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

Symbol Gene name - legless

Synonyms -

Cytological map position - 102B7

Function - scaffold protein

Keywords - wingless pathway

Symbol Symbol - lgs

FlyBase ID: FBgn0039907

Genetic map position -

Classification - homolog of mammalian BCL9

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | UniGene
BIOLOGICAL OVERVIEW

Wnt transduction is mediated by the association of ß-catenin (see Shaggy) with nuclear TCF DNA binding factors (see Pangolin). The products of two newly identified Drosophila segment polarity genes, legless (lgs), and pygopus (pygo: Pygopus refers to a legless lizard with scaly skin) 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).

In an attempt to identify new components of the Wnt signal transduction pathway, a screen was performed for dominant suppressors of the rough eye phenotype caused by a sevenless-wg transgene (sev-wg) that drives ectopic expression of wingless (wg) during eye development. The majority of suppressors found in this screen belong to one of three complementation groups, two of which represent alleles of the previously identified loci armadillo (arm) and pangolin (pan), which encode the Drosophila homologs of ß-catenin and TCF, respectively. However, six mutations were recessive alleles of a locus on chromosome 4 that was designated legless (lgs). In transheterozygous situations, three lgs alleles interact genetically with either arm or pan mutations, causing adult phenotypes characteristic of reduced wg activity (see below). These results suggested that lgs, like arm and pan, encodes a component of the Wg transduction pathway and raised the possibility that these components may participate in a common, critical task (Kramps, 2002).

Chromosome 4 does not permit meiotic mapping because, unlike all other Drosophila chromosomes, it fails to undergo spontaneous recombination during female meiosis. To genetically map lgs, an alternative approach was used. A series of 60 terminal deficiencies of Dp(4;Y)E were used, where Y is a chromosome that carries at its tip an extra copy of chromosome 4. These deficiencies resulted from random breaks of dicentric Dp(4;Y)E chromosomes, which were produced by Flp-mediated unequal sister-chromatid exchange. Each terminal deficiency of Dp(4;Y)E was analyzed cytologically and tested for its ability to rescue a homozygous lgs mutant genotype. Two terminal deficiencies were identified that barely differ cytologically, yet one, PE6.5, is lgs+ whereas the other, PE9.1, is lgs-. Based on these results, the lgs gene must be located at position 102B7 (Kramps, 2002).

A chromosomal walk was initiated with probes flanking P element MS209 to isolate a contiguous stretch of 150 kb DNA covering the breakpoints of both PE6.5 and PE9.1, as determined by chromosome in situ hybridization. Because the region between these two breakpoints must contain at least an essential part of the lgs gene, focus was placed on transcribed regions within this interval and their sequences were identified and compared with those derived from lgs mutant chromosomes. One transcript with an open reading frame encoding a protein of 1464 amino acids contained point mutations in five out of six lgs alleles, two of which, lgs20F and lgs7I, are nonsense mutations predicted to cause a premature termination of translation. This candidate gene must represent lgs, since a full-length cDNA driven by the ubiquitous promoter of the tubulinalpha1 gene was found to completely rescue the lethality and other phenotypes associated with homozygous lgs mutations (Kramps, 2002).

Key to the discovery of a role of lgs in the WNT pathway was a sensitized morphological reporter system for Wg signaling. lgs is required in Wg-receiving cells for the expression of Wg target genes and it encodes a nuclear protein with slight, but significant, structural similarities to human BCL9. Genetic epistasis experiments and protein-protein interaction assays indicate that Lgs and BCL9 bind to Arm and ß-catenin, respectively. The identification of Lgs permitted the isolation of Pygo, which like Lgs, is required for Wg signaling. Pygo and its human homologs bind to Lgs and BCL9, respectively. Lgs/BCL9 and the DNA binding proteins Pan/TCF can bind simultaneously to Arm/ß-catenin. Together, these findings provide the basis for postulating the existence of a nuclear multiprotein complex, in which ß-catenin ties Lgs/BCL9 and thus Pygo to Wnt-inducible promoters (Kramps, 2002).

