF (Pangolin in Drosophila), enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. A ubiquitously expressed protein, Pygopus, is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. These data argue strongly that Pygopus (referring to a legless lizard with scaly skin) is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF (Parker, 2002). Gammy legs, an alternative name for Pygopus, was independently identified by Cardigan (2001).

Drosophila have typical compound insect eyes. Misexpression of wg using the eye-specific GMR-Gal4 driver (the GMR driver is targeted by Glass) in combination with UAS-wg results in a dramatically reduced eye size. This phenotype can be used as the starting point in a misexpression screen for Wg signaling antagonists. If a Wg antagonist such as Axin is co-expressed with wg in the eye, the small eye phenotype is greatly suppressed. 'EP' P elements, which contain a Gal4-dependent promoter, were used to randomly co-express genes with wg in the eye. A collection of 2300 EP lines were crossed to a P[GMR-Gal4]/P[UAS-wg] (GMR/wg) stock and the progeny were scored for suppression of the small eye phenotype (Parker, 2002).

The initial positives (36) from the screen were crossed to a P[GMR-Gal4], P[sev-wg] line. P[sev-wg] eyes lack interommatidial bristles but are otherwise morphologically normal. Two of the positives suppressed the ability of Wg to inhibit bristles, suggesting that they may be bona fide Wg signaling antagonists. One of these lines was inserted adjacent to a known negative regulator of the pathway, shaggy/zw3 (EP(X)1576). Overexpression of zw3 is known to suppress Wg signaling. The second line (EP(3)1076), significantly suppresses the GMR/wg phenotype and corresponds to a novel gene, now termed pygopus (Parker, 2002).

The suppression of the GMR/wg and P[sev-wg] phenotypes by pygo overexpression is consistent with the notion that high levels of Pygo block Wg signaling. However, the data can also be explained by pygo interacting with the targets of Wg in the eye or the promoters driving wg expression. To address this, the effect of pygo overexpression was examined on Wg readouts in other tissues (Parker, 2002).

A total of twelve distinct readouts of Wg signaling from embryos and leg, wing and eye imaginal discs have been found to be significantly (two readouts) or completely (ten readouts) blocked in cells lacking pygo. The effects of pygo loss in clones are completely cell autonomous for Wg and Senseless expression in the wing. In addition, pygo transcripts are ubiquitously expressed at low levels throughout embryonic and larval tissues. It is formally possible that pygo acts to produce a factor that is required for Wg signaling or acts in parallel to the pathway. However, the fact that pygo is required for Wg action in so many contexts favors a model where Pygo acts directly in the signal transduction cascade of cells that receive Wg (Parker, 2002).

In the case of the embryonic Engrailed stripes and Distal-less expression in the wing blade primordia, the loss of pygo activity results in a less severe effect than that observed in wg or Wg signaling component mutants. The current model of Wg action in the wing, where Wg is thought to act as a morphogen, postulates that the Dll promoter requires a low level of Wg signaling for its activation. Perhaps loss of pygo does not completely abolish Wg signaling, so that there is still some activation of Dll and en. Alternatively, there could be a redundant factor that can partially replace pygo, or perhaps specific promoters are less sensitive to loss of pygo than others. However, the ability of pygo mutants to block the high levels of signaling induced by loss of Axin, argues that many targets absolutely require pygo even when Wg signaling is greatly elevated (Parker, 2002).

In the third instar wing imaginal discs, wg is expressed in a stripe of cells along the dorsoventral border. Wg secreted from these cells is thought to act as a morphogen, regulating both short- and long-range targets. In addition, Wg signaling refines the distribution of Wg protein by negative autoregulation of wg expression and downregulation of the Wg receptor Frizzled2 (Fz2). Thus, the wing imaginal disc offers several readouts to monitor Wg signaling (Parker, 2002 and references therein).

Random clones of cells expressing high levels of pygo were generated; GFP was used to mark the clones. If a clone is positioned in the endogenous wg stripe, it blocks Wg expression. This is not seen when Wg signaling is inhibited in these cells, indicating that pygo overexpression has consequences not related to Wg signaling. However, if the clone is adjacent to the wg stripe, then Wg protein is upregulated, consistent with a block in Wg signaling. The extent of Wg expansion is similar to that observed in clones mutant for dishevelled (a positive regulator of Wg signaling); this expansion is due to derepression of Wg synthesis and increased Wg stability produced by high levels of Fz2 (Parker, 2002).

