Bcl9 and Pygo synergise downstream of Apc to effect intestinal neoplasia in FAP mouse models

Bcl9 and Pygo are Wnt enhanceosome components that effect β-catenin-dependent transcription. Whether they mediate β-catenin-dependent neoplasia is unclear. Here we assess their roles in intestinal tumourigenesis initiated by Apc loss-of-function (ApcMin), or by Apc1322T encoding a partially-functional Apc truncation commonly found in colorectal carcinomas. Intestinal deletion of Bcl9 extends disease-free survival in both models, and essentially cures Apc1322T mice of their neoplasia. Loss-of-Bcl9 synergises with loss-of-Pygo to shift gene expression within Apc-mutant adenomas from stem cell-like to differentiation along Notch-regulated secretory lineages. Bcl9 loss also promotes tumour retention in ApcMin mice, apparently via relocating nuclear β-catenin to the cell surface, but this undesirable effect is not seen in Apc1322T mice whose Apc truncation retains partial function in regulating β-catenin. Our results demonstrate a key role of the Wnt enhanceosome in β-catenin-dependent intestinal tumourigenesis and reveal the potential of BCL9 as a therapeutic target during early stages of colorectal cancer.

C olorectal cancer is the second most common cause for cancer mortality in the developed world (http://globocan. iarc.fr). The pathway to this cancer is usually initiated by the mutational inactivation of the Adenomatous Polyposis Coli (APC) tumour suppressor, both in sporadic and hereditary forms 1 . Individuals bearing APC germline mutations develop thousands of benign adenomas in their large intestine by their teenage years one of which, invariably, progresses to carcinoma within the next decades of their lives 2 . Progression to colorectal cancer requires a small number of additional driver mutations, e.g., the activation of the KRAS or PIK3CA oncogenes, and/or the inactivation of the P53 or ARID1A tumour suppressors 3 . Notably, Apc germline mutations in the mouse also cause multiple intestinal neoplasia (Min), and various Apc loss-based models have been developed for pre-clinical studies 4 , whereby the original Apc Min model 5 has been used most widely.
At the molecular level, APC attenuates Wnt signal transduction through the 'canonical' branch, by cooperating with Axin to promote the proteasomal degradation of the key effector βcatenin. This process is inhibited by Wnt signals that promote the stabilisation of β-catenin, allowing it to access T cell factors (TCF) bound to cis-regulatory enhancers of Wnt-responsive genes to operate Wnt-dependent transcription. Key to this process is the Wnt enhanceosome 6,7 , a multiprotein complex tethered via TCF to enhancers of lineage-determining master control genes whose Wnt-responsiveness is conferred by BCL9, or its paralog B9L (BCL9-Like, or BCL9-2) 8,9 : these factors (called BCL9 below unless a specific paralog is referred to) bind to and capture stabilised β-catenin, to convey it to TCF within the enhanceosome via docking to Pygo, a chromatin-binding PHD finger protein 10 . Wnt/β-catenin signalling specifies numerous cell fate decisions during animal development, and controls self-renewal of virtually every adult tissue, including the intestinal epithelium 11 . This stem cell function could explain why β-catenin is a potent oncogene, as revealed by the prevalence, in many cancers, of activating mutations in β-catenin that render it refractory to degradation 3 .
Despite its considerable oncogenic potential, there are no wellvalidated inhibitors of activated β-catenin that could be developed as therapeutics, because of the lack of druggable downstream targets 12 . Nevertheless, several small molecules have been reported to inhibit activated β-catenin directly or indirectly, but most of these produce unspecific (off-target) cell toxicity: e.g., tankyrase inhibitors destabilise β-catenin by boosting the levels of Axin 13 , but also of other targets 14 , which may contribute to their toxicity in mice 15 . Furthermore, cells experiencing chronic Wnt signalling such as APC-mutant cancer cells proved refractory to tankyrase inhibition, mainly because they express high levels of LEF1 (a TCF paralog) or B9L, which shield β-catenin from Axindependent destruction 13 .
The role of BCL9 as an essential co-factor of Drosophila βcatenin was demonstrated by genetic studies 16 . In mice, deletion of both Bcl9 paralogs causes embryonic lethality, while tissuespecific deletion in muscle leads to β-catenin-dependent regeneration defects 17 . Similarly, conditional deletion of both paralogs in the intestine reduces β-catenin-dependent transcription compartment in intestinal crypts, suggesting a role of Bcl9 in specifying 'stemness' in this self-renewing compartment 18,19 . BCL9 and B9L are often overexpressed in colorectal cancer cell lines and carcinomas, maintaining their β-catenin-dependent transcription 13,20 , and overexpressed B9L promotes intestinal tumourigenesis 21 .