Three sources of in vivo evidence for this model have been provided by the nature of the mutations recovered in the sensitized screen. (1) The two alleles found in the pan gene both encode proteins with N-terminal mutations that have a deleterious effect on the affinity to ß-catenin. (2) The lgs17E allele encodes a protein with a mutation in the HD2 domain that abolishes the interaction between Lgs and Arm. (3) The pygo130 allele encodes a protein lacking its PHD finger and hence the ability to bind to Lgs. In all cases, the disruption of only one of the three protein-protein interactions within the tetrapartite ß-catenin complex leads to a common outcome, namely a decrease in Wg signal response (Kramps, 2002).

All previously identified proteins that have been implicated in mediating ß-catenin-dependent activation of Wnt target genes, such as TATA binding protein (TBP; Hecht, 1999), TIP49 (Bauer, 1998), p300/CBP (Drosophila homolog: Nejire), or Brg-1 (Drosophila homolog: Brahma), have pleiotropic roles, and their specific in vivo contribution to Wnt transduction cannot be assessed by genetic means. In sharp contrast, the newly identified proteins Lgs/BCL9 and Pygo appear to represent components that are dedicated to the Wnt/Wg signaling pathway. This argument is based largely on an inability to detect lgs or pygo phenotypes that differ from those caused by reduction of Wg activity. The argument is reinforced, however, by the observation that the PHD finger of Pygo and all but HD2 of Lgs are dispensable if Pygo is allowed to directly interact with ß-catenin in the form of a hybrid Pygo[DeltaPHD]-HD2 protein (Kramps, 2002).

The activities of Pygo and Lgs are specific to the Wg signaling pathway, where they appear to be universally required to mediate all apparent Wg responses during normal Drosophila development. There is no evidence for any tissue in which Arm transduces a Wg signal in the absence of Pygo and Lgs. However, it is noted that the results do not rule out the possibility that there may be situations in mammalian systems where Pygo or BCL9 proteins are specifically absent or limiting (Kramps, 2002).

The results suggest that the primary function of Lgs/BCL9 is to recruit Pygo into the Arm/ß-catenin complex. An understanding regarding the mechanism of action of Pygo is less complete. In reporter gene assays, Pygo effectively enhances the transcription of TCF reporter genes, indicating that Pygo possesses properties of a transcriptional activator. The human and Drosophila Pygo proteins share two domains of structural similarity: the N-terminal homology domain (NHD) and the C-terminal PHD finger. The PHD finger appears to mediate the binding of Pygo to the HD1 domain of Lgs/BCL9. Hence, it is likely the NHD motif by which Pygo exerts its transcriptional activator function. While it is not yet known which proteins are targeted by the NHD, plausible candidates include TBP-associated factors (TAFs), histone acetyl transferases, and components of chromatin-remodeling complexes. A recent study suggested a role for the Brahma chromatin-remodeling complex in repressing Wg target genes, indicating that altering chromatin conformation may be an obligatory step when activating these genes. Firm answers regarding the mode of action of Pygo will have to await the identification of NHD target proteins (Kramps, 2002).

BCL9 has been identified by the translocation t(1;14)(q21;q32) from a patient with precursor-B-cell acute lymphoblastic leukemia (ALL; Willis, 1998). B-cell malignancies and, in particular, the B-cell non-Hodgkin's lymphomas are often associated with chromosomal translocations in which certain genes become overexpressed due to a juxtaposition to immunoglobulin loci. BCL9 transcript levels are normally very low in B cells, but 50-fold higher in the CEMO-1 cell line from which the translocation break point was cloned (Willis, 1998). The finding that the Drosophila homolog of BCL9, Lgs, is a component of the Wg signaling pathway raises the possibility that activation of the Wnt pathway may be causally linked to certain forms of B-cell leukemia or lymphoma (Kramps, 2002).

