Identification of Wnt regulated genes that are repressed by, or independent of, β-catenin

  1. Shiyang Liu
  2. Sara Haghani
  3. Enrico Petretto
  4. Babita Madan
  5. Nathan Harmston
  6. David M Virshup

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Wnt signaling regulates metazoan development and homeostasis, in part by β-catenin dependent activation and repression of a large number of genes. However, Wnt signaling also regulates genes independent of β-catenin, genes that are less well characterized. In this study, using a pan-Wnt inhibitor we performed a comprehensive transcriptome analysis in a Wnt-addicted orthotopic cancer model to delineate the β-catenin-dependent and independent arms of Wnt signaling. We find that while a large percentage of Wnt-regulated genes are regulated by β-catenin, ten percent of these genes are regulated independent of β-catenin. Interestingly, a large proportion of these β-catenin independent genes are Wnt-repressed. Among the β-catenin dependent genes, more than half are repressed by β-catenin. We used this dataset to investigate the mechanisms by which Wnt/β-catenin signaling represses gene expression, revealing the role of a cis-regulatory motif, the negative regulatory element (NRE). The NRE motif is enriched in the promoters of β-catenin repressed genes and is required for their repression. This provides a comprehensive analysis of the β-catenin independent arm of the Wnt signaling pathway in a cancer model and illustrates how a cis-regulatory grammar can determine Wnt-dependent gene activation versus repression.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    Revision Plan

    June 28, 2025

    Manuscript number: RC-2025-02982

    Corresponding author(s): Babita Madan, Nathan Harmston, David Virshup

    General Statements In Wnt signaling, the relative contributions of ‘canonical (β-catenin dependent) and non- canonical (β-catenin independent) signaling remains unclear. Here, we exploited a unique and highly robust in vivo system to study this. Our study is therefore the first comprehensive analysis of the β-catenin independent arm of the Wnt signaling pathway in a cancer model and illustrates how a combination of cis-regulatory elements can determine Wnt-dependent gene regulation.

    We are very pleased with the reviews; it appears we communicated our goal and our findings clearly, and in general the reviewers felt the study provided important information, was well planned and the results were “crystal clear”.

    While more experiments could strengthen and extend the results, we feel our results are already very robust due to the use of multiple replicates in the in vivo system.

    The Virshup lab in Singapore closed July 1, 2025 and so additional wet lab studies are not feasible.

    1. Description of the planned revisions

    *Insert here a point-by-point reply that explains what revisions, additional experimentations and *analyses are planned to address the points raised by the referees.

    Below we address the points raised by the reviewers:

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    …

    The article has the merit of addressing a yet-unsolved question in the field (if beta-catenin can also repress genes) that only a limited number of studies has tried to tackle, and provides useful datasets for the community. The system employed is elegant, and the PORCN-inhibition bypassed by a ____constitutively active beta-catenin is clean and ingenious. The manuscript is clearly written.

    We thank the reviewers for their kind comments on the importance of the data. Our orthotopic model provides the opportunity to exploit robust Wnt regulated gene expression in a more responsive microenvironment than can be achieved in cell culture and simple flank xenograft models.

    Here we propose a series of thoughts and comments that, if addressed, would in our opinion improve the study and its description.

    1. We wonder why a xenograft model is necessary to induce a robust WNT response in these cells.

    The authors describe this set-up as a strength, as it is supposed to provide physiological relevance, yet it is not clear to us why this is the case.

    We welcome the opportunity to expand on our choice of an orthotopic xenograft model. It has been long established that cancer cells behave differently in different in vivo locations (Killion et al., 1998). Building on this, we confirmed this in our system that identical pancreatic cancer cells treated with the same PORCN inhibitor had very different responses in vitro, in the flank and in their orthotopic environment (Madan et al., 2018). To quote from our prior paper, “Looking only at genes decreasing more than 1.5-fold at 56 hours, we would have missed 817/1867 (44%) genes using a subcutaneous or 939/1867 (50%) using an in vitro model. Thus, the overall response to Wnt inhibition was reduced in the subcutaneous model and further blunted in vitro. An orthotopic model more accurately represents real biology.

    The reason for this is presumably the very different orthotopic microenvironment, including tissue appropriate stroma-tumor, vascular-tumor, lymphatic-tumor, and humoral interactions.

    Moreover, as the authors homogenize the tumour to perform bulk RNA-seq, we wonder whether they are not only sequencing mRNA from the cancer cells but also from infiltrating immune cells and/or from the surrounding connective tissue.

    In experiments generating RNA-seq data from xenograft models, the resulting sequences can originate from either human (graft) or mouse (host). In order to account for this, following standard practice, we filtered reads prior to alignment using Xenome (Conway et al., 2012). We have added additional text to the methods to highlight this step in our pipeline.

    1. If, as the established view implies, Wnt/beta-catenin only leads to gene activation, pathway

    inhibition would free up the transcriptional machinery - there is evidence that some of its constituents are rate-limiting. The free machinery could now activate some other genes: the net effect observed would be their increased transcription upon Wnt inhibition, irrespective of beta-catenin's presence. Could this be considered as an alternative explanation for the genes that go up in both control and bcat4A lines upon ETC-159 administration? This, we think, is in part corroborated by the absence of enrichment of biological pathways in this group of genes. The genes that are beta-catenin-dependent and downregulated (D&R) are obviously not affected by this alternative explanation.