BCL9 functions as a scaffold of the Wnt enhanceosome 7 . Its binding to the Pygo PHD finger promotes its recognition of methylated histone H3 tail 10,22 . It also binds to the N-terminus of the Armadillo Repeat Domain (ARD) of β-catenin via a short helical domain called HD2 23,24 , an interaction that can be blocked by individual HD2 point mutations 16,25 . Binding of BCL9 to β-catenin can also be blocked by natural compounds 13,23 , stapled helices mimicking HD2 26 or rationallydesigned small molecules 27 , which attenuate β-catenin-dependent transcription in colorectal cancer cells and β-catenin-dependent tumourigenesis in mouse models. These studies have thus provided proof-of-concept for the druggability of BCL9-β-catenin interaction.
In the light of the well-documented role of BCL9 in facilitating β-catenin-dependent transcription, it was puzzling that the conditional double-knockout of Bcl9 and B9l in the intestinal epithelium (Bcl9/B9l DKO below) did not reduce the tumour numbers in mouse models based on colitis and chemicallyinduced β-catenin-dependent tumours 18 , or on weak attenuation of Apc function 19 . Here, we re-assess the role of Bcl9 in intestinal tumourigenesis in two mouse models bearing Apc mutations, namely Apc Min which essentially abolishes Apc function, and Apc 1322T which retains partial function of Apc in regulating βcatenin and, importantly, mimics the most prevalent APC mutations in human colon cancers 28 . Deletion of Bcl9 and Pygo extends the disease-free life in both models, especially deletion of Bcl9 which essentially cures Apc 1322T mice of their neoplastic disease, restoring a normal life span in these otherwise moribund mice. RNA profiling reveals that Bcl9 loss synergises with Pygo loss downstream of Apc loss to shift the adenomatous gene expression programme from stem cell-like towards differentiation along secretory cell lineages. Our study also uncovers a posttranscriptional effect of Bcl9 deletion in Apc Min adenoma, namely a striking relocation of their nuclear β-catenin to their cell surface, likely increasing their cell adhesion and retention in crypts, which could account for the numerous tiny adenomas seen in this model. Importantly, this undesirable effect is not observed in Apc 1322T adenomas whose cell surface β-catenin appears normal, likely because the Apc 1322T truncation retains binding to, and partial regulation of, β-catenin. Our results from this model therefore illustrate the significant potential of BCL9 and Pygo as targets for therapeutic interference in colorectal cancer.

Results
Loss of Bcl9 or Pygo extends the healthy life of Apc Min mice. Mice bearing the Apc Min germline mutation develop some two dozen adenomas in their small intestines, detectable from 100 days of age, each arising as a result of Apc loss-ofheterozygosity (LOH) in an individual intestinal epithelial cell 29 . Half of these mice succumb to their disease by~120 days (Fig. 1a), turning anemic and losing weight. However, conditional deletion of Pygo or Bcl9 in the intestinal epithelium (with villin. Cre) 30 prolonged the mean disease-free live of these mice by 60-90 days, or even more if all four paralogs were deleted simultaneously (QKO; Fig. 1a). The crypt-villus structure appeared normal in these strains, without any detectable changes in the numbers of dividing or apoptotic cells (Supplementary Figure 1, and A.E.K. Ibrahim, unpublished). Clearly, Apc Min mice benefit from loss of Pygo or Bcl9 in terms of survival, with maximal benefit derived from the simultaneous loss of all four paralogs.
Next, we determined the RNA expression profiles of intestinal crypts from the DKO strains using Illumina Bead Arrays, but found only 96 significant (p < 0.05) gene expression changes compared to matched controls. Of these, 90% showed the same up-or downregulation trends in the two types of DKO (Supplementary Figure 2), as expected since Bcl9 and Pygo act in the same complex 6 . Notably, five histone H2A and four ribosomal protein genes were downregulated in these DKO samples, while five Defensin genes were upregulated, including Defa1 encoding an early differentiation marker for Paneth cells 31,32 . Thus, deletion of Bcl9 or Pygo may promote Paneth cell specification, although there was no detectable increase in the numbers of lysozyme-positive cells (marking fully differentiated Paneth cells) 31,33 in DKO crypt sections (Supplementary Figure 1). Nevertheless, our RNA profiling data suggest that Bcl9 or Pygo loss biases cell fates from proliferative towards differentiated.  Loss of Bcl9 or Pygo increases tumour numbers in Apc Min mice. In light of this, it was surprising that the tumour numbers were increased~3x in small intestines of Pygo-deleted Apc Min mice compared to Apc Min littermates (Fig. 1b). By contrast, there were far fewer large adenomas (>1 mm) in Bcl9-deleted Apc Min intestines, although these were embedded within a lawn of tiny adenomas (< 1 mm; Fig. 1c). In addition, histological analysis of 'swiss rolls' revealed numerous minute adenomas in these intestines buried below the intestinal villi and thus undetectable by surface inspection (Fig. 1d), while the great majority of adenomas in Pygo-deleted and Apc Min control intestines protrude above these villi (Fig. 1e, f). This striking change in size distribution from large to minute adenomas indicates that Bcl9 is required for tumour growth. Note that the increased tumour numbers in Apc Min DKO intestines were unlikely to be caused by elevated LOH, given that the numbers of γ-H2AX positive foci (marking double-stranded DNA breaks and other lesions underlying LOH) 34 are similarly low as in Apc Min controls (Supplementary Figure 3). We conclude that loss of Bcl9 in the intestine, despite elevating tumour numbers, slows down the growth of these tumours, suggesting a role of Bcl9 in promoting their proliferation. This is consistent with the findings by Gay et al. (co-submitted) that Bcl9 is required for crypt regeneration following irradiation, and for efficient clonal expansion in the murine intestine. Furthermore, B9L also appears to be essential for the proliferation of APCmutant cancer cells: its depletion in SW480 cells (expressing APC truncated at amino acid 1338) attenuated their proliferation substantially, and it proved impossible to recover proliferating cell colonies that lack B9L (Supplementary Figure 4). The tumour phenotype of the Apc Min QKO intestines was virtually indistinguishable from that of Bcl9-deleted Apc Min intestines (Fig. 1b, c), suggesting that Bcl9 loss may be epistatic over Pygo loss (but see below). Notably, the weight gain of DKO and QKO mice was normal during their extended life spans, and so their increased tumour loads were well tolerated. Indeed, the morbidity of Apc Min mice is thought to be caused by their anaemia rather than their adenoma burden 5 .