The formation of mature B- and T-lymphocytes and other hematopoetic lineages is guided by complex genetic and environmental cues. Some recent reports have implicated Wnt signaling in these processes. Proliferation and differentiation of CD4-CD8- double-negative thymocytes require an intact Wnt pathway. Hence, in both pro-T and pro-B cells, Wnt signals might provide important mitogenic stimuli at central developmental stages, although some of the effects of these signals may depend on inputs from additional receptor systems. Further support for the notion that deregulated Wnt signaling could play a role in blood cell cancers comes from recent findings that Wnt3A has a mitogenic effect on pro-B cells and that in pre-B cell ALL patients, Wnt 16 is overexpressed (Kramps, 2002 and references therein).

However, in Drosophila the mere overexpression of Lgs does not lead to an activation of the Wg pathway, and compared to hPYGO1 and 2, expression of BCL9 has only a mild stimulatory effect in TCF reporter assays. It is possible, though, that the expression of high BCL9 levels may render certain cell types more responsive to Wnt inputs. Hence, an additional event, such as the upregulation of Wnt expression, could create conditions in which the high levels of BCL9 cause a deregulated cellular behavior resulting in pre-B ALL. Such a scenario could explain the apparent low incidence of deregulated BCL9 expression in tumor samples (Kramps, 2002 and references therein).

An interesting observation in this respect is the finding that Jurkat T cells and normal T lymphocytes exhibit a vast difference in their responsiveness to ß-catenin. Overexpression of ß-catenin leads to a 150-fold induction of reporter gene expression in Jurkat cells but has no discernible effect in normal T cells, although both cells show a comparable response to a VP16 control. This result has been interpreted to indicate that normal T lymphocytes lack a component necessary for gene activation by nuclear ß-catenin. If such a situation occurs in vivo where the amounts of either BCL9 or Pygo are limiting, a transcriptional upregulation of the respective gene might have a significant impact (Kramps, 2002 and references therein).

The most prevalent activation of the Wnt pathway in cancer is caused by the loss of APC. Mutations in APC occur in >85% of inherited and sporadic colorectal cancers. Such mutations result in the accumulation of nuclear ß-catenin and the concomitant overexpression of Wnt target genes. The ß-catenin-TCF complex has therefore emerged as an attractive target for anticancer drugs. The discovery that Lgs/BCL9 and Pygo are required for the transcriptional activity of Arm/ß-catenin raises the possibility that the protein-protein contacts between the three components may represent additional targets for anticancer intervention. As a proof of principle the effect of disrupting one such interaction was tested on the consequences of mutations in Drosophila APC2. Embryos devoid of wild-type maternal and zygotic dAPC2 function die during embryogenesis with a naked cuticle phenotype caused by the constitutive activation of the Wg pathway. This situation is reverted to a lawn-of-denticle phenotype (loss of Wg signaling) if dAPC2DeltaS embryos are also mutant for pygo130. The Pygo130 protein lacks the C-terminal PHD finger. Hence, in dAPC2DeltaS animals, the loss of wild-type APC2 function is largely, if not completely, ineffective if Pygo can no longer interact with Lgs (Kramps, 2002).

Any drug designed to disrupt a protein-protein interaction involving ß-catenin must be highly specific and should not interfere, for example, with the binding of E-cadherin to ß-catenin. E-cadherin has properties of a tumor suppressor, and its loss has been implicated in the transition from adenoma to carcinoma. Recent structural studies indicate that E-cadherin and TCF use many of the same protein-protein contacts to bind to ß-catenin, complicating the strategy to identify substances that specifically disrupt the ß-catenin-TCF interaction. The observation that TCF and BCL9 do not compete for their binding to ß-catenin suggests that the ß-catenin-BCL9 interaction may provide an attractive alternative for therapeutic intervention (Kramps, 2002).

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).


