The Wnt pathway is an evolutionarily conserved cell signaling cascade that acts as major driving force of several developmental processes, as well as for the maintenance of the stem cell populations within adult tissues (Nusse and Clevers, 2017). Deregulation of this signaling pathway results in a spectrum of consequences, ranging from lethal developmental abnormalities to several forms of aggressive cancer (Nusse and Clevers, 2017). Most prominently, colorectal cancer (CRC) is initiated by genetic mutations that constitutively activate Wnt signaling (Kahn, 2014).

Secreted WNT ligands trigger an intracellular biochemical cascade in the receiving cells that culminates in the calibrated expression of target genes (Mosimann et al., 2009). This transcriptional response is orchestrated by nuclear β-catenin, that acts as a ‘scaffold’ to buttress a host of co-factors to cis-regulatory elements occupied by the TCF/LEF transcription factors (Valenta et al., 2012). Among the co-factors, the two paralogs BCL9 and BCL9L (referred to as BCL9/9L) and PYGO1/2 proteins reside within the Wnt/β-catenin transcriptional complex, and their concerted action is required to efficiently activate Wnt-target gene expression (Kramps et al., 2002; Parker et al., 2002; van Tienen et al., 2017; Figure 1A). During vertebrate development, their requirement in the β-catenin-mediated transcription appears to be context-dependent (Cantù et al., 2018; Li et al., 2007), and they also have evolved β-catenin-independent functions (Cantù et al., 2017; Cantù et al., 2014). Curiously however, BCL9 and PYGO always seem to act as a ‘duet’ (Kennedy et al., 2010).

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Importantly, BCL9/9L and PYGO proteins were found to significantly contribute to the malignant traits typical of Wnt-induced CRCs (Deka et al., 2010; Gay et al., 2019; Jiang et al., 2020; Mani et al., 2009; Mieszczanek et al., 2019; Moor et al., 2015; Talla and Brembeck, 2016). These observations provided impetus to consider the BCL9/PYGO axis as relevant ‘targetable’ unit in CRC (Lyou et al., 2017; Mieszczanek et al., 2019; Talla and Brembeck, 2016; Zimmerli et al., 2017).

However, here we noticed an apparent divergence between the roles of BCL9/9L and PYGO proteins. We found that genetic abrogation of Bcl9/9l in mouse CRC cells results in broader consequences than Pygo1/2 deletion, suggesting that BCL9 function does not entirely depend on PYGO1/2. Among the putative β-catenin/BCL9 interactors we identified the developmental transcription factor TBX3. Intriguingly, we show that also during forelimb development, BCL9/9L possess a PYGO-independent role. In this in vivo context, TBX3 occupies β-catenin/BCL9 target loci genome-wide, and mutations in Bcl9/9l affect the expression of TBX3 targets. Finally, TBX3 modulates the expression of Wnt target genes in a β-catenin- and TCF/LEF-dependent manner, and increases the metastatic potential of human CRC cells when overexpressed. We conclude that TBX3 can assist the Wnt/β-catenin mediated transcription in selected developmental contexts, and that this partnership could be aberrantly reactivated in some forms of Wnt-driven CRCs.

We induced intestinal epithelium-specific recombination of Pygo1/2 loxP alleles (Pygo1/2-KO), that efficiently deleted these genes in the whole epithelium, including the stem cells compartment (Figure 1—figure supplement 1A and B). Consistently with recent reports (Mieszczanek et al., 2019; Talla and Brembeck, 2016), and similarly to deletion of Bcl9/9l (Deka et al., 2010; Mani et al., 2009; Moor et al., 2015), Pygo1/2-KO displayed no overt phenotypic defects (Figure 1B; Figure 1—figure supplement 1C–E). We were surprised in noticing that the expression of Lgr5, the most important intestinal stem cell marker and Wnt target gene (Barker et al., 2007), was heavily downregulated upon loss of Bcl9/9l but unaffected in Pygo1/2-KO (Figure 1C). To address the functionality of the stem cell compartment in these two conditions, we subjected both Bcl9/9l and Pygo1/2 compound mutants (KO) to a model of intestinal regeneration by DSS treatment (Kim et al., 2012; Figure 1D). While Bcl9/9l-KO mice showed a defect in regeneration after insult (Deka et al., 2010), Pygo1/2-KO proved indifferent when compared to control littermates (Figure 1D). While we cannot exclude that PYGO1/2 also contributes to the Wnt/β-catenin-dependent transcriptional regulation, our results highlight that the BCL9/9L function in the intestinal epithelium homeostasis and regeneration does not entirely depend on PYGO1/2. This was surprising, since BCL9/9L proteins were thought to act as mere ‘bridge’ proteins that tethered PYGO to the β-catenin transcriptional complex (Figure 1A; Fiedler et al., 2015; Mosimann et al., 2009). Both BCL9 and PYGO proteins have been implicated in colorectal carcinogenesis (Gay et al., 2019; Jiang et al., 2020; Mieszczanek et al., 2019; Talla and Brembeck, 2016). We tested if the consequence of the deletion of Bcl9/9l and Pygo1/2 genes was also different in the context of carcinogenesis. Specifically, we looked at the contribution to gene expression in chemically-induced AOM/DSS colorectal tumors (Figure 1E). As previously observed, Bcl9/9l-KO tumors exhibit a massive decrease in Wnt target gene expression, epithelial-to-mesenchymal transition (EMT) and stemness traits (Deka et al., 2010; Moor et al., 2015), which was not observed in Pygo1/2-KO tumors (Figure 1E, Figure 1—figure supplement 2A and B). This phenotypic difference is consistent with a recent study in which Bcl9/9l but not Pygo1/2 loss reduced the activation of Wnt target genes induced by APC loss-of-function (Mieszczanek et al., 2019); we interpret this as an independent validation of our observation. All these experiments open up the question of how BCL9/9L imposes its function independently of PYGO.

