TBX3 acts as tissue-specific component of the Wnt/β-catenin enhanceosome

BCL9 and PYGO are β-catenin cofactors that enhance the transcription of Wnt target genes. They have been proposed as therapeutic targets to diminish Wnt signalling output in intestinal malignancies. Here we find that, in colorectal cancer cells and in developing mouse forelimbs, BCL9 proteins sustain the action of β-catenin in a largely PYGO-independent manner. Our genetic analyses implied that BCL9 necessitates other interaction partners in mediating its transcriptional output. We identified the transcription factor TBX3 as a candidate tissue-specific member of the β-catenin transcriptional complex. In developing forelimbs, TBX3 and BCL9 co-occupy a large number of Wnt-responsive regulatory elements, genome-wide. Moreover, mutations in Bcl9 affect the expression of TBX3 targets in vivo, and modulation of TBX3 abundance impacts on Wnt target genes transcription in a β-catenin- and TCF/LEF-dependent manner. Finally, TBX3 overexpression exacerbates the metastatic potential of Wnt-dependent human colorectal cancer cells. Our work implicates TBX3 as a new, context-dependent component of the Wnt/β-catenin-dependent enhanceosome.


Introduction
The Wnt pathway is an evolutionarily conserved cell signalling 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 signalling 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 signalling (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 so-called Wnt enhanceosome, 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 b-catenin-mediated transcription appears to be context-dependent (Cantù et al., 2018;Li et al., 2007), and they also have evolved b-catenin-independent functions (Cantù et al., , 2014. Curiously however, BCL9 and PYGO always seem to act as a "duet" (Kennedy et al., 2010).
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 b-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 co-occupies b-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 b-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/b-catenin mediated transcription in selected developmental contexts, and that this partnership could be aberrantly reactivated in some forms of Wnt-driven CRCs.

Results and discussion
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 supplement 1A and 1B). 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 not overt phenotypic defects (Figure 1B and Figure supplement 1C). 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 have subtle transcriptional contribution, 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 b-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, EMT and stemness traits (Deka et al., 2010;Moor et al., 2015), which was not observed in 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), induced i) beneficial phenotypic changes that are not observed upon deletion of Pygo1/2 ( Figure 2B) and ii) a strong downregulation of Wnt target, EMT and stemness genes ( Figure 2C, figure supplement 2C). 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 2E) and selected it for further validation. TBX3 appeared as the most interesting candidate, since the malformations induced by Bcl9/9l loss-of-function are strikingly similar to those induced by Tbx3 loss (Frank et al., 2013). Indeed, the in vivo deletion of the HD1 domain (in Bcl9/9l-DHD1 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.
We confirmed cytological vicinity between transfected tagged versions of BCL9 and TBX3 by proximity ligation assay (PLA) (Figure supplement 3A). 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 supplement 3B). 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 3C).
Surprisingly, we discovered that BCL9 co-occupies a large fraction (ca. 2/3 rd ) of the TBX3-bound regions ( Figure 3D). Suggestive of a role for TBX3 within the Wntdependent transcriptional apparatus, motif analysis of the co-occupied loci identified statistical prevalence for TCF/LEF and Homeobox transcription factors consensus sequences, but not that for any TBX transcription factor ( Figure E). This suggests that TBX3 interacts with the DNA in these locations via affinity to the Wnt enhanceosome rather than via direct contact with DNA. Accordingly, TBX-specific motifs were detected within the group of TBX3 exclusive peaks (which are not co-occupied by BCL9, Figure   supplement 4). Notably, TBX3 and BCL9 co-occupancy was detected at virtually all previously described Wnt-responsive-elements (WRE) within known Wnt target genes ( Figure 3F).
So far, we have presented genetic evidence that BCL9 proteins require additional cofactors, and that TBX3 associates with the b-catenin/BCL9 bound regions on the genome. We reasoned that our hypothesis -in which BCL9 functionally tethers TBX3 to the b-catenin transcriptional complex -raises several testable predictions that will be addressed below. First, if TBX3 is physically tethered by BCL9 on its targets, mutations in Bcl9/9l should influence the expression of genes associated with TBX3 peaks. Second, our model implies that TBX3 could impact on Wnt target genes expression and its activity should be dependent on the presence of both b-catenin and TCF/LEF. Finally, as for BCL9, TBX3 should be capable of enhancing the metastatic potential of colorectal cancer cells.
We then addressed our second prediction implying a potential role of TBX3 in the transcription of Wnt target genes. We overexpressed it into HEK293T cells and monitored the activation status of Wnt signalling 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 4C). Upon Wnt signalling 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 4C). Importantly, TBX3 proved transcriptionally incompetent on the STF if the cells carried mutations in TCF/LEF or CTNNB1 (encoding for b-catenin; Doumpas et al., 2018), strongly supporting the notion of its cell-autonomous involvement in the activation of canonical Wnt target gene transcription ( Figure 4C, central and right panels, respectively). Endogenous Wnt targets showed a similar expression behaviour to that of STF upon TBX3 overexpression (Figure supplement 5). Consistently, TBX3 was bound to Axin2 promoter both in "OFF" and in "ON" conditions ( Figure 4D).
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 4E). Three days after injection, TBX3-OE cells displayed a marked increase in number, both at the injection site and in the caudal hematopoietic plexus ( Figure 4F and 4G), the main metastatic site for cells migrating from the perivitelline space (Rouhi et al., 2010). This indicated that TBX3 increases proliferation and migratory capability of human CRC cells bearing a constitutively active Wnt signalling.
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 b-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/b-catenin signalling. 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.
Induction of tumors: 6-8-week-old female mice were treated with five tamoxifen injections (i.p., 1mg/day) for five consecutive days. 10 days later they were injected i.p. with 44mg/kg body weight DMH 2HCl (N,N' Dimethylhydrazine dihydrochloride). After another 7 days later 2% DSS was administered, ad libitum, in the drinking water for 7 days.
Mice were monitored clinically for rectal bleeding, prolapse and general signs of morbidity, including hunched posture, apathetic behavior and failure to groom.
The relative body weight (in %) was calculated as follows: 100 X weight at a certain day / weight at the first day of DSS treatment. Epithelial damage of DSS treated mice was defined as percentage of distal colon devoid of epithelium.