Loss of Bcl9 or Pygo shifts cells towards differentiation. To test this apparent epistasis between Bcl9 and Pygo, we determined the RNA profiles in adenomas excised from Apc Min DKO and QKO small intestines (pooled from 2-3 RNA extractions). Comparing Apc Min adenomas to normal crypt samples, we found 8852 significant changes (p < 0.01), most of which likely represent indirect gene expression changes downstream of master transcription regulators (such as Ascl2, Math1 or c-myc; see below). They include upregulation of known Wnt targets (such as Lgr5), H2A and ribosomal protein genes, and downregulation of Defensin genes, e.g., Defa1 ( Fig. 2a and Supplementary Figure 2). Consistent with this, acute inactivation of Apc in the intestinal epithelium causes crypt expansions, and cell-fate shifts from differentiated to proliferative 35 . Hierarchical clustering based on Euclidean distance confirmed that profiles within samples of the same genotypic cohort were more similar to one another than to those from different cohorts (Fig. 2b, top).
Comparing Apc Min to QKO adenomas, we found 2806 significant changes of which 62.3% were dependent on Apc, i.e., the corresponding gene probes showed differential expression in adenomas versus (vs) normal crypts; of these, 82.9% showed opposite trends in QKO vs Apc Min controls (Supplementary Figure 5). Furthermore, a substantial fraction of the QKOdependent changes were trend reversals towards the RNA profile of normal crypts (Fig. 2b), including most Wnt target genes ( Fig. 2c) except for Axin2, a gene constituting a negative feedback loop with elevated expression in Apc-mutant adenomas 36 which was unaffected by loss of Pygo and Bcl9. Indeed, these 'normalisations' also extended to previously identified stem cell signatures downstream of Wnt/TCF 37 , EphB2 or Lgr5 38,39 (Supplementary Figure 6), consistent with previous results 18,19 . Thus, simultaneous deletion of Bcl9 and Pygo shifts the transcription programme of Apc Min adenomas from stem celllike towards normal crypts.
Synergy between loss of Bcl9 and Pygo in normalising tumours. Our RNA profiling data suggested that Bcl9 loss contributed rather more than Pygo loss to the QKO-dependent trend reversals, perhaps explaining why the tumour phenotypes of the QKO mice resembled those of Bcl9/B9l rather than Pygo1/2 DKO. To test this, and to address whether Bcl9 is epistatic over Pygo at the level of transcription, we conducted systematic Pearson's correlations of the significant changes between QKO and control adenomas across all five cohorts.
Two conclusions can be drawn: firstly, a large fraction of the Bcl9-and Pygo-regulated genes are downstream of Apc, consistent with Bcl9 and Pygo acting predominantly on Apcregulated genes in these tumours. Secondly, although more Apcregulated genes are affected by Bcl9 rather than Pygo loss, simultaneous deletion of both paralog pairs affects vastly more Apc-regulated genes than deletion of either alone. An important corollary is that Bcl9 loss strongly synergises with Pygo loss in shifting the adenoma gene expression programme towards that of normal crypts, and argues against an epistatic relationship between the two enhanceosome factors.
Loss of Bcl9 or Pygo promotes differentiation towards secretory cell fates. Defa1 was our top hit amongst the changes shared between all three DKO and QKO cohorts ( Supplementary Figure 7). Indeed, lysozyme and other Paneth cell markers behaved similarly, showing a tendency to be upregulated in both types of DKO vs control adenomas (Fig. 4a). Immunohistochemistry (IHC) revealed 2 × 3 more lysozyme-positive cells in DKO adenoma vs Apc Min control sections ( Fig. 4b-e), confirming that the former contain more Paneth cells than adenomas with functional Bcl9 or Pygo.