Surprisingly, the intestine-specific deletion of the homology domain 1 (HD1) of BCL9/9L (Figure 2A), that was previously annotated to interact only with PYGO1/2 (Cantù et al., 2014; Kramps et al., 2002), i) suppressed the metastatic phenotype of the AOM/DSS tumors while deletion of Pygo1/2 did not (Figure 2B) and ii) induced a strong downregulation of Wnt target, EMT and stemness genes (Figure 2C, Figure 2—figure supplement 1). The discrepancy between the gene expression changes induced by recombining Pygo1/2 or deleting the HD1 domain of Bcl9/9l implies that currently unknown proteins assist BCL9/9L function. We set out to identify new candidate BCL9 partners that might be responsible for the different phenotypes. To this aim, we performed a pull-down of tumor proteins expressing either a full-length or a HD1-deleted variant of BCL9, followed by mass spectrometry (Figure 2D). Among the proteins differentially pulled down by control but not by mutant BCL9 we detected TBX3 (Figure 2D and E) and selected it for further validation. The in vivo deletion of the HD1 domain (in Bcl9/9l-ΔHD1 embryos) leads to severe forelimb malformations, while Pygo1/2-KO embryonic forelimbs are unaffected (Figure 2F)(also see Schwab et al., 2007). Limb development, thus, represents another context where BCL9/9L appear to act independently of PYGO. Of note, TBX3 plays a fundamental role in the development of this structure (Frank et al., 2013).

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We confirmed cytological vicinity between transfected tagged versions of BCL9 and TBX3 by proximity ligation assay (PLA) (Figure 2—figure supplement 2A,B). However, overexpression-based in vitro co-immunoprecipitation experiments could not detect any stable interaction between these two proteins, suggesting absence of direct binding or a significantly lower affinity than that between BCL9 and PYGO (Figure 2—figure supplement 2C). Hence, we aimed at testing the functional association between TBX3 and BCL9 in a more relevant in vivo context. To this aim, we collected ca. 500 forelimbs from 10.5 dpc wild-type mouse embryos and subjected the crosslinked chromatin to immunoprecipitation using antibodies against BCL9 (Salazar et al., 2019) or TBX3, followed by deep-sequencing of the purified DNA (ChIP-seq, Figure 3A). By using stringent statistical parameters, and filtering with Irreproducible Discovery Rate (IDR), we extracted a list of high confidence BCL9 and TBX3 peaks (Figure 3B and C). Surprisingly, we discovered that BCL9 occupies a large fraction (ca. 2/3^rd) of the TBX3-bound regions (Figure 3D). Suggestive of a role for TBX3 within the Wnt-dependent transcriptional apparatus, motif analysis of the common TBX3-BCL9 target loci identified statistical prevalence for TCF/LEF and Homeobox transcription factor consensus sequences, but not for any TBX transcription factor (Figure 3E). This suggests that TBX3 interacts with the DNA in these locations via affinity to the Wnt/β-catenin co-factors rather than via direct contact with DNA. Accordingly, TBX-specific motifs were detected within the group of TBX3 exclusive peaks (which do not display BCL9 binding, Figure 3—figure supplement 1). Notably, TBX3 and BCL9 occupancy was detected at virtually all previously described Wnt-responsive-elements (WRE) within known Wnt target genes (Figure 3F).