Real-time PCR genotyping
To determine the deletion rate, the intestinal epithelium was separated from the underlining muscle. The intestine was dissected, flushed with PBS, cut open longitudinally and incubated in 3mM ethylenediamine tetraacetic acid (EDTA) and 0.05mM dithiothreitol (DTT) in PBS for 1.5h at RT on a rotor. The tubes were shaken vigorously, the muscle removed, and the epithelium centrifuged and used for genomic DNA extraction. SYBR green real-time PCR assays were performed on each sample analyzed. Genotyping primers are available upon request.

Chromatin Immunoprecipitation
Forelimb buds were manually dissected from ca. 250 RjOrl:SWISS outbred 10.5 dpc mouse embryos. Chromatin immunoprecipitation was performed as previously described (Cantù et al., 2018). Briefly, the tissue was dissociated to a single cell suspension with collagenase (1 ug/ml in PBS) for 1 hr at 37° C, washed and crosslinked in 20 ml PBS for 40 min with the addition of 1.5 mM ethylene glycol-bis(succinimidyl succinate) (Thermo Scientific, Waltham, MA, USA), for protein-protein crosslinking (Salazar et al., 2019), and 1% formaldehyde for the last 20 min of incubation, to preserve DNA-protein interactions. The reaction was blocked with glycine and the cells were subsequently lysed in 1 ml HEPES buffer (0.3% SDS, 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES). Chromatin was sheared using Covaris S2 (Covaris, Woburn, MA, USA) for 8 min with the following set up: duty cycle: max, intensity: max, cycles/burst: max, mode: Power Tracking. The Data analysis: Overall sequencing quality of the acquired fastq files was assessed using FastQC (version 0.11.5). Because all the samples exhibited good quality (MAPQ > 30) and had no adapter contamination > 0.1% trimming of reads was not deemed necessary. In addition, test alignments against several reference genomes were done using the FastQ Screen tool (version 0.13.0). Reference genomes were downloaded from UCSC (http://hgdownload.cse.ucsc.edu/goldenpath/mm10/bigZips/). Quality results were summarized using MultiQC (version 1.7). Fastq files were mapped to mouse reference genome (mm10) using the read aligner Bowtie2 (version 2.3.4.1).
The resulting alignment files were then adjusted (conversion to binary format, removal of read aligned to mitochondrial DNA and indexing) using SamTools (version 1.9). To identify genomic regions enriched with aligned reads the peak calling tool MACS2 (version 2.2.6) was used. Calculated p-values were adjusted for false discovery rate (FDR) using Benjamini-Hochberg procedure, generating q-values. A cutoff q < 0.05 was used to assess significance. IgG sample were used as enrichment-normalization control. MACS2 generated peak files were further filtered by removal of blacklisted regions according to the ENCODE project using bedtools (version 2.26.0). Annotation and visualization were made with the R programming language (version 3.4.4) and Rstudio (version 1.1.463), using R packages ChIPpeakAnno (version 3.12.7), ChIPseeker (version 1.14.2) and ggplot2 (version 3.2.1). CIRCOS plots was produced using the R package Circlize (version 0.4.8) and genomic track visualization was done with Integrative Genomics Viewer (IGV) (version 2.4.17). Motif analysis was performed with HOMER. The data have been deposited at ArrayExpress with accession number E-MTAB-8997.