If pygo is a core component of Wg signaling in the fly, where does it act in the pathway? This question was approached using epistasis analysis. Initially, this was achieved via overexpression. In the absence of Wnt signaling, ß-catenin (and by extension Arm) is believed to be phosphorylated at serine and threonine residues at its N terminus via the GSK3ß/Axin/APC complex. If these residues are deleted or substituted, ß-catenin becomes resistant to degradation. In flies, these mutant forms of Arm (Arm*) activate Wg signaling independently of Wg. When placed under the control of the GMR promoter, Arm* causes a small eye phenotype similar to that of GMR-wg. Co-expression of pygo severely suppresses this phenotype. This strongly suggests that pygo overexpression blocks Wg signaling downstream of Wg-induced Arm stabilization (Parker, 2002).

To examine the position of pygo in the pathway using loss-of-function genetics, Axin;pygo double mutants were created. In Axin mutants, the signaling pathway is constitutively activated because of stabilization of Arm. As found in vertebrate systems, Axin functions in a complex with Sgg to phosphorylate Arm. The Wg target gene Senseless (Sens) was used as a readout in wing imaginal discs. In pygo clones, Sens expression adjacent to the dorsal/ventral Wg stripe is lost. In Axin clones, Sens is activated, no matter where in the presumptive wing blade the clones are located, since loss of Axin constitutively activates Wg signaling. In Axin;pygo double mutant clones, Sens expression is always lost. Thus, pygo acts downstream of Axin in this assay (Parker, 2002).

Epitasis analysis was also performed in the eye. At the beginning of the third larval instar, a wave of apical constriction of the columnar epithelial cells, called the morphogenetic furrow (MF) sweeps across the presumptive eye from the posterior to the anterior. Behind the MF, ordered clusters of photoreceptors develop. When Wg signaling is activated in the primordial eye, such as in Axin mutant clones, no photoreceptors are specified. Thus, the eye offers another test of whether pygo is epistatic to Axin (Parker, 2002).

Photoreceptor development, as judged by Elav staining, appears normal in pygo mutant cells. Even at higher magnification, no detectable difference was observed in the photoreceptor clusters between pygo-positive and pygo mutant cells. Clones that lacked Axin lack any evidence of photoreceptor development. This dramatic phenotype is completely rescued in Axin;pygo double mutant clones, clearly demonstrating that pygo is epistatic to (acts downstream of) Axin. This is consistent with the overexpression studies that suggest pygo acts downstream of Arm stabilization (Parker, 2002).

When Wg signaling is activated, Arm is stabilized and translocates to the nucleus. In Drosophila, it has proved very difficult to detect nuclear Arm, even in cells receiving high levels of endogenous Wg. However, Axin maternal and zygotic mutant embryos display high levels of nuclear Arm. Because attempts to make Axin;pygo germline clones were unsuccessful, clones in the wing disc were generated to investigate Arm levels and localization. In clones of cells lacking pygo, Arm is present at low levels at the cell periphery, consistent with its role in adherence junctions. In Axin clones, Arm protein levels are greatly increased in both the nucleus and cytoplasm. Axin;pygo double mutant clones also have high levels of cytosolic and nuclear Arm, though the nuclear levels of Arm appear slightly less than in Axin clones. These data are interpreted to mean that Arm is still stabilized in the absence of pygo (as would be expected if pygo acts downstream of Axin) and that, for the most part, pygo is not required for Arm nuclear import (Parker, 2002).

Consistent with these data, a GFP-Pygo fusion protein localizes to the nucleus in a Drosophila cell line. Thus pygo acts genetically downstream of Arm stability and nuclear import, consistent with the nuclear localization of the Pygo fusion protein (Parker, 2002).

These experiments indicate that pygo acts downstream of Axin, an activated form of Arm and Arm nuclear import. Consistent with this, a tagged form of Pygo is nuclear. Taken together these data strongly suggests that Pygo acts in the nucleus, probably at the transcriptional level (Parker, 2002).