    This is an interesting suggestion, and we will incorporate this thought into our discussion of potential mechanisms.

    1. The authors mention that HPAF-II are Wnt addicted. Do they die upon ETC-159 administration, and is this effect rescued by exogenous WNT addition?

    We and several others have previously reported that Wnt-addicted cells differentiate and/or senesce upon Wnt withdrawal in vivo but not in vitro. This is related to the broader changes in gene expression in the orthotopic tumors. The effect of PORCN inhibition has been demonstrated by us and others and is rescued by Wnt addition, downstream activation of Wnt signaling by e.g. APC mutation, and, as we show here, stabilized β-catenin.

    1. Line 120: the authors write about Figure 1C: "This demonstrates that the growth of β-cat4A cells in vitro largely requires Wnts to activate β-catenin signaling." The opposite is true: control cells require WNT and form less colony with ETC159, while β-cat4A are independent from Wnt secretion.

    We appreciate the reviewer pointing out our mis-statement. This error has now been corrected in the revised manuscript.

    1. Lines 226-229: "The β-catenin independent repressed genes were notably enriched for motifs bound by homeobox factors including GSC2, POU6F2, and MSGN1. This finding aligns with the known role of non-canonical Wnt signaling in embryonic development" This statement assumes that target genes, or at least the beta-catenin independent ones, are conserved across tissues, including developing organs. This contrasts with the view that target genes in addition to the usual suspects (e.g., AXIN2, SP5 etc.) are modulated tissue-specifically - a view that the authors (and in fact, these reviewers) appear to support in their introduction.

    We agree with the reviewer that a majority of Wnt-regulated genes are tissue specific. Indeed, the β-catenin independent Wnt-repressed genes may also be tissue specific. In other tissues, we speculate that other β-catenin independent Wnt-repressed genes may also have homeobox factor binding sites as well and so the general concept remains valid. We do not have sufficient data in other tissues to resolve this issue.

    1. The luciferase and mutagenesis work presented in Figure 5 are crystal-clear. One important aspect that remains to be clarified is whether beta-catenin and/or TCF7L2 directly bind to the NRE sites. Or do the authors hypothesize that another factor binds here? We suggest the authors to show TCF7L2 binding tracks at the NRE/WRE motifs in the main figures.

    A major question of the reviewers was, can we provide additional evidence that the NRE is bound by LEF/TCF family members. Our initial analysis of more datasets indicates TCF7L2 peaks are enriched on NREs in Wnt-β-catenin responsive cell lines like HCT116 and PANC1. These analyses appear to further support the model that the NRE binds TCF7L2, but we fully agree these analyses can neither prove nor disprove the model.

    In our revision, we will analyze additional cut and run datasets as suggested and look at the HEPG2 datasets suggested by reviewer 1. We are concerned about tissue specificity as some of the genes are not expressed in e.g. HEPG2 or HEK293 cells where datasets are available. However, our data continues to support a functional role for the NRE in the modulation of β-catenin regulated genes. The best analysis would be more ChIP-Seq or Cut and Run assays on tissues, not cells, but these studies are beyond what we can do.

    What about other TCF/LEFs and beta-catenin? Are there relevant datasets that could be explored to test whether all these bind here during Wnt activation?

    As above, We will analyze additional ChIP and Cut & Run datasets to address this question looking at β-catenin and other LEF/TCF family members. We also reflect on the fact that ChIP-Seq does not necessarily imply that the targeted factor (e.g.,TCF7L2) is bound in the target site in all the cells.

    The repression might be mediated by beta-catenin partnering with other factors that bind the NRE even by competing with TCF7L2.

    We appreciate the insightful comments and now incorporate this into our discussion.

    1. In general, while we greatly appreciate the github page to replicate the analysis, we feel that the methods' description is lacking, both concerning analytical details (e.g., the cutoff used for MACS2 peak calling) or basic experimental planning (e.g, how the luciferase assays were performed).

    We thank reviewers for the suggestions and will add further details regarding the analysis

    and experimental planning in the method sections.

    1. The paper might benefit from the addition of quality metrics on the RNA-seq. Interesting for example would be to see a PCA analysis - as a more unbiased approach - rather than the kmeans clustering.

    We have this data and will add it to the revised manuscript.

    1. It seems that in Figure 3A the clusters are mislabelled as compared to Figure 3B and Figure 1. Here the repressor clusters are labelled DR5, DR6 and DN7 whereas in the rest of the paper they are labelled DR1, DR2 and DN1.

    Thank you for pointing out this issue. This has now been corrected in Figure 3.

    1. The siCTNNB1 in Figure 5E is described to be a significant effect in the text whereas in Figure 5E this has a p value of 0.075.

    Thank you for pointing out the p value did not cross the 0.05 threshold. We have modified the text to remove the word ‘significant’.

    1. Line 396: 'Here we confirm and extend the identification of a TCF-dependent negative regulatory element (NRE), where beta-catenin interacts with TCF to repress gene expression'. We suggest caution in stating that beta-catenin and TCF directly repress gene expression by binding to NRE. In the current state the authors do not show that TCF & beta-catenin bind to these elements. See our previous point 7.