GENE STRUCTURE

cDNA clone length - 5307

Bases in 5' UTR - 447

Exons - 6

Bases in 3' UTR - 450


PROTEIN STRUCTURE

Amino Acids - 1464

Structural Domains

The Lgs protein sequence contains neither a recognizable protein motif nor does it show sequence homologies to any other Drosophila protein. Also, no proteins encoded in the genomes of Caenorhabditis elegans, mouse, and humans were predicted to share extensive sequence similarities with Lgs. However, a few stretches of ~30 amino acids were identified in Lgs that show a statistically significant match to sequences in human and mouse BCL9 proteins (Willis, 1998). Although short, these patches of protein similarities are arranged in a colinear fashion in these proteins. These regions are referred to as homology domains 1-3 (HD1-3). Intriguingly, all three missense mutations isolated in lgs map within, or in the immediate vicinity of, HD2 (Kramps, 2002).

BCL9 was identified as the gene overexpressed in a cell line derived from a patient with precursor B cell acute lymphoblastic leukemia. It was found that a translocation caused the juxtaposition of the BCL9 gene with regulatory elements of an immunoglobulin locus (Willis, 1998). In both mouse and humans there is an additional BCL9-related gene, referred to as mLgs2 and hLGS2, respectively. No data has been reported describing the normal function of these genes (Kramps, 2002).

To explore the possibility that, despite the low degree of sequence similarity, BCL9 could represent the functional homolog of Lgs, a full-length BCL9 cDNA was assembled from human EST clones and used to generate a tubulinalpha1 promoter-driven transgene. This transgene is able to rescue viability and limb pattern of lgs17E/lgs21L animals, which normally die as pharate legless adults. Even animals homozygous for the putative null allele lgs20F, which causes larval lethality and encodes a severely truncated protein (amino acids 1-383), are rescued to adulthood, although the majority of these individuals (80%) fail to hatch from their pupal cases. Finally, the segment polarity phenotype of embryos lacking both maternal and zygotic lgs function is fully rescued by expression of BCL9. Together, these results provide convincing evidence that lgs encodes the Drosophila homolog of human BCL9 and suggest that the role of these two proteins in Wnt signaling is evolutionarily conserved (Kramps, 2002).


EVOLUTIONARY HOMOLOGS

Wnt signaling plays a crucial role in a number of developmental processes and in tumorigenesis. beta-Catenin is stabilized by Wnt signaling and associates with the TCF/LEF family of transcription factors, thereby activating transcription of Wnt target genes. Constitutive activation of beta-catenin-TCF-mediated transcription resulting from mutations in adenomatous polyposis coli (APC), beta-catenin, or Axin is believed to be a critical step in tumorigenesis among divergent types of cancers. The transactivation potential of the beta-catenin-TCF complex is enhanced by its interaction with a BCL9-like protein, B9L, in addition to BCL9. B9L is required for enhanced beta-catenin-TCF-mediated transcription in colorectal tumor cells and for beta-catenin-induced transformation of RK3E cells. Furthermore, expression of B9L is aberrantly elevated in about 43% of colorectal tumors, relative to the corresponding noncancerous tissues. These results suggest that B9L plays an important role in tumorigenesis induced by aberrant activation of Wnt signaling (Adachi, 2004).

Essential role of BCL9-2 in the switch between ß-catenin's adhesive and transcriptional functions

ß-Catenin controls both cadherin-mediated cell adhesion and activation of Wnt target genes. The ß-catenin-binding protein BCL9-2, a homolog of the human proto-oncogene product BCL9, induces epithelial-mesenchymal transitions of nontransformed cells and increases ß-catenin-dependent transcription. RNA interference of BCL9-2 in carcinoma cells induces an epithelial phenotype and translocates ß-catenin from the nucleus to the cell membrane. The switch between ß-catenin's adhesive and transcriptional functions is modulated by phosphorylation of Tyr 142 of ß-catenin, which favors BCL9-2 binding and precludes interaction with alpha-catenin. During zebrafish embryogenesis, BCL9-2 acts in the Wnt8-signaling pathway and regulates mesoderm patterning (Brembeck, 2004).