RNA-seq data analysis
Quality control of fastq files were done using FastQC (version 0.11.5). Trimming of reads to remove adapter remnants and low quality read (MAPQ < 30) was performed with BBDuk, part of the BBMap suite (version 38.58). Test alignments against different reference genomes were done with the FastQ Screen tool (version 0.13.0).
Reference genome data (FASTA and GTF) were downloaded from GENCODE, release M24 (https://www.gencodegenes.org/mouse/). Annotation and visualization were made with the R programming language (version 3.4.4) and Rstudio (version 1.1.463). Hierarchical clustered heatmap was produced with the pheatmap R package, using Ward's Hierarchical Agglomerative Clustering Method. A complete list of bioinformatic tools and references is listed in the accompanying Supplementary Table. The RNA-seq experiment has been deposited at ArrayExpress with accession number E-MTAB-9000.

Protein Immunoprecipitation and Mass Spectrometry
Dissected mouse tumors were minced in cold PBS and treated with a hypotonic lysis buffer (20 mM tris-HCl, 75 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 0.5% NP-40, and 5% glycerol). Protein extracts obtained were incubated with 1 μg of anti-BCL9 antibody (ab54833 or ab37305, Abcam) and protein A-conjugated Sepharose beads (GE Healthcare); they were then diluted in lysis buffer to a final volume of 1 ml. After 4 hours of incubation at 4°C on a rotating wheel, the beads were spun down and washed three times in lysis buffer. All steps were performed on ice, and all buffers were supplemented with fresh protease inhibitors (cOmplete, Roche) and 1 mM phenylmethylsulfonyl fluoride. For detecting the proteins in Western blot, the pulldown reactions were treated with Laemmli buffer, boiled at 95°C for 15 min, and subjected to SDS-PAGE separation and blotting on a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was probed with the anti-TBX3.

For liquid chromatography-MS/MS analysis, the protein samples, already dissolved in
Laemmli buffer, were submitted to a filter-aided sample preparation (FASP) and digested with trypsin in 100 mM triethylammonium bicarbonate buffer overnight.
Desalted samples were dried completely in a vacuum centrifuge and reconstituted with 50 μl of 3% acetonitrile and 0.1% formic acid. Each peptide solution (4 μl) was analyzed on both Q Exactive and Fusion mass spectrometers (Thermo Scientific) coupled to EASY-nLC 1000 (Thermo Scientific). Spectra acquisition and peptide count was