How pygo influences transcription of Wg target genes in the nucleus could occur in several ways. Simple explanations include the possibility that pygo could simply be required for the interaction of Arm with TCF, or for TCF to bind to DNA. However, the fact that Arm still accumulates to high levels in the nuclei of Axin;pygo mutant cells may indicate that the Arm/TCF/DNA complex still forms in the absence of pygo. It has been shown that expression of a dominant-negative version of TCF (which lacks the Arm-binding domain but retains its ability to bind DNA) prevents Arm nuclear accumulation. This supports the idea that TCF acts as a nuclear tether for stabilized Arm. Using this line of reasoning, Arm is still found in the nuclei of Axin;pygo mutant cells because it is still bound by TCF, which is still localized properly on the DNA. It should be noted a subtle reduction in nuclear Arm accumulation is seen in Axin;pygo versus Axin mutant cells. However, the small difference suggests that this effect may be indirect (Parker, 2002).

Another line of evidence suggesting that pygo is not required for TCF to bind to DNA comes from a detailed analysis of the pericardial enhancer of the eve gene. Eve expression in the pericardial cells is absent in wg and pygo mutants. Mutation of a single high-affinity site in the pericardial enhancer significantly reduces expression. However, mutation of all the sites bound by TCF in vitro reveals a depression of the enhancer throughout the dorsal mesoderm, suggesting that in the absence of Wg signaling, Tcf represses the eve pericardial enhancer. Since no such derepression of Eve expression was observed in pygo mutants, this suggests that TCF can bind to the eve enhancer and repress transcription in the absence of pygo (Parker, 2002).

If Pygo does not promote DNA binding of TCF or formation of the Arm/TCF/DNA complex, what might it be doing? Pygo could help positive factors like Pontin52 (Bauer, 2000) to complex with Arm/TCF, or it could normally prevent negative factors like Groucho or Osa from localizing to Wg target genes. Pontin52 (also called TIP49a) is a DNA helicase that binds to the N-terminus of ß-catenin and synergizes with it in the reporter gene assay. Pontin52 can also bind to the TATA box binding factor TBP, suggesting it links ß-catenin/Tcf to the basal transcription complex (Parker, 2002).

In addition, there are a multitude of additional negative regulators of TCF activity identified in vertebrates (see Hecht, 2000 for a noncomprehensive list). Pygo could negatively regulate any of these factors (Parker, 2002).

While the above possibilities must be addressed, the presence of the PHD domain in the Pygo protein suggests another model. PHD domains are often found in chromatin remodeling factors. These complexes are thought to alter chromatin structure to allow activation or repression of specific genes. The PHD domain is a zinc-binding domain that does not bind DNA, and is thought to be involved in protein-protein interactions. Therefore, it is possible that pygo is a member of such a chromatin remodeling complex (Parker, 2002).

The finding that overexpression of full-length Pygo inhibits Wg signaling is consistent with Pygo acting in a multisubunit complex. For example, a heterotrimeric complex consisting of A/Pygo/B could be disrupted by an abundance of Pygo, shifting the equilibrium to A/Pygo and B/Pygo heterodimers. While this is speculation, examples of similar situations are known for histone octomers, Apterous/Chip tetramers and cytoskeletal complexes (Parker, 2002 and references therein).

Does pygo have any functions other than regulation of Wg signaling? The phenotypes obtained in clonal analysis in the wing and eye suggests that pygo is highly specific for Wg signaling in these tissues. The fact that clones of pygo in the eye have no detectable defects in morphogenetic furrow progression and photoreceptor recruitment or in the morphology of the adult eye is especially impressive. Eye development requires a cadre of transcription factors, including Eyeless, Sine oculis and Eyes absent, that act in concert with Hedgehog, Dpp, Notch and Ras signaling to specify eye identity in the growing eye-antennal disc. However, while the data in the embryo suggests that pygo primarily affects Wg signaling, it is also required for non Wg-dependent processes. For example, when pygo germline clones are zygotically rescued, they have cuticles that appear, at least on a superficial level, to be of the pair-rule class. Such phenotypes are not seen in wg, dsh or arm mutant embryos. Pygo is not a pair-rule mutant in the classical sense, since even in the complete absence of pygo, En and Wg stripes are normal at cellular blastoderm. Rather, the decrease in alternative En stripes begins during germband extension. pygo maternal/zygotic mutants also have morphological abnormalities not seen in wg mutants, such as incomplete germ-band extension and less organized epithelium, another indication of a wider role for pygo. Thus, while pygo is highly dedicated to Wg signaling, it clearly has other roles as well (Parker, 2002).