Vertebrate BCL9-2 proteins show an overall amino acid sequence identity of 60%, and 35% identity to the human proto-oncogene product BCL9. Up to 90% sequence identity was found in seven short clusters of 20-30 amino acids, which are also conserved in Legless from Drosophila: the ß-catenin-binding domain (ß-catBD), the Pygopus (PyBD)-binding domain, a domain that contains a classical nuclear localization signal (NLS, KRRK motif), three C-terminal homology domains (C-HD1 to C-HD3), and a novel N-terminal homology domain (N-HD) that contains a lysine-rich potential sumoylation motif (K*K*KXE/D; in amino acid residues 108-137) and a sequence similar to classical nuclear localization signals (PRSKRRC; in amino acids 138-173) (Brembeck, 2004).

BCL9-2 was expressed in epithelial MDCK cells and cell clones were established that had stably incorporated the expression vector. Control cells formed round compact colonies at subconfluency, and exhibited the typical cobblestone morphology of epithelial cells. In contrast, BCL9-2 expressing colonies were flattened and irregularly shaped, and contained scattered cells. Moreover, BCL9-2 expressing colonies were completely scattered in the presence of suboptimal dosages of Hepatocyte Growth Factor (HGF), which activates the receptor tyrosine kinase Met. Control clones were not scattered at these low concentrations of HGF. BCL9-2-expressing cells also exhibited a threefold increased cell migration. These results suggest that BCL9-2 induces epithelial-mesenchymal transitions, and that tyrosine phosphorylation by Met collaborates with BCL9-2 action (Brembeck, 2004).

Epithelial-mesenchymal transitions correlated with complex formation of BCL9-2 and ß-catenin in the nucleus of the scattered cells; BCL9-2 was located in the nucleus (the nucleoli were excluded) and translocated ß-catenin to the nuclear compartment, whereas ß-catenin located at the plasma membrane, but not in the nucleus in control cells. A fragment of BCL9-2 that contains only the N-terminal domain (amino acids 1-175) also located to the nucleus. In contrast, BCL9-2 with a deletion of the nuclear localization signal of the N-terminal domain (Delta amino acids 138-173) was located in the cytoplasm. Mutations of the putative sumoylation motif in the N-terminal domain of BCL9-2 (e.g., the K110R mutation) favored accumulation in nuclear bodies. The BCL9/Legless homologs, which lack the nuclear localization signal in the N-terminal domain, did not localize to the nucleus (Brembeck, 2004).

Tyrosine phosphorylation of ß-catenin also contributed to nuclear translocation. A deletion fragment of BCL9-2 that contained only the ß-catenin-binding domain (amino acids 387-530) located to the cytoplasm. Upon HGF treatment, ß-catenin was partially released from the plasma membrane, translocated to the nucleus, where it colocalized with the BCL9-2 fragment (Brembeck, 2004).

Colon cancer cell lines were used that exhibit activated Wnt/ß-catenin signaling by mutation of the tumor suppressor gene APC. SW480 cells are fibroblast-like and show high levels of nuclear ß-catenin, and they express high levels of BCL9-2. Transfection with specific siRNAs against BCL9-2 reduced endogenous BCL9-2 (but not BCL9) RNA levels almost completely within 24 h, and abolished protein expression of transfected BCL9-2 in HEK293 cells. Remarkably, the morphology of the BCL9-2 siRNAs-treated SW480 cells became epithelial-like, and ß-catenin was translocated from the nucleus to the cell membrane. Moreover, cell migration of the treated SW480 cells was drastically reduced. BCL9-2 siRNA's treatment of DLD-1 colon cancer cells also induced loss of nuclear ß-catenin, and the cells showed reduced colony formation in soft agar (Brembeck, 2004).