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To test whether the in vivo abrogation of the simultaneous interactions mediated by BCL9/9L would influence the expression of genes associated with TBX3 peaks, we set out to mutate the Bcl9/9l interaction domains, while leaving TBX3 protein unaffected. We combined different Bcl9/9l alleles in which the HD2 (β-catenin-interacting) and HD1 (PYGO/new co-factor-interacting) motifs are deleted (Cantù et al., 2018). Double heterozygous animals for the HD1 (Bcl9^ΔHD1/+; Bcl9l^ΔHD1/+) or the HD2 (Bcl9^ΔHD2/+; Bcl9l^ΔHD2/+) deletions are viable and fertile. The cross between them leads to a trans-heterozygous genetic configuration in which both domain deletions are present (Bcl9^ΔHD1/ΔHD2; Bcl9l^ΔHD1/ΔHD2, referred to as Bcl9/9l-Δ1/Δ2, Figure 1A). As in these mice BCL9/9L retain both the HD1 and the HD2 domains in heterozygosity, this allelic combination is a way of testing the consequences of abrogating the tripartite complex mediated by the two interacting motifs of BCL9/9L without causing a full loss-of-function of these proteins. Bcl9/9l-Δ1/Δ2 embryos also display forelimb malformations, the cause of which cannot be due to PYGO (Figure 2F) but must be caused by the failure of recruiting the new HD1-interacting partner by BCL9/9L onto the β-catenin transcriptional complex. We collected forelimbs from control and Bcl9/9l-Δ1/Δ2 mutant embryos at 10.5, and measured gene expression via RNA-seq (Figure 3G). We found a significant enrichment (Hypergeometric test, p=1.4e-6) of TBX3 targets among the genes differentially expressed in Bcl9/9l-Δ1/Δ2 mutants (Figure 3H). The enrichment was particularly significant when considering down-regulated genes in Bcl9/9l-Δ1/Δ2 mutants, indicating that the BCL9-TBX3 partnership sustains transcriptional activation (Figure 3H). Of note, the design of our experiment directly implicates that these TBX3 transcriptional targets are also β-catenin-dependent. The overlap list includes several regulators of limb development, such as Meis2 (Capdevila et al., 1999), Irx3 (Li et al., 2014) and Eya2 (Grifone et al., 2007; Figure 3H, heat map on the right). Despite being of correlative nature, this analysis supports a model in which BCL9/9L and TBX3 cooperate to the activation of target genes.

So far, we have presented genetic evidence that BCL9 proteins require additional co-factors, and that TBX3 associates with the β-catenin/BCL9 bound regions on the genome possibly influencing the expression of target genes. However, the similarity of genomic binding profiles between TBX3 and BCL9 might be due to their binding in different cells, and the decreased expression of genes with nearby enhancers bound by BCL9 and TBX3 might imply a requirement for BCL9/9L, but not necessarily for TBX3. We reasoned that our hypothesis - in which BCL9 functionally tethers TBX3 to the β-catenin transcriptional complex - raises several testable predictions that will be addressed below.

First, our model implies that TBX3 could impact on Wnt target gene expression and its activity should be dependent on the main constituents of the Wnt/β-catenin transcriptional complex. Second, if TBX3 is tethered by BCL9 to its targets, mutations in BCL9/9L should influence the ability of TBX3 to physically associate with WREs. Finally, as for BCL9, TBX3 should be capable of enhancing the metastatic potential of colorectal cancer cells.

To test our first prediction, implying a potential role of TBX3 in the transcription of Wnt target genes, we overexpressed it in HEK293T cells and monitored the activation status of Wnt signaling using the transcriptional reporter SuperTopFlash (STF). Consistent with its role as repressor, TBX3 led to a moderate but significant transcriptional downregulation that was, importantly, specific to the STF but not the control reporter plasmid (Figure 4A). Upon Wnt signaling activation achieved via GSK3 inhibition, TBX3-overexpressing cells exhibited a markedly increased reporter activity when compared to control cells, in particular at non-saturating pathway stimulating conditions (Figure 4A, left panel). Importantly, TBX3 proved transcriptionally incompetent on the STF if the cells carried mutations in TCF/LEF (Δ4TCF) or CTNNB1 (encoding for β-catenin, Δβ-CAT; Doumpas et al., 2019), strongly supporting the notion of its cell-autonomous involvement in the activation of canonical Wnt target gene transcription (Figure 4A, central and right panels, respectively). Endogenous Wnt targets showed a similar expression behaviour to that of STF upon TBX3 overexpression (Figure 4—figure supplement 1). While our experiments show that TBX3 can influence the expression of Wnt target genes, the mechanisms by which this occurs remain to be elucidated.