Structure and function of Pygo in organ development dependent and independent Wnt signalling

Pygo is a nuclear protein containing two conserved domains, NHD and PHD, which play important roles in embryonic development and carcinogenesis. Pygo was first identified as a core component of the Wnt/β-catenin signalling pathway. However, it has also been reported that the function of Pygo is not always Wnt/β-catenin signalling dependent. This review summarises the functions of both domains of Pygo and show that their functions are synergetic. The PHD domain mainly combines with transcription co-factors, including histone 3 and Bcl9/9l. The NHD domain mainly recruits histone methyltransferase/acetyltransferase (HMT/HAT) to modify lysine 4 of the histone 3 tail (H3K4) and interacts with Chip/LIM-domain DNA-binding proteins (ChiLS) to form enhanceosomes to regulate transcriptional activity. Furthermore, chromatin modification differences of Pygo in Drosophila (dPygo) and vertebrates are summarized; Pygo displays a chromatin silencing function in Drosophila, while in vertebates, Pygo has a chromatin-activating function due to the two substitution of two amino acid residues. Next, the relationship between Pygo and Bcl9/9l was confirmed; Pygo-Bcl/9l are specifically partnered both in the nucleus and in the cytoplasm. Finally, whether transcriptional activity of Pygo is Wnt/β-catenin dependent during embryonic development is discussed. Available information indications that the transcriptional activity of Pygo in embryonic development is either Wnt/β-catenin dependent or independent in both tissue-specific and cell-specific-modes (Shi, 2020).

GENE STRUCTURE

The position of EP(3)1076 has been mapped by the Berkeley Drosophila Genome Project using inverse PCR. It is located in a small intron in the 5' UTR of a gene referred to as pygopus. Northern blot analysis reveals the major isoform of the gene to be approximately 5 kb in length. Sequencing of a 4 kb cDNA has confirmed the predicted splice sites. More recent searches have revealed an EST that extends the 5' end of the transcript, indicating the pygo transcript overlaps (but does not encompass) the transcript of rough deal (rod) (Scaerou, 1999; Basto, 2000) by at least 14 bases. This start site gives a transcript length of approximately 4.5 kb, which agrees with the data from Northern blots when polyadenylation is taken into consideration (Parker, 2002).

The EP(3)1076 transposon is inserted in the proper orientation to misexpress full length pygo, and this was confirmed experimentally. (1) RT-PCR shows that expression of pygo, but not of rod, increases dramatically when a heat shock promoter is used to drive Gal4 expression. (2) Random clones of cells expressing Gal4 under the control of an Actin promoter in a EP(3)1076 background cause a dramatic increase in pygo transcript levels. (3) Most directly, a P[UAS-pygo] transgene strongly suppresses the GMR/wg phenotype. These data argue that pygo is responsible for the antagonistic effects of EP1076 on Wg signaling (Parker, 2002).

cDNA clone length - 4079

Bases in 5' UTR - 551

Exons - 3

Bases in 3' UTR - 1080

PROTEIN STRUCTURE

Amino Acids - 815

Structural Domains

The predicted Pygo protein contains 815 amino acids and possesses two recognized motifs, a predicted NLS at the N terminus (residues 39-44) and a PHD domain at the C terminus (residues 747-811). The function of PHD domains is unclear. They are zinc finger-binding motifs, and are found in many transcription factors and chromatin remodeling proteins (Parker, 2002 and references therein).

Database searches reveal that there are potential vertebrate Pygo homologs. An embryonic mouse cDNA (accession number, AK011208) and human cDNA (XM_034083) have C-terminal PHD domains similar to that of Pygo (42-47% identity; 63-67% similarity). No other PHD domains in the human or fly genomes are more closely related. There is only one other region of sequence similarity outside this domain, encompassing the predicted NLS of the three genes. Further experiments will be required to determine if these vertebrate proteins function in Wnt signaling (Parker, 2002).

pygo encodes a novel and evolutionarily conserved protein. The most strikingly homologous domain is located in the C-terminal region that contains a PHD finger domain. The PHD finger is a domain of 60 amino acids characteristically defined by seven cysteines and a histidine, spatially arranged in a consensus of C4HC3 of varying lengths and composition. This evolutionarily conserved domain is predicted to chelate two zinc ions and is similar to, but distinct from, other zinc-binding motifs such as the RING finger (Cys3-His-Cys4) and LIM domain (Cys2-His-Cys5). PHD finger domains have been found in many different proteins, including transcription factors and are the targets of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In many cases, they serve as protein-protein interaction motifs involved in the formation of multi-protein complexes (Belenkaya, 2002).