BCL9-2 was initially identified as an interaction partner of tyrosine-phosphorylated ß-catenin in a yeast two-hybrid screen. BCL9-2 interacted efficiently with ß-catenin in yeast when the armadillo domains were fused to kinase-active, but not to kinase-defective Met. Armadillo repeats 1 and 2 of ß-catenin are required to bind BCL9-2, which contains only one tyrosine residue at position 142. Mutation of Y142 to alanine indeed abrogates BCL9-2 binding, whereas mutation to glutamic acid (Y142E), which can mimic tyrosine phosphorylation, increases binding efficiency (Brembeck, 2004).

The presence of a nonphosphorylated Tyr 142 in ß-catenin is required for alpha-catenin binding. The mutations Y142A and Y142E both abrogated binding to alpha-catenin. Interaction of BCL9-2 with tyrosine-phosphorylated ß-catenin was confirmed by coimmunoprecipitation from tissue culture cells. HGF treatment of cells induced tyrosine phosphorylation of ß-catenin and promoted interaction with BCL9-2, as did the Y142E mutation of ß-catenin in GST pull-down experiments. In contrast, a strongly reduced binding between ß-catenin and alpha-catenin was observed when the Y142A and Y142E mutations were present in ß-catenin (Brembeck, 2004).

HEK293 cells, which contain low levels of endogenous BCL9-2 and ß-catenin, were used for testing the transcriptional activity of BCL9-2. Full-size BCL9-2 enhanced ß-catenin-dependent transcription threefold. BCL9-2 had no effect in the absence of ß-catenin. Moreover, mutation of the conserved lysines in the potential sumoylation motif of the N-terminal domain of BCL9-2 (K108R, K110R, or K112R) activated ß-catenin-dependent transcription up to ninefold. Remarkably, expression of the BCL9-2 fragment that contains the ß-catenin-binding domain only (amino acids 387-530) and does not affect binding of ß-catenin to Lef/Tcfs abolished ß-catenin-dependent transcription. Deletion of the nuclear localization signal in the N-terminal domain of BCL9-2 (Delta amino acids 138-173) abolished transcriptional activation. Internal deletion of the previously identified Pygopus-binding domain of BCL9 proteins showed that this domain is not required in BCL9-2 to potentiate ß-catenin's transcriptional activity. BCL9/Legless stimulated transcription to a similar degree, but this activation was dependent on the Pygopus-binding domain. BCL9-2 expression increased ß-catenin-dependent transcription also in other assays, as assessed for the expression levels of the Wnt target gene conductin. BCL9-2 siRNA treatment of SW480 and DLD-1 cells significantly reduced transcriptional activity of ß-catenin. The coactivator function of BCL9-2 depended on Y142 of ß-catenin; in the presence of ß-catenin that contains a Y142A mutation, BCL9-2 had a reduced effect on transcription . alpha-Catenin blocked ß-catenin's transcriptional activity. This inhibition was overcome by cotransfected BCL9-2, but only in the presence of wild-type and not of Y142A mutant ß-catenin. Tyrosine phosphorylation of ß-catenin was forced by HGF treatment of cells and by cotransfection of the receptor tyrosine kinase trk-Met. BCL9-2 cofactor function was increased, but only in the case of wild-type, and not Y142A mutant ß-catenin (Brembeck, 2004).