Xenograft tumor zebrafish model
HCT116 cells were transfected with pCS2 (empty vector) or TBX3-expressing plasmid and labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, cat. no. D3899; Invitrogen) according to previously described method (Rouhi et al., 2010). Briefly, tumor cells were washed twice with PBS, followed by labeling with a final concentration of 5 μg/ml of DiI for 30 min at 37 °C. After rigorous washing with PBS, tumor cells were trypsinized for 5 minutes, counted under a phase contrast microscope, centrifuged at 300 g for 5 min and resuspended at a final concentration of 30-40 cells per µl in medium. Human cells were injected into the perivitelline space of 72 h old fli1:EGFP transgenic zebrafish embryos. The eggs were fertilized, collected and dechorionated. After that, HCT116 cells were injected into the vitreous cavity of zebrafish embryo with non-filamentous borosilicate glass capillary needles attached to the microinjector under the stereomicroscope (Leica Microsystems). After tumor cell injection, zebrafish embryos were further selected under fluorescent microscopy to ensure that tumor cells were located only within the cavity and then incubated in aquarium water for consecutive 3 days at 36.0 °C. Primary tumor growth, invasion and metastasis in the zebrafish body were monitored at day 3 with a fluorescent microscope (Nikon Eclipse C1) as previously published (Rouhi et al., 2010). Briefly, each zebrafish embryo was picked up and monitored to detect tumor cell distribution.
Two different sets of images from the head region and the trunk region were collected separately from each zebrafish embryo. Disseminated tumor cells in the caudal hematopoietic plexus of zebrafish embryos were counted (in a double-blind manner) and the primary tumor areas were measured using Image J software. At least 15-20 embryos were included in each experimental group.

Super-TOP/FOP-Flash reporter assay
The β-catenin reporter plasmid (TOP-flash) and its mutant control (FOP-flash) were constructed by Addgene (12456)  (right panels), respectively. (C) Quantitative RT-PCR detecting Lgr5 mRNA extracted from colonic epithelium of control (black), Bcl9/9l (blue) or Pygo1/2 (red) conditional mutants (KO). (D) 6-8-week-old male mice were treated with five tamoxifen (Tam) injections (i.p., 1mg/day) for five consecutive days. 10 days later mice were treated with 2.5% dextran sodium sulfate (DSS) ad libitum in the drinking water for 9 days. While 17% of control mice (n=30) were severely affected or died due to the DSS treatment (red lines), 65% of conditional Bcl9/9l-KO (n=34) mice performed poorly in this test. Deletion of Bcl9/9l increased statistically significantly the death rate after DSS treatment (p-value = 0.00013 in Fisher's Exact Test). No difference between Pygo1/2-KO and control mice could be measured: 27% of control mice (n=22) and 25% of Pygo1/2-KO (n=24) were affected upon DSS treatment (p-value = 0.5626 in Fisher's Exact Test). (E) 6-8-week-old female mice were exposed to a single dose of the carcinogenic agent azoxymethane (AOM), followed by 7 days of DSS administration in the drinking water. This regimen results in the emergence of dysplastic adenomas that are collected for RNA extraction and analysis of the indicated targets via RT-PCR: Wnt target genes and genes expressed during epithelial-to-mesenchymal transition (EMT), associated with cancer metastasis.  showing the genomic distribution of high-confidence BCL9 peaks (B, 5303 total) and TBX3 peaks (C, 2369 total). (D) Overlap of the high-confidence peak groups between BCL9 and TBX3. (E) Selected result entries from motif analysis performed on the BCL9-TBX3 overlapping high-confidence peaks. Significant enrichment was found for TCF/LEF and Homeobox motifs. No TBX consensus sequence was detected in this analysis. (F) Select genomic tracks demonstrating co-occupancy of BCL9 and TBX3 within the Wnt Responsive Element (WRE) of known Wnt-target genes (Axin2, Ccnd1, Nkd1 and Lef1) and genes important in limb morphogenesis (Hand1 and Hand2). The scale of peak enrichment is indicated in the top-left corner of each group of tracks. In light blue the BCL9 (Salazar et al., 2019), in orange the TBX3 replicates, and in green the control track (IgG). Genomic tracks are adapted for this figure upon visualization with IGV Integrative Genomic Viewer (https://igv.org/). mutation of Bcl9/9l (Bcl9/9l-Δ1/Δ2 vs CTRL). DEGs were a total of 1143 (p < 0.05), with 606 up-regulated and 537 down-regulated. (B) A significant portion (28.4%) of DEGs exhibited overlap with TBX3 ChIP-seq peaks. The overlap with TBX3 ChIP-seq peaks appeared statistically significant, in particular, when the down-regulated genes were considered. (D) Hierarchical clustering of samples (3 CTRL versus 3 Bcl9/9l-Δ1/Δ2) based on genes overlapping between DEGs and genes annotated for TBX3 ChIP-seq peaks (normalized RNA-seq read counts, Ward's clustering method, Euclidian distance). Annotation added for genes associated by Gene Ontology to Wnt signaling (Fgf10,Ptk7,Kremen1,Zfp703,Bmp2 and Gli3) and genes known as regulators of limb development (Meis2, Irx3 and Eya2). (C) β-Catenin/TCF luciferase reporter STF assay in parental, β-catenin knockout and TCF knockout HEK293T cells. Cells were treated with the indicated concentration of CHIR or DMSO, overnight. Expression of TBX3 (black bars) compared to control (empty vector, white bars) showed that TBX3 acts as an activation switch for Wnt/TCF pathway. Only   does not recapitulate the effects of deleting Bcl9/9l (Bcl9/9l-KO) in dysplastic adenomas. The expression of genes associated with intestinal stem cell function was analyzed via quantitative RT-PCR, and the expression in the mutant cells (red) was compared to that in the control (CTRL, black). (C) Quantitative RT-PCR of target genes associated with intestinal stem cell function (compare it with the same analysis of Pygo1/2-KO in panel B) of RNA extracted from control (CTRL, black) or Bcl9/9l-DHD1 (red) tumors.