Evolutionary Homologs

Two Xenopus mRNAs have been identified that encode proteins homologous to a component of the Wnt/ß-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2alpha having an additional 21 N-terminal residues. Xpygo-2alpha messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2ß mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2ß mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) causes both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2alpha and Xpygo-2ß by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm causes brain defects that are prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2alpha and Xpygo-2ß morpholinos reduce the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2alpha morpholino also eliminates expression of the mid–hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2alpha/ß in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain (Lake, 2003).

Recent studies in Drosophila identified Pygopus a PHD finger protein as an additional nuclear component of the canonical Wingless(Wg)/Wnt signaling pathway. A mouse pygopus gene, mpygo2, has been identified. mpygo2 transcripts are detected in almost all adult mouse tissue, whereas transcripts of another mouse pygopus gene, mpygo1, are detected only in heart tissue. Abundant mpygo2 transcripts are observed during embryogenesis in multiple developmental sites. Consistent with the demonstrated role of the Wnt-β-catenin-LEF/TCF signaling pathway in mammalian skin development, mpygo2 expression is detected in the developing epidermis and hair follicles, which suggests that mpygo2 might mediate the effect of this signaling pathway in mouse skin (Li, 2004).

β-catenin is the central signalling molecule of the canonical Wnt pathway, where it activates target genes in a complex with LEF/TCF transcription factors in the nucleus. The regulation of β-catenin activity is thought to occur mainly on the level of protein degradation, but it has been suggested that β-catenin nuclear localization and hence its transcriptional activity may additionally be regulated via nuclear import by TCF4 and BCL9 and via nuclear export by APC and axin. Using live-cell microscopy and fluorescence recovery after photobleaching (FRAP), the impact of these factors on the subcellular localization of β-catenin, its nucleo-cytoplasmic shuttling and its mobility within the nucleus and the cytoplasm were directly analysed. TCF4 and BCL9/Pygopus recruit β-catenin to the nucleus, and APC, axin and axin2 enrich β-catenin in the cytoplasm. Importantly, however, none of these factors accelerates the nucleo-cytoplasmic shuttling of β-catenin, i.e. increases the rate of β-catenin nuclear import or export. Moreover, the cytoplasmic enrichment of β-catenin by APC and axin is not abolished by inhibition of CRM-1-dependent nuclear export. TCF4, APC, axin and axin2 move more slowly than β-catenin in their respective compartment, and concomitantly decrease β-catenin mobility. Together, these data indicate that β-catenin interaction partners mainly regulate β-catenin subcellular localization by retaining it in the compartment in which they are localized, rather than by active transport into or out of the nucleus (Krieghoff, 2006).

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

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 arm^2a9 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 arm^2a9 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 (arm^S7X-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).

Canonical Wnt signaling involves complex intracellular events culminating in the stabilization of β-catenin, which enters the nucleus and binds to LEF/TCF transcription factors to stimulate gene expression. Pygopus was identified as a genetic modifier of Wg (Wnt homolog) signaling in Drosophila, and encodes a PHD domain protein that associates with the β-catenin/LEF/TCF complex. Two murine pygopus paralogs, mpygo1 and mpygo2, have been identified, but their roles in development and Wnt signaling remain elusive. This study reports that ablation of mpygo2 expression in mice. Defects were apparent in morphogenesis of both ectodermally and endodermally derived tissues, including brain, eyes, hair follicles, and lung. However, no gross abnormality was observed in embryonic intestine. Using a BAT-gal reporter, Wnt signaling at most body sites was found to be reduced in the absence of mpygo2. Taken together, these studies show that mpygo2 deletion affects embryonic development of some but not all Wnt-requiring tissues (Li, 2007).

The pygopus gene of Drosophila encodes an essential component of the Armadillo (β-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2. Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, die shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, are viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants show no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud is altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there is an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeds normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development. It is concluded that the mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity (Schwab, 2007; full text of article).