We also found upregulation trends of markers for the other two cell types of the secretory lineage, enteroendocrine and goblet cells 31 , in DKO vs control adenomas (Fig. 4a). We confirmed this for goblet cells, using Period-Acid Schiff (PAS) diastase (PAS-D) staining 33 : we found about twice as many PAS-D-positive cells in Bcl9/B9l DKO vs control adenomas ( Fig. 4f-h), although the adenomatous PAS-D signals (labelling intracellular mucus) were smaller than those from normal goblet cells (Fig. 4f, g; highmagnification views), suggesting that these metaplastic goblet cells are not fully differentiated. Thus, loss of Pygo or Bcl9 promotes secretory cell metaplasia, apparently at the expense of stem cell-like fates, given the downregulation of Lgr5 and stem cell signature genes in these adenomas (Supplementary Figure 6). In contrast, markers for absorptive enterocytes (constituting the bulk of intestinal villi) were not significantly changed (Fig. 4a). This is consistent with the notion that enterocytes represent the 'default' cell fate within the intestinal epithelium 31 whose specification requires neither β-catenin nor Math1 32,33 .
Redistribution of β-catenin in Bcl9-deleted Apc Min adenomas. The above-described RNA profile changes do not fully explain why the tumour phenotypes caused by Bcl9 loss differed from those in Pygo-deleted Apc Min intestines. We thus surmised that the underlying cause may be post-transcriptional. One key determinant for tumour multiplicity is the β-catenin-dependent cell adhesion between tumour and adjacent normal cells which, if elevated, decreases the rate of tumour loss through the normal epithelial renewal process 40 1 2 3 1 2 3 1 2 3 4 5 1 2 3 4 5 6 7 8  Notably, APC regulates the junctional pool of β-catenin 42 , and complete Apc loss-of-function causes a drastic increase of nucleocytoplasmic at the expense of junctional β-catenin 35 . We thus examined the subcellular distribution of β-catenin in Bcl9deleted Apc Min adenomas by immunofluorescence (IF).
As expected, Apc Min adenoma cells exhibited high levels of cytoplasmic and nuclear β-catenin (Fig. 5a, red signal), but low levels of junctional β-catenin compared to adjacent normal epithelial cells. In contrast, Bcl9/B9l DKO adenoma cells showed very little nucleocytoplasmic β-catenin, which explains the low  levels of Wnt target gene expression in these Bcl9-deleted adenomas. Instead, we observed prominent β-catenin staining at the cell surface of these tumours, coinciding with strong Ecadherin staining (Fig. 5b, yellow). Indeed, in these sections, the membraneous β-catenin staining patterns barely differed between adenomas (which showed slightly elevated nucleocytoplasmic βcatenin, allowing their identification) and adjacent normal epithelium (Fig. 5b, insets). In contrast, the β-catenin staining patterns in Pygo-deleted adenomas looked similar to those in Apc Min controls (Fig. 5a, insets), and so even the tiniest nascent tumours stand out by their strong nucleocytoplasmic β-catenin signal 43 . The total levels of β-catenin and E-cadherin are comparable in DKO and Apc Min control adenomas (Supplementary Figure 8). It thus appears that, in the absence of its nuclear binding partner Bcl9, β-catenin cannot be retained in the nucleus and is thus sequestered at the cell surface by its alternative binding partner E-cadherin (see Discussion).
Loss of Bcl9 cures Apc 1322T mice of their neoplastic disease. While our analysis was in progress, a new model for colorectal cancer became available 28 , based on mice bearing an Apc allele (Apc 1322T ) that mimics one of the most commonly observed APC mutations observed in human carcinomas. The Apc 1322T truncation is substantially longer than the Apc Min truncation and, unlike the latter, retains multiple β-catenin binding sites (Fig. 6a) and, thus, partial function in downregulating β-catenin. Notably, a wealth of genetic data from human colorectal cancers indicates a strong positive selection for the retention of β-catenin binding sites 44 , i.e., for the preservation of partial APC function ('justright signalling') 45 . Indeed, colorectal carcinomas that express Apc Min -like truncations without any β-catenin binding sites are exceedingly rare 44 .
We thus repeated our analyses in the Apc 1322T model. RNAseq analysis of adenomas from 100-120 day old Apc Min and Apc 1322T mice revealed only 178 significant changes, a minute fraction of the > 8000 significant changes caused by the Apc Min mutation itself. We thus expect the above-described RNA profiling data from Apc Min adenomas to pertain also to Apc 1322T adenomas, at least qualitatively. However, Wnt target genes tended to be less upregulated in Apc 1322T compared to Apc Min adenomas (Supplementary Figure 9), consistent with the Apc 1322T truncation retaining partial function in downregulating β-catenin.