Figure supplement 3: (A) Proximity
Ligation Assay (PLA) confirms vicinity between BCL9 and TBX3 when expressed in human HEK293T cells: red signal is observed upon protein-protein proximity. Nuclear GFP and BCL9 do not give rise to PLA signal (left panel, negative control). PYGO2 is used as positive interactor with BCL9 (middle panel) and gives rise to PLA signal (red). TBX3 and BCL9 produce nuclear PLA signal in a subset of transfected cells (detected with GFP; right panel). (B) Immunoprecipitation assay using a GFP-trap strategy. BCL9 reliably interacts with HAtagged PYGO2 in high-salt washing conditions (400 mM NaCl). In this stringent condition, FLAG-tagged TBX3 is not observed when GFP-BCL9 is immunoprecipitated, suggesting absence of direct interaction or affinity inferior to that between BCL9 and PYGO2. IP of GFP is used a control.

Figure supplement 4:
Overlap of the high-confidence BCL9 and TBX3 peaks in developing murine limbs reveals the existence of BCL9 exclusive (one example displayed in the genomic tracks on the left) and TBX3 exclusive peaks (one example in the genomic track on the right). The greatest group of BCL9 exclusive peaks, surprisingly, does not display high enrichment of TCF/LEF motifs, suggesting that a predominant genomic function of BCL9 is not related to Wnt/b-catenin signalling, as previously suggested by our work and that of others (Cantù et al., 2018(Cantù et al., , 2014Jiang et al., 2020   Figure supplement 5: AXIN2 and NKD1 are here considered as representative Wnt transcriptional targets. mRNA expression in HEK293T cells upon TBX3 overexpression (black bars) is measured via qRT-PCR and compared to control condition (CTRL, transfection with empty vector, white bars). Consistently with the STF/Luciferase assay ( Figure 4C), TBX3 represses the transcription of endogenous Wnt targets. Upon suboptimal Wnt stimulation however (achieved via addition of 1 μM of CHIR), AXIN2 and NKD1 are only mildly activated in CTRL condition. On the other hand, TBX3 overexpression induces a substantial statistical increase in AXIN2 and NKD1 mRNA abundance; the Wnt-ON (CHIR 1 µM) condition is compared to the Wnt-OFF basal condition (ON/OFF ration; right panel). The data are presented as the mean ± SD of three independent experiments. Parametric unpaired Student t-test was used to measure the statistical significance (p-values above the charts). GAPDH mRNA was used to normalize transcript levels.