Crystal structure analysis of the PHD domain of the transcription co-activator Pygopus

The Wnt/β-catenin signaling pathway plays important roles in animal development and cancer. Pygopus (Pygo) and Legless (Lgs) are recently discovered core components of the Wnt/β-catenin transcription machinery complex, and are crucially involved in the regulation of the transcription of the Arm/β-catenin and T cell factors (TCF). Lgs/Bcl9 functions as an adaptor between Pygo and Arm/β-catenin. The crystal structure has been solved of the PHD finger of Pygopus (Pygo1 PHD), a Pygo family member, which is essential for the association with Lgs/Bcl9. The Pygo1 PHD structure forms a canonical PHD finger motif, stabilized by two Zn ions coordinated in a cross-brace scheme. Surprisingly, the Pygo1 PHD domain forms a dimer in both the crystals and solution. This is the first structural evidence for dimerization among the known PHD domain structures. The dimer formation occurs by the interactions of antiparallel β-sheets between the symmetry-related β3 strands of the monomers. The Pygo1 PHD dimer interface mainly comprises hydrophobic residues. Interestingly, some of the interface residues, such as Met372, Thr373, Ala376 and Leu380, are reportedly important for the association with Lgs/Bcl9 and are also critical for transcriptional activation. The M372A and L380D mutants, and several surrounding mutants such as S385A and A386D, showed decreased ability to form dimers and to interact with the homology domain 1 (HD1) of Lgs/Bcl9. These results suggest that the Pygo1 PHD dimerization is functionally important for Lgs/Bcl9 recognition as well as for the regulation of the Wnt/β-catenin signaling pathway (Nakamura, 2007).

The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes

The APC tumor suppressor controls the stability and nuclear export of β-catenin (β-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. β-cat and APC have opposing actions at Wnt target genes in vivo. The β-cat C-terminal activation domain associates with TRRAP/TIP60 (see Tip60) and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and β-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and ubiquitin is found necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that β-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and βTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, βTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. It is concluded that, in addition to regulating the stability of β-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells (Sierra, 2006).

The data presented here support a model in which the APC tumor suppressor functions directly to counteract β-cat-mediated transcription at Wnt target genes in vivo. This possibility was first suggested by the finding that full-length APC cycles on and off the c-Myc enhancer in conjunction with β-cat and associated coactivators in LiCl-treated C2C12 cells. In contrast, the enhancer complex appears to be stable and does not cycle in HT29 CRC cells, which contain a Class II APC mutant protein that is unable to degrade β-cat. Most strikingly, the binding of the full-length APC protein to the c-Myc gene in HT29-APC cells correlates with the rapid disassembly of the Wnt enhancer complex in vivo and the subsequent decline in steady-state c-Myc mRNA levels, both of which significantly precede the drop in β-cat protein levels that occurs as a result of proteolytic degradation in the cytoplasm. Thus, the effect of APC on c-Myc transcription appears to be immediate and direct, and may serve to coordinate the switch between the β-cat coactivator and TLE1 corepressor complexes (Sierra, 2006).

The β-cat enhancer complex includes the Wnt coactivators Pygopus and Bcl-9/Lgs, which control the retention of β-cat in the nucleus and may also function directly in transcription. The observation that APC can also regulate nuclear transport of β-cat raises the possibility that these factors may reside within a larger regulatory complex that chaperones β-cat in and out of the nucleus and mediates its release from the DNA. Indeed, sequential ChIP (re-ChIP) data indicate that the mutant APC in HT29 colorectal cancer cells exists in a stable complex with β-cat and LEF-1 at the active c-Myc gene. This finding is unexpected because β-cat cannot bind simultaneously to APC and LEF-1, and thus, if the full-length APC is part of a larger β-cat:LEF enhancer complex, it may interact with other subunits. Alternatively, the full-length APC and β-cat may exist in different complexes that rapidly exchange at the enhancer. The current data indicate that targeting is mediated by the N-terminal half of the APC protein, and that CtBP and βTrCP appear only in conjunction with the full-length APC protein. How APC is recruited to Wnt enhancers remains an open and important question (Sierra, 2006).