In zebrafish embryos, BCL9-2 and BCL9 mRNAs are contributed maternally and are highly expressed during gastrulation. Injection of antisense BCL9-2 morpholinos (MOs) into zebrafish embryos resulted in severe defects of trunk and tail developmental. BCL9-2 MOs directed against the start codon were highly effective at minimal dosages. The mutant phenotype was observed in 80% of the cases. In contrast, different MOs against the homolog BCL9/Legless had no effect. Moreover, coinjection of BCL9-2 and BCL9 MOs did not alterate the developmental defects induced by BCL9-2 MOs alone. The loss-of-function of BCL9-2 suggested a deficit in Wnt8 signaling. At 70% epiboly of wild-type embryos, floating head was expressed in the dorsal axial mesoderm, and tbx6 in the ventro-lateral mesoderm. After injections of BCL9-2 MOs, the expression of floating head was broadened, and tbx6 expression was lost. Moreover, injection of low concentrations of MOs against BCL9-2 and Wnt8 synergized to suppress tbx6 expression in the ventro-lateral mesoderm. No change of expression of pax2.1 or otx2 in the neuroectoderm was observed. Loss of tbx6 expression induced by BCL9-2 MOs was completely rescued by mouse BCL9-2 mRNA, indicating that the MOs specifically target zebrafish BCL9-2. Injection of mouse BCL9-2 mRNA alone expanded tbx6 expression. Injection of BCL9-2 mRNA that encodes a fragment lacking the N-terminal domain did not expand tbx6 expression, nor did such injection rescue the phenotypes of BCL9-2 MOs. The epistasic relationship between Wnt8, Diversin (a negative regulator of ß-catenin signaling and BCL9-2 was examined in zebrafish embryos. Injection of Wnt8 DNA or stabilization of ß-catenin by injection of Diversin MOs lead to an expansions of tbx6 expression. This was completely blocked by coinjection of BCL9-2 MOs (Brembeck, 2004).

Epithelial-mesenchymal transitions occur during critical phases of embryonic development. Such transitions are also observed late in the progression of carcinomas and provide a possible metastatic mechanism. Several signaling systems can induce epithelial-mesenchymal transitions, such as Wnt/ß-catenin, TGFß/BMPs, or tyrosine kinases. Epithelial-mesenchymal transitions are initiated by a breakdown of the E-cadherin/ß-catenin/alpha-catenin complex at the plasma membrane and a dissociation of this adhesive complex from the cytoskeleton, which can be induced by tyrosine phosphorylation of ß-catenin. This study demonstrates that BCL9-2 forms a complex with ß-catenin that is phosphorylated at Tyr 142, which precludes interaction with alpha-catenin. Phosphorylation of Tyr 142 of ß-catenin and interaction with BCL9-2 allows location of the complex in the nucleus and increases transcription of Wnt/ß-catenin target genes. Thus, BCL9-2 appears to contribute to oncogenicity by two mechanisms: (1) interfering with cadherins, which act as tumor suppressor genes, and (2) by increased signaling of family members of the Wnt pathway, which contains many oncogenes and tumor suppressor genes (Brembeck, 2004).

During vertebrate embryogenesis, Wnt/ß-catenin signals control formation of the dorso-anterior axis and patterning of the mesoderm at early stages, and organ specification at later stages of development. In zebrafish embryos, Wnt8/ß-catenin signaling is required to pattern ventro-lateral mesoderm and to posteriorize neural ectoderm. This study has shown that BCL9-2, but not the homolog BCL9/Legless, is essential for mesoderm patterning in early zebrafish embryogenesis, and that BCL9-2 acts downstream of Wnt8/ß-catenin. However, BCL9-2 does not appear to be required for other early functions of the canonical Wnt pathway, such as formation of the dorsal organizer or posteriorization of anterior neuroectoderm. These data therefore suggest that BCL9-2 acts as a specific modulator of canonical Wnt signaling at particular developmental stages, rather than as a general component of Wnt signaling (Brembeck, 2004).

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 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).

LATS2 suppresses oncogenic Wnt signaling by disrupting beta-catenin/BCL9 interaction

Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. This study reports that LATS2 inhibits oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacts with β-catenin and is present on Wnt target gene promoters. Mechanistically, LATS2 inhibits the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 is downregulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an antimicrotubule drug, potently induces LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. These results suggest that LATS2 is not only a key tumor suppressor in human cancer but may also be an important target for anticancer therapy (Li, 2013).


legless:
Regulation | Developmental Biology | Effects of Mutation | References

date revised: 30 January 2005

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.