    We appreciate the suggestion of the reviewers. We will be more cautious in our interpretation.

    Further suggestions - or food for thoughts:

    1. A frequently asked question in the field concerns the off-target effects of CHIR treatment as opposed to exposure to WNT ligands. CHIR treatment - in parallel to bcat4A overexpression - would allow the authors to delineate WNT independent effects of CHIR treatment and settle this debate.

    We thank the reviewers for suggesting this interesting experiment to sort out the non- Wnt effects of GSK3 inhibition. Such a study would require a new set of animal experiments and a different analysis; we think this is beyond the scope of this manuscript.

    1. We think that Figure 4C could be strengthened by adding more public TCF-related datasets (e.g., from ENCODE) to confirm the observation across datasets from different laboratories. In particular, the HEPG2 could possibly be improved as there is an excellent TCF7L2 dataset available by ENCODE.

    Many more datasets are easily searchable through: https://www.factorbook.org/.

    As above, we will analyze the HEPG2 dataset. We plan on updating Fig 4 with data from analysis from different datasets such as (Blauwkamp et al., 2008; Zambanini et al., 2022).

    1. The authors show that there is no specific spacing between NREs and WREs. This implies that it is not likely that TCF7L2 recognizes both at the same time through the C-clamp. Do the authors think that there might be a pattern discernible when comparing the location of WRE and NRE in relation to the TCF7L2 ChIP-seq peak summit? This would allow inferring whether TCF7L2 more likely directly binds the WRE (presumably) and if the NRE is bound by a cofactor.

    This is an interesting suggestion and we will conduct this analysis as suggested on available datasets (as the result may be different in different tissue types with varying degrees of Wnt/β-catenin signaling).

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Overall, the study provides a solid framework for understanding noncanonical transcriptional ____outputs of Wnt signaling in a cancer context. The majority of the conclusions are well supported by the data. However, there are a few substantive points that require clarification before the manuscript is ready for publication.

    Major Comments

    The authors' central claim-that their findings represent a comprehensive analysis of the β-catenin- independent arm of Wnt signaling and uncover a "cis-regulatory grammar" governing Wnt-dependent gene activation versus repression-is overstated based on the presented data.

    We appreciate the reviewers concern and will temper our language.

    Specifically:

    • Figure 3B identifies TF-binding motifs enriched among different Wnt-responsive gene clusters, but the authors only functionally investigate the role of NRE in β-catenin-dependent repression, particularly in the context of TCF motif interaction.

    • To support a broader claim regarding cis-regulatory grammar, additional analyses are required:

    o What is the distribution of NREs across all clusters? Are they exclusive to β-catenin-dependent repressed clusters, or more broadly present?

    The distribution of the NREs is a statistically significant enrichment; they are observed in the repressed clusters more frequently than expected by chance alone, but they are present elsewhere as well. We have tempered our language around the cis-regulatory grammar.

    o Do NREs interact with other enriched motifs beyond TCF? Is this interaction specific to repression or also involved in activation?

    This is an interesting question beyond the scope of this analysis. Our dataset uses multiple interventions; The NREs may interact with other motifs but we would need more transcriptional analysis data with biological intervention to assess this.

    o A more comprehensive analysis of cis-element combinations is needed to draw conclusions about their collective influence on gene regulation across clusters.

    We agree; This would be a great question if we had TCF binding data in our orthotopic xenograft model. It’s a dataset we do not have, nor do we have the resources to pursue this.

    Other important clarifications:

    • The use of the term "wild-type" to describe HPAF-II cells is potentially misleading. These cells are not genetically wild-type and harbor multiple oncogenic alterations.

    Thank you for pointing this out. We will use the word “parental” in the text

    • The manuscript does not clearly present the kinetics of Wnt target downregulation upon ETC-159 treatment of HPAF-II cells. Understanding whether repression mirrors activation dynamics (e.g., delay or persistence of Wnt effects) is essential to interpreting the system's temporal behavior.

    We previously addressed the temporal dynamics of activation and repression in our more comprehensive time course papers (Harmston et al., 2020; Madan et al., 2018); there are differences in the dynamics that are difficult to tease out in this new dataset as the density of time points is less. Having said that, we will compare the time course and annotate the sets of genes identified in this current study with the data from our original study to provide more information on the temporal dynamics of this system.

    Minor Comment

    • The statement in Figure 1C (lines 119-120) that "growth of β-cat4A cells in vitro largely requires Wnts to activate β-catenin signaling" is inconsistent with the data. As the β-cat4A allele encodes a constitutively active form of β-catenin, Wnts should not be required. Please revise this conclusion for clarity.

    We thank the reviewers for pointing out this mis-statement. We have corrected this.

    Reviewer #2 (Significance (Required)):

    This study offers a systematic classification of Wnt-responsive gene expression dynamics, differentiating between β-catenin-dependent and -independent mechanisms. The insights into temporal expression patterns and the potential role of the NRE element in transcriptional repression add depth to our understanding of Wnt signaling. These findings have relevance for developmental biology, stem cell biology, and cancer research-particularly in understanding how Wnt-mediated repression may influence tumor progression and therapeutic response.

    Nice review; thank you.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    … The work advances understanding of Wnt mediated repression via cis regulatory grammar.