The ChIP experiments also suggest that APC-mediated inhibition of c-Myc transcription in HT29 cells occurs in two steps, initiated by transient binding of APC, βTrCP, CtBP, and YY1 to the enhancer, and followed by stable binding of the TLE-1 and HDAC1 corepressors. The transient recruitment of APC and CtBP, at the time when β-cat, Bcl-9, Pygo, and other Wnt enhancer factors leave the DNA, strongly suggests a role for these factors in the exchange of Wnt coactivator and corepressor complexes. In this respect it is interesting that CtBP was shown recently to associate with APC, both in vivo and in vitro. The results confirm a high-affinity interaction between CtBP and the full-length APC protein induced in HT29-APC cells, as well as with the native (full-length) APC protein in 293 cells. Consequently, APC may function to recruit CtBP to Wnt enhancers. Although both CtBP and TLE-1 are well-established corepressors of Wnt target genes, the different functions of the two types of corepressors remain unclear, and the ChIP data suggest that they act at distinct steps. Together, these data suggest that APC counteracts β-cat function in the nucleus, as well as in the cytoplasm, and may facilitate turnover of the enhancer complex at responsive genes by recruiting βTrCP and CtBP (Sierra, 2006).

Pygopus 2 has a crucial, Wnt pathway-independent function in lens induction

Drosophila Pygopus was originally identified as a core component of the canonical Wnt signaling pathway and a transcriptional coactivator. This study investigated the microophthalmia that arises in mice with a germline null mutation of pygopus 2. This phenotype is a consequence of defective lens development at inductive stages. Using a series of regionally limited Cre recombinase transgenes for conditional deletion of Pygo2^flox, it has been shown that Pygo2 activity in pre-placodal presumptive lens ectoderm, placodal ectoderm and ocular mesenchyme all contribute to lens development. In each case, Pygo2 is required for normal expression levels of the crucial transcription factor Pax6. Finally, multiple lines of evidence are provided that although Pygo2 can function in the Wnt pathway, its activity in lens development is Wnt pathway-independent (Song, 2007).

A model is proposed for the function of Pygo2 in development of the lens. This is primarily a genetic model but can be superimposed on the tissue structures to indicate likely tissue interactions. In Pygo2^-/- embryos, reduced Pax6 immunofluorescence and Le-cre(GFP) reporter expression suggest that Pygo2 is upstream of the Pax6 ectoderm enhancer in regulating the placodal phase of Pax6 expression. By contrast, in the Pygo2 germline null, the pre-placodal phase of Pax6 expression (head surface ectoderm) is unchanged. In a reciprocal experiment, it was shown that in Pax6^Sey/Sey embryos (which represent the pre-placodal phase of Pax6), Pygo2 expression was unchanged. These data argue for a genetic model in which Pygo2 and Pax6^pre-placode converge on the EE to regulate Pax6 expression. Previous analysis has shown that Pax6^placode depends on the ectoderm enhancer and at least one additional enhancer. With the information currently available, the involvement of another Pax6 lens enhancer in Pygo2-dependent regulation cannot be excluded. The best candidate for a second lens enhancer in Pax6 is the SIMO element. It has been suggested that Pax6 can directly bind the ectoderm element. Pygo2 influence on the ectoderm element could be direct or indirect (Song, 2007).

Regional deletion of the Pygo2 conditional allele has indicated that Pygo2 in multiple tissues contributes to lens development. Deletion of Pygo2^flox with Wnt1-cre indicates that Pygo2 in neural crest-derived ocular mesenchyme positively influences lens development. In one model, this could occur by direct signaling of ocular mesenchyme to presumptive lens or, conceivably, indirectly through enhancement of the ability of the optic vesicle to induce lens. Either way, the end result is Pygo2-dependent upregulation of Pax6^placode. The more-severe lens phenotype occurring when Wnt1-cre is combined with the post-induction placodal ectoderm-driver Le-cre indicates that mesenchymal and placodal Pygo2 cooperate. A comparison of that outcome with the Ap2alpha-cre conditional (where deletion also occurs in pre-placodal ectoderm) suggests that this domain is also involved. This is consistent with a function for Pygo2 in parallel with Pax6^pre-placode. It is interesting to note that Pygo2 influences lens development through the ectoderm enhamcer, as do both Fgf receptor and Bmp7 signaling. It will be interesting to determine whether the non-Wnt activity of Pygo2 resides in one of these pathways (Song, 2007).

lncRNA-dependent mechanisms of androgen-receptor-regulated gene activation programs

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behaviour of prostate cancer cells, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy. This study reports that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor-mediated gene activation programs and proliferation in prostate cancer cells. Binding of PRNCR1 to the carboxy-terminally acetylated androgen receptor on enhancers and its association with DOT1L appear to be required for recruitment of the second lncRNA, PCGEM1, to the androgen receptor amino terminus that is methylated by DOT1L. Unexpectedly, recognition of specific protein marks by PCGEM1-recruited pygopus 2 PHD domain enhances selective looping of androgen-receptor-bound enhancers to target gene promoters in these cells. In 'resistant' prostate cancer cells, these overexpressed lncRNAs can interact with, and are required for, the robust activation of both truncated and full-length androgen receptor, causing ligand-independent activation of the androgen receptor transcriptional program and cell proliferation. Conditionally expressed short hairpin RNA targeting these lncRNAs in castration-resistant prostate cancer cell lines strongly suppressed tumour xenograft growth in vivo. Together, these results indicate that these overexpressed lncRNAs can potentially serve as a required component of castration-resistance in prostatic tumours (Yang, 2013).