Despite this, Apc 1322T mice develop more rapid and severe polyposis, and succumb to their neoplastic disease earlier, than Apc Min mice 28 . Under our conditions, Apc 1322T mice typically showed overt disease by~110 days, but their disease-free life was extended to~180 days upon Pygo deletion (Fig. 6b). Furthermore, the mean tumour numbers in Pygo-deleted Apc 1322T intestines were < 10, compared to~18 in control littermates  Figure 10). Thus, the loss of Pygo alleviated the neoplastic disease in this model considerably.
Strikingly, the Bcl9-deleted Apc 1322T intestines showed no adenomas whatsoever at 77 days, and about half of these mice remained disease-free beyond~450 days (Fig. 6b). Indeed, two mice were still healthy at our experimental end-point (day 500). Examining each of the 9 mice aged > 400 days, we found < 11 adenomas per intestine, which probably developed after~200 days since younger mice (2 aged 77, and 2 aged 200 days) showed only one adenoma between them (at 200 days), while the remaining 3 intestines showed none. Intestinal swiss rolls of 3 mice aged > 400 days confirmed that these were completely normal, without any adenomas. Thus, Bcl9 loss essentially cures Apc 1322T mice of their neoplastic disease.
Apc 1322T adenomas show normal cell surface β-catenin. Given the similar RNA profiles between the two models, we surmised that the root cause for their different responses to Bcl9 loss might be post-transcriptional. Indeed, we detected only a subtle increase in nucleocytoplasmic β-catenin staining in Apc 1322T adenomas, barely delineating these tumours (Fig. 7a), consistent with previous results 28 and with the notion that the Apc 1322T truncation retains moderate function in downregulating β-catenin. As might be expected, there was no detectable discontinuity in cell surface β-catenin between adenomatous and adjacent normal epithelial cells in Apc 1322T Bcl9/B9l DKO intestines (Fig. 7b), in stark contrast to the overt discontinuities in Apc Min adenomas (Fig. 7c).

Discussion
We used two mouse tumour models based on different Apc germline mutations (Apc Min and Apc 1322T ) to assess the roles of the Wnt enhanceosome components Bcl9 and Pygo in intestinal neoplasia. In both models, deletion of these factors suppressed tumourigenesis, and extended the disease-free life spans, possibly owing to a shift in the gene expression of Apc-mutant adenomas from stem cell-like towards differentiated. Importantly, simultaneous deletion of Bcl9 and Pygo proved vastly more effective in reversing the neoplastic transcription programme towards normal than deleting either of them alone. Our results underscore the notion that these factors synergise to facilitate the docking of βcatenin to the Wnt enhanceosome 6,7 , to co-activate transcription of Apc target genes. In addition, we uncovered evidence for increased tumour retention in Bcl9-deleted Apc Min intestines, but Several of our results support the Wnt enhanceosome model and, indeed, can be explained by it even though this model, derived from biochemical and functional data from Drosophila and human cells (including APC-mutant colorectal cancer cells) has not been validated specifically for murine cells. For example, we observed a strong synergy between Bcl9 loss and Pygo loss in the suppression of neoplastic disease (Fig. 1a), and in the shift of adenomatous gene expression from neoplastic towards normal (Figs 2b and 3a), consistent with this model. However, we also uncovered clear quantitative differences between the two conditions in that the effects of Bcl9 loss on gene expression and tumour phenotypes were consistently stronger than those of Pygo loss, indicating that Bcl9 is more important than Pygo in enabling β-catenin to co-activate TCF-dependent transcription. This is consistent with its scaffold function within the Wnt enhanceosome 7 , whereby Bcl9 contacts Pygo as well as the Chip/LDB-SSDP (ChiLS) core complex of the enhanceosome and so only partially relies on Pygo for its association with the enhanceosome.
Also, the Wnt enhanceosome may retain partial integrity and function in the absence of Pygo owing to its linkage to the BAF chromatin remodelling complex 46 whose two PHD fingers might substitute for Pygo's PHD finger in conferring chromatin association. Crucially, Bcl9 confers Wnt responsiveness on this multiprotein complex as its only component that can bind to and recruit β-catenin. This, together with the scaffolding role of Bcl9, readily explains why the transcriptional profile of the QKO tumours is largely determined by Bcl9 loss (Fig. 3b, c) and, by implication, why the latter has such a profound impact on βcatenin-dependent intestinal neoplasia in both Apc models (Figs 1a and 6b).