    Major Concerns

    1. Statistical thresholds and clustering - The criteria for classifying β catenin-dependent versus - independent genes rely on FDR cutoffs above or below 0.1. If the more stringent cutoff of 0.05 was used, how many genes would still be considered Wnt regulated?

    We can readily address this in a revised manuscript.

    1. Validation of selected β catenin-dependent and -independent Wnt target genes - While the authors identify β catenin-dependent and -independent Wnt target genes (4 selected genes from different clusters in Fig.2), RT-qPCR based validation of Axin2 has been performed in Fig. S3. Authors should also validate other 3 genes as well.

    We had considered performing qPCR to re-validate some of our gene-expression changes but qPCR analyses is intrinsically more error prone than RNAseq, and we believe the literature shows that qPCR from the same samples will not add any extra utility. Previous studies that have examined this question have reported excellent correlation between the RNAseq and pPCR (Asmann et al., 2009; Griffith et al., 2010; Wu et al., 2014).

    1. NRE mechanistic insight - The most important contribution of this manuscript is the extension of the importance of the NRE motif in Wnt regulated enhancers. But the mutagenesis data provided is insufficient to conclusively nail down that the NREs are responsible for the repression. The effects in the synthetic reporters in Fig. 4D are small - it's not clear that there is much activity in the MimRep to be repressed by the NREs. The data in Fig. 5 is a better context to test the importance of the NREs, but the authors use deletion analysis which is too imprecise and settle for single nucleotide mutants in individual NREs in the ABHD11-AS1 reporter. In the Axin2 report, they mutate sequences outside of the NRE. It's too inconsistent. They should mutate 3 or 4 positions within the NRE in BOTH motifs in the context of the ABHD11-AS1 reporter. Same for the Axin2 reporter.

    We feel our analysis, coupled with the Kim paper (Kim et al., 2017), support the role of the NRE. We agree that more data is always desirable, but in our current circumstances are we cannot add additional wetlab experiments.

    Regarding Figure 4D, this is a synthetic system lacking the endogenous elements in the promoter. We agree with the reviewer that the effect is small but we would also like to point out that adding the well-established 2WRE in front of the MinRep increased the transcription activity to 1.5 fold, which is of similar magnitude change of the 2NRE deceasing the transcriptional activity 1/1.5 = 0.6.

    In Kim et al, it was shown that mutating the 11st nucleotide of the NRE motif showed the strongest effect, so we followed their lead in only mutated the 11st nucleotide in ABHD11- AS1 NRE.

    As for the putative NRE sequence present in AXIN2 promoter, its downstream sequence is polyT (__GTGTTTTTTTT__TTTTTTTTTT), if we only mutate 11st nucleotide to G/C, we could create similar sequence to NRE, so we mutated sequences outside of the NRE to fully disrupt it.

    1. Even if the mutagenesis is done more completely, the results simply replicate that of the Goentoro group. In Kim et al 2017, they provide suggestive (not convincing) evidence that TCFs directly bind to the NRE. The authors of this manuscript should explore that in more detail, e.g., can purified TCF bind to the NRE sequence? Can the authors design experiments to directly test whether beta-catenin is acting through the NRE - their data currently only demonstrates that the NRE provide a negative input to the reporters - that's an important mechanistic difference.

    We point out that our minimal reporter studies with the NRE showed a repressive effect in HCT116 (colorectal cancer cells with stabilized β-catenin) but not HT1080 (sarcoma cells with low Wnt) supporting the importance of β-catenin acting through the NRE (Figs. 4D, 4E).

    We fully agree with the reviewers that additional study of TCF interaction with the NRE would be of value. While EMSA and culture-based ChIP assays would be of some value, the best study should be done in vivo where the system is most robust. We are not in a position to do these studies, but we will add in a discussion of this as a limitation of the current study.

    1. In vertebrates, some TCFs are more repressive than others and TLEs have been implicated in repressive. Exploring these factors in the context of the NRE would increase the value of this story.

    This is an interesting idea but beyond the scope of the current manuscript. It is likely this would be dependent on tissue specific expression, local expression levels, and local binding of co-factors. As we look at other TCF members in other datasets we may be able to address this. Further wetlab experiments are beyond the scope of this work.

    **Referees cross-commenting**

    I respectfully disagree that the luciferase assays are sufficient. Using deletion analysis to understand the function of specific binding sites is insufficient and the more specific mutations of NREs are incomplete. Regarding this paper extending our knowledge of direct transcriptional repression by Wnt/bcat signaling, I don't agree that it adds much - there are numerous datasets where Wnt signaling activates and represses genes - the trick is determining whether any of the repressed genes are the result and direct regulation by TCF/bcat. They don't explore that. The main finding is an extension of the work by Lea Goentoro on the importance of the NRE motif, but they don't address whether TCF directly associates with this sequence. Goentoro argued in the 2017 paper that it does, but that data is unconvincing to me. Can purified TCF bind the NRE? Without that information (done carefully) this manuscript is very limited.

    We respectfully disagree with the reviewer regarding the contribution of this manuscript. There are certainly many datasets looking at Wnt-regulated genes in tissue culture, but these cell-based studies are underpowered to really understand Wnt biology. There are only two papers, ours and Cantú’s, that address Wnt repressed genes in any depth. No prior papers have differentiated β-catenin dependent from β-catenin independent genes before, and certainly not in an orthotopic animal model.