To address whether the PYGO2 PHD domain might itself be instrumental for its function in mediating chromatin looping, PYGO2 was depleted by shRNA treatment followed by overexpression in LNCaP-cds2 cells of either shRNA-resistant wild-type PYGO2 or a W352A mutant defective for H3K4me3 recognition. In 3C assays, knockdown of PYGO2 reduced FASN enhancer-promoter interactions, which could be rescued by overexpression of wild-type, but not W352A, PYGO2, even though there was equal recruitment of wild-type PYGO2 or the W352A mutant to enhancers, and no altered promoter H3K4me3 levels. Knockdown of PYGO2 curtailed the expression of canonical AR target genes TMPRSS2, KLK2, PSA, FKBP5 and NKX3-1, and overexpression of wild-type, but not W352A, PYGO2 was able to robustly rescue the induction of these genes. These data indicate that PYGO2 exerts a quantitatively important role in DHT-dependent enhancer-promoter interactions and coding target gene activation. For 220 AR-regulated coding gene promoters under regulation of an enhancer recruitment of PYGO2 was observed by ChIP followed by high-throughput sequencing (ChIP-Seq), but ligand-induced increase in the next adjacent, non-AR-regulated transcription unit (~204 promoters) was not observed; indicating that the H3K4me3 mark cannot alone be sufficient to effectively recruit PYGO2 and suggesting a role for other similarly modified proteins in prostate cancer cell gene activation events (Yang, 2013).

This study found a mechanistic link between prostate cancer-upregulated lncRNAs and AR transcriptional activity, revealing the biological importance of the lncRNAs, PRNCR1 and PCGEM1, in licensing C-terminally truncated, as well as full-length, AR-dependent gene activation events in prostate cancer cells. Considering the regulatory potential of enhancer-derived RNAs identified in recent studies, lncRNAs may also be part of a broad transcription regulatory network (Yang, 2013).

Pygopus and cancer

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for β-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. This study reports the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein is overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but is not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 is required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. It is concluded that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells (Andrews, 2007).

The Pygopus proteins are critical elements of the canonical Wnt/β-catenin transcriptional complex. In epithelial ovarian cancer, constitutively active Wnt signaling is restricted to one (endometrioid) tumor subtype. The purpose of this study was to determine the level of expression and growth requirements of human Pygopus2 (hPygo2) protein in epithelial ovarian cancer. Expression and subcellular localization of hPygo2 was determined in epithelial ovarian cancer cell lines and tumors using Northern blot, immunoblot, and immunofluorescence. Immunohistochemistry was done on 125 archived patient epithelial ovarian cancer tumors representing all epithelial ovarian cancer subtypes. T-cell factor-dependent transcription levels were determined in epithelial ovarian cancer cells using TOPflash/FOPflash in vivo assays. Phosphorothioated antisense oligonucleotides were transfected into cell lines and growth assayed by cell counting, anchorage-independent colony formation on soft agar, and xenografting into severe combined immunodeficient mice. All six epithelial ovarian cancer cell lines and 82% of the patient samples overexpressed nuclear hPygo2 compared with control cells and benign disease. Depletion of hPygo2 by antisense oligonucleotides in both Wnt-active (TOV-112D) and Wnt-inactive serous (OVCAR-3, SKOV-3) and clear cell (TOV-21G) carcinoma cell lines halted growth, assessed using tissue culture, anchorage-independent, and xenograft assays. It is concluded that hPygo2 is unexpectedly widely expressed in, and required in the absence of, Wnt signaling for malignant growth of epithelial ovarian cancer, the deadliest gynecologic malignancy. These findings strongly suggest that inhibition of hPygo2 may be of therapeutic benefit for treating this disease (Popadiuk, 2006).

date revised: 8 June 2002

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