We also observed qualitative differences between Bcl9 and Pygo loss regarding the tumour phenotypes. The most striking one was a marked shift in adenoma size from large to tiny in Bcl9deleted Apc Min mice, while Pygo loss had no effect on the tumour size distribution, implying a role of Bcl9 but not Pygo in promoting tumour growth. However, this apparent qualitative difference might simply reflect a threshold effect: in the absence of Bcl9, expression of master regulators of tumour growth such as cmyc 47 might be reduced sub-critically, which could translate into an off-switch of downstream effector programmes, while the effect of Pygo loss on these genes might be too weak to toggle this switch. This would explain why far fewer genes were differentially expressed in Pygo-deleted compared to Bcl9-deleted tumours. It is also conceivable that some Apc target genes are controlled only by Bcl9 but not Pygo, but the synergy of their simultaneous deletion argues against this. Also, the qualitative differences in tumour phenotypes between the two DKO strains were only apparent in the Apc Min model (for reasons discussed below) while the Apc 1332T mice responded to Bcl9 loss similarly albeit more profoundly than to Pygo loss.
Many of the Wnt enhanceosome components including its ChiLS core and Groucho/TLE co-repressor were discovered as Notch signalling effectors in Drosophila 48 . ChiLS is recruited to target enhancers via direct binding to members of the basic helixloop-helix (bHLH) proteins, such as the mammalian Notch effectors Ascl (Achaete/scute-like) and Math/Atoh (Atonal homolog); these positively-acting enhancer-binding proteins compete with negatively-acting Hairy/enhancer-of-split (Hes) factors, Notch effectors whose expression is universally induced by Notch signalling, and which silence downstream enhancers by recruiting Groucho/TLE 49 . We thus proposed that the Wnt enhanceosome integrates Wnt and Notch signalling inputs 6 , relying on Bcl9-Pygo and positively-acting Notch effectors for its activation, and on Notch-dependent Hes repressors for its rerepression.
This notion is reinforced by our finding that loss of Bcl9 or Pygo tends to shift cell fates from stem-cell like towards differentiation along all three secretory cell lineages of the intestinal epithelium. Each of these cell types requires opposing inputs from Wnt and Notch signalling 50 , and their fates appear to be specified stochastically in transit-amplifying cells located above the crypt base, descending from Lgr5-positive crypt cells 51 . Their specifications depend on β-catenin 33 and the master regulator Math1 32 , while their differentiation along specific secretory lineages requires repressive inputs from Hes1 and other redundant Hes factors 52 whose localised accumulation depends on Notch signalling from neighbouring cells 31 . If Math1 were controlled by the Wnt enhanceosome, a molecular device for integrating opposing inputs from β-catenin and Notch > Hes signalling, this would provide a mechanistic explanation for the results by Tian et al. 50 .
Paneth cells serve as stem cell niches that provide Wnt and Notch signals to reinforce Lgr5-positivity of adjacent cells 53 . This seemed at odds with the observed reductions of Lgr5 itself, and its downstream signatures, in Pygo-or Bcl9-deleted adenomas 18 (Supplementary Figure 6) despite their Paneth cell metaplasia. However, these metaplastic Paneth cells may only be partially differentiated (Fig. 4a) and, thus, not fully functional stem cell niches. Other differentiation markers also failed to be upregulated in metaplastic secretory cells (Fig. 4a), e.g., metaplastic goblet cells seemed partially differentiated judging by their petite PAS-D signals in DKO adenomas (Fig. 4f, g). However, the differentiation of enterocytes appeared unaffected (Fig. 4a), as expected since these absorptive cells constitute the 'default' fate adopted by intestinal epithelial cells in the absence of β-catenin and Math1 31-33 .
Our results thus underscore the notion that the Wnt enhanceosome operates in cellular contexts, and on target genes encoding cell lineage-determinants whose regulation requires integration of Wnt and Notch signalling, such as Math1 (whose expression was under our detection threshold; Fig. 2a). Another candidate for an enhanceosome-controlled gene is Ascl2, a lineage-determining gene for crypt stem cell fates 54 . Finally, cmyc responds to Notch via a remote enhancer 55 and to TCF/βcatenin via proximal cis-regulatory sequences 56 , which may be integrated by the Wnt enhanceosome whose ChiLS core confers communication between promoters and remote enhancers 6 . Note that upregulation of c-myc by Wnt/TCF is subtle 56 albeit functionally relevant 47 which explains why this gene did not feature in our lists of significant expression changes ( Fig. 2 and Supplementary Figure 5).
Our comparative analysis of the two Apc models revealed an unexpected post-transcriptional effect of Bcl9 loss on β-catenin. Tumourigenesis in each of these models is initiated by an Apc truncation of which the longer (Apc 1332T ) retains binding to βcatenin and, thus, partial function regarding β-catenin's roles in transcription and adhesion 57 whereas the shorter Apc Min truncation does not, and so Apc Min is equivalent to a null allele with regard to β-catenin regulation. The tumours produced by two models are morphologically indistinguishable, and exhibit very similar RNA profiles 28,58 , although the Wnt and stem cell signature genes tended to be more upregulated in Apc Min than in Apc 1332T adenomas (Supplementary Figure 9). This is consistent with results from the human colorectal cancer cell line COLO320 whose APC truncation is Apc Min -like and atypically short for human cancers 59 and which exhibit 2-3 fold higher levels of βcatenin-dependent transcription compared to typical colorectal cancer cells 60 whose longer APC truncations retain moderate function in downregulating β-catenin. The low levels of junctional β-catenin in Apc Min tumours may be explained if their dysfunctional Apc Min truncation were unable to maintain junctional β-catenin 42 . By contrast, the partially functional Apc 1332T truncation may maintain near-normal junctional β-catenin in adenomas (Fig. 7a) which could boost their adhesion to neighbouring cells, thereby enhancing their retention within intestinal crypts 41 , and resisting their upward displacement to the villar apices and shedding into the gut lumen. It would explain why Apc 1332T mice develop more severe, and faster, intestinal polyposis than Apc Min mice 28 , despite their lower Wnt and stem cell signatures.