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We then addressed our second hypothesis, in which BCL9/9L are required for tethering TBX3 onto WREs. We performed ChIP of TBX3 in HEK293T cells followed by quantitative PCR to detect enrichment on the WRE present in the AXIN2 promoter (Jho et al., 2002). Consistent with an effect on transcription in the absence as well as in the presence of Chir99021 (Chir) (Figure 4A), TBX3 was bound to this region both in ‘OFF’ and in ‘ON’ conditions (Figure 4B). We then exploited a HEK293T clone devoid of both BCL9 and BCL9L (ΔB9/9L; van Tienen et al., 2017), and tested if TBX3 was capable of physical association with the WRE. Of note, enrichment of TBX3 in ΔB9/9 L cells was dramatically reduced to background levels (Figure 4B). While we cannot exclude that TBX3 might act independently of BCL9/9L on several of its targets, this observation supports the notion that BCL9/9L are responsible for TBX3 recruitment on classical WREs (Figure 4C). This also suggests that the previously identified targets of both BCL9 and TBX3 (Figure 3D–F) must display simultaneous co-occupancy of these two factors. In agreement with the notion that BCL9/9L are themselves recruited by the TCF-β-catenin axis, the physical association of TBX3 with the AXIN2 promoter was also lost in Δ4TCF and Δβ-CAT cells (Figure 4B).

Finally, we evaluated the effects of TBX3 overexpression (OE) on growth and metastatic potential of HCT116 human colorectal tumor cells – a representative model of CRC driven by activating mutations in CTNNB1 (Mouradov et al., 2014) –, using a in vivo zebrafish xenograft model (Rouhi et al., 2010). Approximately 200–500 labelled control or TBX3-OE HCT116 cells were implanted in the perivitelline space of 72 hours post-fertilization (hpf) zebrafish embryos (Figure 4D). Three days after injection, TBX3-OE cells displayed a marked increase in number in the caudal hematopoietic plexus (Figure 4E–F), the main metastatic site for cells migrating from the perivitelline space (Rouhi et al., 2010). Of note, TBX3-OE HCT116 cells maintained consistently high expression of TBX3 within fish embryos throughout the experiment, and this was accompanied by increased Wnt/β-catenin-dependent transcription, as measured by AXIN2 expression (Figure 4G). While this experiment does not allow to exclude that TBX3 might also act independently of BCL9/β-catenin in this context, it shows that increased expression of TBX3 enhances proliferation and migratory capability of human CRC cells bearing constitutively active Wnt signaling, and this is associated with simultaneous enhancement of the Wnt/β-catenin-dependent transcription (Figure 4G).

Taken together, our experiments show that, in specific developmental and disease contexts, the transcription factor TBX3 can take active part in the direct regulation of Wnt target genes by functional interplay with the β-catenin/BCL9-dependent transcriptional complex. Our study suggests a new paradigm in which tissue-specific co-factors might be the key to understand the spectrum of possible transcriptional outputs observed downstream of Wnt/β-catenin signaling (Nakamura et al., 2016). Moreover, TBX3 has been linked to different cancer types (Willmer et al., 2017). Our observations suggest that TBX3, or its downstream effectors, could be considered as new relevant targets to dampen CRC progression.

The ChIP-seq data have been deposited at ArrayExpress with accession number E-MTAB-8997. The RNA-seq experiment has been deposited at ArrayExpress with accession number E-MTAB-9000.

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Author details

1. Dario Zimmerli

   Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

   Present address

   Division of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, Netherlands

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Costanza Borrelli and Amaia Jauregi-Miguel

   Competing interests

   No competing interests declared

2. Costanza Borrelli

   Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Dario Zimmerli and Amaia Jauregi-Miguel

   Competing interests

   No competing interests declared

3. Amaia Jauregi-Miguel

   1. Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden
   2. Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology; Faculty of Medicine and Health Sciences; Linköping University, Linköping, Sweden

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Dario Zimmerli and Costanza Borrelli

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0938-7734

4. Simon Söderholm

   1. Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden
   2. Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology; Faculty of Medicine and Health Sciences; Linköping University, Linköping, Sweden

   Contribution

   Resources, Data curation, Software, Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5350-7102

5. Salome Brütsch

   Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

6. Nikolaos Doumpas

   Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

7. Jan Reichmuth

   Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

   Contribution

   Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

8. Fabienne Murphy-Seiler

   Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, Lausanne, Switzerland

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

9. MIchel Aguet

   Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL), School of Life Sciences, Lausanne, Switzerland

   Contribution

   Data curation, Supervision, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

10. Konrad Basler

    Department of Molecular Life Sciences, University of Zurich, Zürich, Switzerland

    Contribution

    Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing - review and editing

    For correspondence

    konrad.basler@imls.uzh.ch

    Competing interests

    No competing interests declared

11. Andreas E Moor

    Department of Biosystems Science and Engineering, ETH Zürich, Basel, Switzerland

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    andreas.moor@bsse.ethz.ch

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8715-8449

12. Claudio Cantù

    1. Wallenberg Centre for Molecular Medicine, Linköping University, Linköping, Sweden
    2. Department of Biomedical and Clinical Sciences, Division of Molecular Medicine and Virology; Faculty of Medicine and Health Sciences; Linköping University, Linköping, Sweden

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    claudio.cantu@liu.se

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1547-5415

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