    A major impact of our study is the finding that only 10% of Wnt regulated genes are independent of β-catenin, at least in pancreatic cancer. We feel this is a major contribution. We further add to this analysis by re-enforcing/extend the prior evidence on the NRE in humans (and correct the motif sequence!) for Wnt-repressed genes. Our data supports the fine-tuning of the Wnt/β-catenin regulated genes by a cis-regulatory grammar.

    Reviewer #3 (Significance (Required)):

    Overall, this study advances our understanding of the dual roles of Wnt signaling in gene activation and repression, highlighting the role of the NRE motif. But this is an extension of the original NRE paper (Kim et al 2017) with no mechanistic advance beyond that original work. The transcriptomics in the first part of the manuscript have some value, but similar data sets already exist.

    We respectfully but strongly disagree with the reviewer. First, our work examines the NRE in a large-scale in vivo transcriptome dataset, significantly extending the candidate gene approach of Kim et al. Secondly, we disagree with the comment that “similar data sets already exist.” Indeed, reviewer 1 (C. Cantú) specifically pointed out we had addressed an “yet-unsolved question in the field” on whether and how β-catenin repressed genes.

    __3. __Description of the revisions that have already been incorporated in the transferred manuscript

    To date we have only corrected several typographical errors.

    1. Description of analyses that authors prefer not to carry out

    We fully agree with the reviewers that additional study of TCF interaction with the NRE would be of value. While EMSA and cell culture-based ChIP assays would be of some modest value, they have already been done in vitro by Kim et al. (Kim et al., 2017) and the best next study should be done in vivo in Wnt-responsive cancers or tissues where the biology is most robust (Madan et al., 2018) . We are not in a position to do these studies, but we will add this into the discussion as a limitation of the current study. We also acknowledge that the NRE may interact with other currently unidentified factors.

    Reviewer 1 asked about considering experiments to determine non-Wnt effects of GSK3 inhibitors like CHIR. Such a study, while interesting, would require a new set of animal experiments and a different analysis; we think this is beyond the scope of this manuscript.

    Finally, we note that the Virshup lab at Duke-NUS Medical School in Singapore, where these in vivo studies were performed, has closed as of July 1, 2025 and the various lab members have moved on to new adventures. Because of this, we are unable to undertake new wet-lab studies.

    Thank you for your consideration,

    For the authors,

    David Virshup

    References:

    Asmann YW, Klee EW, Thompson EA, Perez EA, Middha S, Oberg AL, Therneau TM, Smith DI,

    Poland GA, Wieben ED, Kocher J-PA. 2009. 3’ tag digital gene expression profiling of human

    brain and universal reference RNA using Illumina Genome Analyzer. BMC Genom 10:531–

    1. doi:10.1186/1471-2164-10-531

    Blauwkamp TA, Chang MV, Cadigan KM. 2008. Novel TCF-binding sites specify transcriptional

    repression by Wnt signalling. The EMBO Journal 27:1436–1446. doi:10.1038/emboj.2008.80

    Conway T, Wazny J, Bromage A, Tymms M, Sooraj D, Williams ED, Beresford-Smith B. 2012.

    Xenome—a tool for classifying reads from xenograft samples. Bioinformatics 28:i172–i178.

    doi:10.1093/bioinformatics/bts236

    Griffith M, Griffith OL, Mwenifumbo J, Goya R, Morrissy AS, Morin RD, Corbett R, Tang MJ, Hou

    Y-C, Pugh TJ, Robertson G, Chittaranjan S, Ally A, Asano JK, Chan SY, Li HI, McDonald H,

    Teague K, Zhao Y, Zeng T, Delaney A, Hirst M, Morin GB, Jones SJM, Tai IT, Marra MA.

    1. Alternative expression analysis by RNA sequencing. Nat Methods 7:843–847.

    doi:10.1038/nmeth.1503

    Harmston N, Lim JYS, Arqués O, Palmer HG, Petretto E, Virshup DM, Madan B. 2020.

    Widespread Repression of Gene Expression in Cancer by a Wnt/β-Catenin/MAPK Pathway.

    Cancer Res 81:464–475. doi:10.1158/0008-5472.can-20-2129

    Killion JJ, Radinsky R, Fidler IJ. 1998. Orthotopic models are necessary to predict therapy of

    transplantable tumors in mice. Cancer metastasis reviews 17:279–284.

    Kim K, Cho J, Hilzinger TS, Nunns H, Liu A, Ryba BE, Goentoro L. 2017. Two-Element

    Transcriptional Regulation in the Canonical Wnt Pathway. Current Biology 27:2357-2364.e5.

    doi:10.1016/j.cub.2017.06.037

    Madan B, Harmston N, Nallan G, Montoya A, Faull P, Petretto E, Virshup DM. 2018. Temporal

    dynamics of Wnt-dependent transcriptome reveals an oncogenic Wnt/MYC/ribosome axis. J

    Clin Invest 128:5620–5633. doi:10.1172/jci122383

    Wu AR, Neff NF, Kalisky T, Dalerba P, Treutlein B, Rothenberg ME, Mburu FM, Mantalas GL,