The same could also explain why the Bcl9-deleted Apc Min intestines exhibited hundreds of tiny adenomas (Fig. 1): evidently, their growth was stunted, owing to their dysfunctional β-catenindependent transcription ( Fig. 2 and Supplementary Figure 6), but they appeared to be retained more efficiently within intestinal crypts owing to their high levels of cell surface β-catenin (Fig. 6b). Our evidence suggests that the latter reflects sequestration of βcatenin at the cell surface by its alternative binding partner Ecadherin, following its leakage from the nucleus in the absence of Bcl9: it is well established that β-catenin shuttles freely between nucleus and cell surface, regardless of its phosphorylation status, and that its partitioning between these subcellular compartments is determined by retention via its binding partners in these locations 40,[61][62][63] . This redistribution of β-catenin from the nucleus to the cell surface owing to Bcl9 loss was not detectable in Pygo-deleted Apc Min adenomas, likely because β-catenin remains associated with nuclear Bcl9 in this case. We note that loss of Ecadherin alone does not necessarily result in activation of βcatenin-dependent transcription 64 although the levels of Ecadherin can certainly modulate the nuclear activity of βcatenin in some cellular contexts, e.g., impacting on tumourigenesis in the murine colon 40 .
Therefore, this undesirable effect of Bcl9 deletion on tumour retention in the Apc Min model partially negates the beneficial effects of Bcl9 loss in curbing β-catenin-dependent neoplastic gene expression. Importantly, there was no detectable relocation of β-catenin in Apc 1332T adenomas, likely because the Apc 1332T truncation retains partial activity in downregulating transcriptionally active β-catenin, keeping its nucleocytoplasmic levels relatively low and its cell surface levels normal. This truncation therefore buffers the negative effects of nuclear βcatenin efflux in the absence of Bcl9. The same is expected in the great majority of colorectal carcinomas whose APC truncations, like Apc 1332T , retain binding to β-catenin and regulating its activity. The Apc 1332T model has allowed us to discover a crucial role of Bcl9 and Pygo in effecting intestinal neoplasia in a model with high relevance for colorectal cancer. Similar conclusions were reached by Gay et al. (co-submitted) who also found Bcl9 to be critical for tumourigenesis in different β-catenin-dependent murine models. Furthermore, our results are broadly consistent with evidence that excess Bcl9-2 or Pygo2 promote tumour progression in the intestine 21,65,66 and in other tissues 67,68 . Our study also revealed that Bcl9 is more critical for intestinal neoplasia than Pygo, although both proteins synergise to effect βcatenin-dependent transcription. Thus, BCL9 and B9L have significant potential as targets for therapeutic intervention in colorectal cancer. Since proof-of-concept has been obtained for the discovery of reagents that block their binding to β-catenin 23,26,27 , it might be possible to identify inhibitors of this interaction that can be developed into therapeutics.
For genotyping, tissue from ear biopsies was used. DNA was extracted using TaqMan Sample-to-SNP Kit (ThermoFisher Scientific). For genotyping of Apc Min , the Custom TaqMan SNP Genotyping Assay with the primer set AHX1JET and GTXpress Taqman was used. All other alleles were genotyped by standard PCR with KOD DNA polymerase (Merck Millipore), using the following primers: Apc Apc Min and Apc 1322T mice were monitored for signs of neoplastic disease; their end point was upon presentation of two signs of the disease (anaemia, weight loss, or reduced mobility). Tumours were scored in dissected small intestines at day 77 (Apc 1322T ) or 120 (Apc Min ). To label proliferating cells, mice were injected intraperitoneally with 150 μl of Bromo-deoxyuridine (BrdU) at 10 mg ml −1 in saline, and small intestines were dissected 2 h post injection.
Intestinal tissue preparations. Intestines were flushed with phosphate-buffered saline (PBS) and cut open lengthwise, to remove any remaining mucosa with Kimcare tissue (Kimberly-Clark, Irving TX, USA). For tumour scoring, the colons were removed at the cecum, and the remaining small intestines were fixed in methacarn solution (60% absolute methanol, 30% chloroform, 10% glacial acetic acid, each v/v) for 24 h. Fixed intestines were inspected under a stereo dissecting microscope, for classification of adenomas into macro-and miniadenomas (see Results).
For protein and RNA extraction, individual adenomas were excised, flashfrozen in liquid nitrogen, and stored at −80°C for <24 months. To isolate intestinal crypts, small intestines were cut into 5 mm fragments and washed 10x with ice-cold PBS. Fragments were then incubated in 25 ml PBS containing 2 mM EDTA at 4°C for 30 min, washed with ice-cold PBS, and transferred into 10 ml PBS. Epithelial cells were extracted through vigorous pipetting. The extraction step was repeated 4x with fresh PBS to collect 4 fractions, after which the fractions were pelleted at 300 × g at 4°C for 5 min. Fractions 2-4 were pooled and stored at −80°C for <24 months.
For western blots, intestinal epithelia were separated from their underlying mucosa by scraping small intestines with a blade, flash-frozen in liquid nitrogen and stored at −80°C for <24 months.
For IF and IHC, small intestines were fixed in formalin for 24 h and stored in 80% ethanol for <7 days. Swiss rolls were dehydrated by the following successive incubations: 1x in 70% ethanol for 45 min; 1x in 90% ethanol for 1 h; 4x in 100% ethanol for 75 min; 3x in xylene for 90 min. Subsequently, they were embedded in paraffin by incubating 3x in Tek Wax (SAKURA, no. 4659) for 90 min at 60°C. Sections were cut at 3.5 μm with a Leica microtome RM255.
IF and IHC. For IHC, slides were de-paraffinized and rehydrated by overnight incubation at 65°C and subsequently washed as follows: 2 × 3 min in 100% xylene, homogenized with a Qiagen TissueRuptor followed by QIAshredder columns according to the manufacturer's instructions. RNA was extracted from tissue lysates with the RNeasy Kit, and treated with RNase-free DNase I (Qiagen). Typically, 2-3 RNA extractions from one mouse was pooled for a single sample. RNA concentration and quality were determined with a NanoDrop 2000 (Thermo Scientific, Waltham MA, USA) and a 2100-Bioanalyzer (Agilent Technologies, Santa Clara CA, USA).
Gene expression analysis. Two batches of quality-controlled RNA samples were generated (Supplementary Table 1), for microarray analysis by Cambridge Genomic Services (University of Cambridge). RNA was linearly amplified with the Illumina Totalprep RNA Amplification Kit, and the resulting cRNA was hybridized to the MouseWG-6 v2.0 Expression BeadChip (Illumina, San Diego CA, USA) overnight; subsequently, the BeadChip was washed, stained, and scanned with the Illumina BeadArray Reader. Sample and control probe profiles were exported from GenomeStudio software v2011.1 (Illumina) without background correction or normalisation, and subsequently loaded into the R environment. Relevant subsets of the data were imported separately for each comparison into a LumiBatch object, and filtered by keeping probes that were detected in at least one sample (using a detection p-value < 0.01). The data were then quality-controlled, transformed (Variance Stabilising Transformation, VST) and quantile-normalised with the lumi v2.30.0 package 70 , and non-biological experimental variation between the two microarray sample batches was eliminated with the ComBat function from the SVA v3.26.0 package. Differentially expressed genes were identified with the limma v3.34.9 package 71 , and p-values were corrected for using the Benjamini & Hochberg False Discovery Rate (FDR). Throughout our study, an FDR-adjusted p-value of p < 0.01 was used as the threshold for statistical significance unless otherwise stated.
For RNA-seq analysis, quality-controlled RNA from each sample was used to generate RNA-seq libraries with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich MA, USA) according to the manufacturer's instructions. Briefly, mRNA was isolated from 1 μg of total RNA from each sample. RNA was fragmented enzymatically for 15 min at 94°C and used to generate cDNA libraries with an average size of 361 bp ± 12 bp (SD) barcoded with NEBNext Multiplex Oligos for Illumina Set 1 (New England Biolabs). Sequencing was subsequently performed on one lane of an Illumina HiSeq 4000 at the CRUK Cambridge Institute Genomics Core to generate~30 × 10 6 single-end stranded 50 base pair reads per sample. Reads for each sample were demultiplexed according to the barcodes, and transcript-level abundances were quantified by quasi-mapping the reads against an index build from the GRCm38 transcriptome using Salmon v0.9.1 72 . These quantifications were subsequently imported into the R environment using the tximport v1.6.0 package 73 , and differentially expressed genes were identified using the DEseq2 v1.18.1 package 74 . An FDR-adjusted p < 0.01 was used as a threshold for statistical significance during subsequent analysis. Quantitative set analysis for gene expression (QuSAGE) 75 was performed on rlog transformed read counts using previously published Wnt and stem cell marker gene sets (see Supplementary Figure 9, for references).
Reagents and resources. The reagents and resources used in this study are listed in Supplementary Table 2.

Data availability
The microarray and RNA sequencing data that support the findings of this study are available in NCBI's Gene Expression Omnibus and are accessible through GEO Series reference GSE121145.