    Sim S, Clarke MF, Quake SR. 2014. Quantitative assessment of single-cell RNA-sequencing

    methods. Nat Methods 11:41–46. doi:10.1038/nmeth.2694

    Zambanini G, Nordin A, Jonasson M, Pagella P, Cantù C. 2022. A new cut&run low volume-

    urea (LoV-U) protocol optimized for transcriptional co-factors uncovers Wnt/b-catenin tissue-

    specific genomic targets. Development 149. doi:10.1242/dev.201124

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The authors use a PORCN inhibitor (ETC 159) in an orthotopic RNF43 mutant pancreatic cancer model to distinguish β catenin-dependent from -independent Wnt target genes. They find that ~90% of Wnt regulated genes in this system are β catenin dependent. Approximately half of these genes are activated by Wnt signaling, half repressed. Clustering and functional enrichment link dependent versus independent targets to distinct pathways. They observe enrichment of sequence motifs similar to the 11 bp Negative Regulatory Element (NRE) previously identified by Lea Goentoro's group in the region around the TSS of β catenin-repressed genes. Using reporter constructs, both synthetic and regulatory DNA from Wnt targets (e.g., ABHD11 AS1, AXIN2), they provide evidence that the NREs are a negative input on expression. The work advances understanding of Wnt mediated repression via cis regulatory grammar.

    Major Concerns

    1. Statistical thresholds and clustering - The criteria for classifying β catenin-dependent versus -independent genes rely on FDR cutoffs above or below 0.1. If the more stringent cutoff of 0.05 was used, how many genes would still be considered Wnt regulated?
    2. Validation of selected β catenin-dependent and -independent Wnt target genes - While the authors identify β catenin-dependent and -independent Wnt target genes (4 selected genes from different clusters in Fig.2), RT-qPCR based validation of Axin2 has been performed in Fig. S3. Authors should also validate other 3 genes as well.
    3. NRE mechanistic insight - The most important contribution of this manuscript is the extension of the importance of the NRE motif in Wnt regulated enhancers. But the mutagenesis data provided is insufficient to conclusively nail down that the NREs are responsible for the repression. The effects in the synthetic reporters in Fig. 4D are small - it's not clear that there is much activity in the MimRep to be repressed by the NREs. The data in Fig. 5 is a better context to test the importance of the NREs, but the authors use deletion analysis which is too imprecise and settle for single nucleotide mutants in individual NREs in the ABHD11-AS1 reporter. In the Axin2 report, they mutate sequences outside of the NRE. It's too inconsistent. They should mutate 3 or 4 positions within the NRE in BOTH motifs in the context of the ABHD11-AS1 reporter. Same for the Axin2 reporter.
    4. Even if the mutagenesis is done more completely, the results simply replicate that of the Goentoro group. In Kim et al 2017, they provide suggestive (not convincing) evidence that TCFs directly bind to the NRE. The authors of this manuscript should explore that in more detail, e.g., can purified TCF bind to the NRE sequence? Can the authors design experiments to directly test whether beta-catenin is acting through the NRE - their data currently only demonstrates that the NRE provide a negative input to the reporters - that's an important mechanistic difference.
    5. In vertebrates, some TCFs are more repressive than others and TLEs have been implicated in repressive. Exploring these factors in the context of the NRE would increase the value of this story.

    Referees cross-commenting

    I respectfully disagree that the luciferase assays are sufficient. Using deletion analysis to understand the function of specific binding sites is insufficient and the more specfic mutations of NREs are incomplete. Regarding this paper extending our knowledge of direct transcriptional repression by Wnt/bcat signaling, I don't agree that it adds much - there are numerous datasets where Wnt signaling activates and represses genes - the trick is determining whether any of the repressed genes are the result and direct regulation by TCF/bcat. They don't explore that. The main finding is an extension of the work by Lea Goentoro on the importance of the NRE motif, but they don't address whether TCF directly associates with this sequence. Goentoro argued in the 2017 paper that it does, but that data is unconvincing to me. Can purified TCF bind the NRE? Without that information (done carefully) this manuscript is very limited.

    Significance

    Overall, this study advances our understanding of the dual roles of Wnt signaling in gene activation and repression, highlighting the role of the NRE motif. But this is an extension of the original NRE paper (Kim et al 2017) with no mechanistic advance beyond that original work. The transcriptomics in the first part of the manuscript have some value, but similar data sets already exist.

    Addressing the major points-especially gaining a deeper mechanistic insight into NRE function-would elevate the manuscript's impact. Major revisions are recommended.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This manuscript by Liu et al. explores lesser-known aspects of Wnt signaling, particularly focusing on genes that are repressed by β-catenin and those regulated independently of β-catenin. While canonical Wnt signaling is well characterized through the stabilization and nuclear translocation of β-catenin to activate TCF/LEF target genes, the mechanisms underlying gene repression and β-catenin-independent regulation remain relatively underexplored.

    The authors leverage the Wnt-addicted HPAF-II cancer cell line, combining PORCN inhibitor (ETC-159) treatment with ectopic expression of a stabilized β-catenin mutant (β-cat4A) in orthotopic xenograft models. Through RNA-sequencing analysis, they systematically identify Wnt-responsive gene clusters that are either dependent or independent of β-catenin stabilization. They further demonstrate that a specific cis-regulatory element, termed the Negative Regulatory Element (NRE), contributes to β-catenin-mediated transcriptional repression.

    Overall, the study provides a solid framework for understanding noncanonical transcriptional outputs of Wnt signaling in a cancer context. The majority of the conclusions are well supported by the data. However, there are a few substantive points that require clarification before the manuscript is ready for publication.

    Major Comments

    The authors' central claim-that their findings represent a comprehensive analysis of the β-catenin-independent arm of Wnt signaling and uncover a "cis-regulatory grammar" governing Wnt-dependent gene activation versus repression-is overstated based on the presented data.

    Specifically:

    • Figure 3B identifies TF-binding motifs enriched among different Wnt-responsive gene clusters, but the authors only functionally investigate the role of NRE in β-catenin-dependent repression, particularly in the context of TCF motif interaction.
    • To support a broader claim regarding cis-regulatory grammar, additional analyses are required:
      • What is the distribution of NREs across all clusters? Are they exclusive to β-catenin-dependent repressed clusters, or more broadly present?
      • Do NREs interact with other enriched motifs beyond TCF? Is this interaction specific to repression or also involved in activation?
      • A more comprehensive analysis of cis-element combinations is needed to draw conclusions about their collective influence on gene regulation across clusters.

    Other important clarifications:

    • The use of the term "wild-type" to describe HPAF-II cells is potentially misleading. These cells are not genetically wild-type and harbor multiple oncogenic alterations.
    • The manuscript does not clearly present the kinetics of Wnt target downregulation upon ETC-159 treatment of HPAF-II cells. Understanding whether repression mirrors activation dynamics (e.g., delay or persistence of Wnt effects) is essential to interpreting the system's temporal behavior.

    Minor Comment

    • The statement in Figure 1C (lines 119-120) that "growth of β-cat4A cells in vitro largely requires Wnts to activate β-catenin signaling" is inconsistent with the data. As the β-cat4A allele encodes a constitutively active form of β-catenin, Wnts should not be required. Please revise this conclusion for clarity.

    Significance

    This study offers a systematic classification of Wnt-responsive gene expression dynamics, differentiating between β-catenin-dependent and -independent mechanisms. The insights into temporal expression patterns and the potential role of the NRE element in transcriptional repression add depth to our understanding of Wnt signaling. These findings have relevance for developmental biology, stem cell biology, and cancer research-particularly in understanding how Wnt-mediated repression may influence tumor progression and therapeutic response.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Shiyang Liu and colleagues investigate the transcription induced by Wnt/beta-catenin by employing PORCN inhibition (ECT-159, blocking the secretion of WNTs) in the Wnt-addicted HPAF-II cell line. Classical targets, such as AXIN2, are downregulated by PORCN inhibition (as expected), while many other genes are upregulated, suggesting that Wnt/beta-catenin represses them. Overexpression of a GSK3/CK1-insensitive beta-catenin variant leads to the re-established upregulation of AXIN2 and the concomitant repression of the other group of repressed genes, demonstrating that the repression is mediated by beta-catenin. Other genes are repressed (activated by ECT-159) irrespective of the presence of activated beta-catenin, and the authors conclude that they are beta-catenin-independent Wnt-repressed genes. The authors observe that beta-catenin-dependent repressed genes present enrichment, in their promoters, of the Negative Regulatory Element (NRE) previously identified by the Goentoro lab. In elegant Luciferase assays, the authors now confirm that individual NRE elements are causally involved in target gene repression by -catenin. The article has the merit of addressing a yet-unsolved question in the field (if beta-catenin can also repress genes) that only a limited number of studies has tried to tackle, and provides useful datasets for the community. The system employed is elegant, and the PORCN-inhibition bypassed by a constitutively active beta-catenin is clean and ingenious. The manuscript is clearly written.

    Here we propose a series of thoughts and comments that, if addressed, would in our opinion improve the study and its description.

    1. We wonder why a xenograft model is necessary to induce a robust WNT response in these cells. The authors describe this set-up as a strength, as it is supposed to provide physiological relevance, yet it is not clear to us why this is the case. Moreover, as the authors homogenize the tumour to perform bulk RNA-seq, we wonder whether they are not only sequencing mRNA from the cancer cells but also from infiltrating immune cells and/or from the surrounding connective tissue.
    2. If, as the established view implies, Wnt/beta-catenin only leads to gene activation, pathway inhibition would free up the transcriptional machinery - there is evidence that some of its constituents are rate-limiting. The free machinery could now activate some other genes: the net effect observed would be their increased transcription upon Wnt inhibition, irrespective of beta-catenin's presence. Could this be considered as an alternative explanation for the genes that go up in both control and cat4A lines upon ETC-159 administration? This, we think, is in part corroborated by the absence of enrichment of biological pathways in this group of genes. The genes that are beta-catenin-dependent and downregulated (D&R) are obviously not affected by this alternative explanation.
    3. The authors mention that HPAF-II are Wnt addicted. Do they die upon ETC-159 administration, and is this effect rescued by exogenous WNT addition?
    4. Line 120: the authors write about Figure 1C: "This demonstrates that the growth of β-cat4A cells in vitro largely requires Wnts to activate β-catenin signaling." The opposite is true: control cells require WNT and form less colony with ETC159, while β-cat4A are independent from Wnt secretion.
    5. Lines 226-229: "The β-catenin independent repressed genes were notably enriched for motifs bound by homeobox factors including GSC2, POU6F2, and MSGN1. This finding aligns with the known role of non-canonical Wnt signaling in embryonic development." This statement assumes that target genes, or at least the beta-catenin independent ones, are conserved across tissues, including developing organs. This contrasts with the view that target genes in addition to the usual suspects (e.g., AXIN2, SP5 etc.) are modulated tissue-specifically - a view that the authors (and in fact, these reviewers) appear to support in their introduction.
    6. The luciferase and mutagenesis work presented in Figure 5 are crystal-clear. One important aspect that remains to be clarified is whether beta-catenin and/or TCF7L2 directly bind to the NRE sites. Or do the authors hypothesize that another factor binds here? We suggest the authors to show TCF7L2 binding tracks at the NRE/WRE motifs in the main figures. What about other TCF/LEFs and beta-catenin? Are there relevant datasets that could be explored to test whether all these bind here during Wnt activation? We also reflect on the fact that ChIP-Seq does not necessarily imply that the targeted factor (e.g., TCF7L2) is bound in the target site in all the cells. The repression might be mediated by beta-catenin partnering with other factors that bind the NRE even by competing with TCF7L2.
    7. In general, while we greatly appreciate the github page to replicate the analysis, we feel that the methods' description is lacking, both concerning analytical details (e.g., the cutoff used for MACS2 peak calling) or basic experimental planning (e.g, how the luciferase assays were performed).
    8. The paper might benefit from the addition of quality metrics on the RNA-seq. Interesting for example would be to see a PCA analysis - as a more unbiased approach - rather than the kmeans clustering.
    9. It seems that in Figure 3A the clusters are mislabelled as compared to Figure 3B and Figure 1. Here the repressor clusters are labelled DR5, DR6 and DN7 whereas in the rest of the paper they are labelled DR1, DR2 and DN1.
    10. The siCTNNB1 in Figure 5E is described to be a significant effect in the text whereas in Figure 5E this has a p value of 0.075.
    11. Line 396: 'Here we confirm and extend the identification of a TCF-dependent negative regulatory element (NRE), where beta-catenin interacts with TCF to repress gene expression'. We suggest caution in stating that beta-catenin and TCF directly repress gene expression by binding to NRE. In the current state the authors do not show that TCF & beta-catenin bind to these elements. See our previous point 7.

    Further suggestions - or food for thoughts:

    1. A frequently asked question in the field concerns the off-target effects of CHIR treatment as opposed to exposure to WNT ligands. CHIR treatment - in parallel to bcat4A overexpression - would allow the authors to delineate WNT independent effects of CHIR treatment and settle this debate.
    2. We think that Figure 4C could be strengthened by adding more public TCF-related datasets (e.g., from ENCODE) to confirm the observation across datasets from different laboratories. In particular, the HEPG2 could possibly be improved as there is an excellent TCF7L2 dataset available by ENCODE. Many more datasets are easily searchable through: https://www.factorbook.org/.
    3. The authors show that there is no specific spacing between NREs and WREs. This implies that it is not likely that TCF7L2 recognizes both at the same time through the C-clamp. Do the authors think that there might be a pattern discernible when comparing the location of WRE and NRE in relation to the TCF7L2 ChIP-seq peak summit? This would allow inferring whether TCF7L2 more likely directly binds the WRE (presumably) and if the NRE is bound by a cofactor.

    Review by Claudio Cantù and Yorick van de Grift

    Why we sign: we believe that peer review should be a transparent dialogue. We strive to be critical but honest and professional, and care that our opinions and criticisms are formulated as if we were meeting the authors in person.

    Our expertise lies in the genomics impact of Wnt/beta-catenin activation, and in the search of mechanisms that drive the tissue-specific functions of this pathway across developmental and disease contexts.

    Significance

    Shiyang Liu and colleagues investigate the transcription induced by Wnt/beta-catenin by employing PORCN inhibition (ECT-159, blocking the secretion of WNTs) in the Wnt-addicted HPAF-II cell line. Classical targets, such as AXIN2, are downregulated by PORCN inhibition (as expected), while many other genes are upregulated, suggesting that Wnt/beta-catenin represses them. Overexpression of a GSK3/CK1-insensitive beta-catenin variant leads to the re-established upregulation of AXIN2 and the concomitant repression of the other group of repressed genes, demonstrating that the repression is mediated by beta-catenin. Other genes are repressed (activated by ECT-159) irrespective of the presence of activated beta-catenin, and the authors conclude that they are beta-catenin-independent Wnt-repressed genes. The authors observe that beta-catenin-dependent repressed genes present enrichment, in their promoters, of the Negative Regulatory Element (NRE) previously identified by the Goentoro lab. In elegant Luciferase assays, the authors now confirm that individual NRE elements are causally involved in target gene repression by -catenin. The article has the merit of addressing a yet-unsolved question in the field (if beta-catenin can also repress genes) that only a limited number of studies has tried to tackle, and provides useful datasets for the community. The system employed is elegant, and the PORCN-inhibition bypassed by a constitutively active beta-catenin is clean and ingenious. The manuscript is clearly written.

  5. Version published to 10.1101/2025.04.06.647504v1 on bioRxiv