Wnt signaling dosage controlled by a Ctnnb1 enhancer balances homeostasis and tumorigenesis of intestinal epithelia

Reviewer #1 (Public Review):

Summary:

Ctnnb1 encodes β-catenin, an essential component of the canonical Wnt signaling pathway. In this study, the authors identify an upstream enhancer of Ctnnb1 responsible for the specific expression level of β-catenin in the gastrointestinal tract. Deletion of this promoter in mice and analyses of its association with human colorectal tumors support that it controls the dosage of Wnt signaling critical to the homeostasis in intestinal epithelia and colorectal cancers.

Strengths:

This study has provided convincing evidence to demonstrate the functions of a gastrointestinal enhancer of Ctnnb1 using combined approaches of bioinformatics, genomics, in vitro cell culture models, mouse genetics, and human genetics. The results support the idea that the dosage of Wnt/β-catenin signaling plays an important role in the pathophysiological functions of intestinal epithelia. The experimental designs are solid and the data presented are of high quality. This study significantly contributes to the research fields of Wnt signaling, tissue-specific enhancers, and intestinal homeostasis.

Weaknesses:

One weakness of this manuscript is an insufficient discussion on the Ctnnb1 enhancers for different tissues. For example, do specific DNA motifs and transcriptional factors contribute to the tissue-specificity of the neocortical and gastrointestinal enhancers? It is also worth discussing the potential molecular mechanisms controlling the gastrointestinal expression of Ctnnb1 in different species since the identified human and mouse enhancers don't seem to share significant similarities in primary sequences.

Reviewer #2 (Public Review):

Wnt signaling is the name given to a cell-communication mechanism that cells employ to inform on each other's position and identity during development. In cells that receive the Wnt signal from the extracellular environment, intracellular changes are triggered that cause the stabilization and nuclear translocation of β-catenin, a protein that can turn on groups of genes referred to as Wnt targets. Typically these are genes involved in cell proliferation. Genetic mutations that affect Wnt signaling components can therefore affect tissue expansion. Loss of function of APC is a drastic example: APC is part of the β-catenin destruction complex, and in its absence, β-catenin protein is not degraded and constitutively turns on proliferation genes, causing cancers in the colon and rectum. And here lies the importance of the finding: β-catenin has for long been considered to be regulated almost exclusively by tuning its protein turnover. In this article, a new aspect is revealed: Ctnnb1, the gene encoding for β-catenin, possesses tissue-specific regulation with transcriptional enhancers in its vicinity that drive its upregulation in intestinal stem cells. The observation that there is more active β-catenin in colorectal tumors not only because the broken APC cannot degrade it, but also because transcription of the Ctnnb1 gene occurs at higher rates, is novel and potentially game-changing. As genomic regulatory regions can be targeted, one could now envision that mutational approaches aimed at dampening Ctnnb1 transcription could be a viable additional strategy to treat Wnt-driven tumors.

Reviewer #3 (Public Review):

The authors of this paper identify an enhancer upstream of the Ctnnb1 gene that selectively enhances expression in intestinal cells. This enhancer sequence drives expression of a reporter gene in the intestine and knockout of this enhancer attenuates Ctnnb1 expression in the intestine while protecting mice from intestinal cancers. The human counterpart of this enhancer sequence is functional and involved in tumorigenesis. Overall, this is an excellent example of how to fully characterize a cell-specific enhancer. The strength of the study is the thorough nature of the analysis and the relevance of the data to the development of intestinal tumors in both mice and humans. A minor weakness is that the loss of this enhancer does not completely compromise the expression of the Ctnnb1 gene in the intestine, suggesting that other elements are likely involved. Adding some discussion on that point would be helpful.

Author response:

eLife assessment

This important study identifies a novel gastrointestinal enhancer of Ctnnb1. The authors present convincing evidence to support their claim that the dosage of Wnt/β-catenin signaling controlled by this enhancer is critical to intestinal epithelia homeostasis and the progression of colorectal cancers. The study will be of interest to biomedical researchers interested in Wnt signaling, tissue-specific enhancers, intestinal homeostasis, and colon cancer.

We greatly appreciate editors’ and reviewers’ extensive and constructive comments and suggestions. We will do our utmost to revise the manuscript accordingly.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Ctnnb1 encodes β-catenin, an essential component of the canonical Wnt signaling pathway. In this study, the authors identify an upstream enhancer of Ctnnb1 responsible for the specific expression level of β-catenin in the gastrointestinal tract. Deletion of this promoter in mice and analyses of its association with human colorectal tumors support that it controls the dosage of Wnt signaling critical to the homeostasis in intestinal epithelia and colorectal cancers.

Strengths:

This study has provided convincing evidence to demonstrate the functions of a gastrointestinal enhancer of Ctnnb1 using combined approaches of bioinformatics, genomics, in vitro cell culture models, mouse genetics, and human genetics. The results support the idea that the dosage of Wnt/β-catenin signaling plays an important role in the pathophysiological functions of intestinal epithelia. The experimental designs are solid and the data presented are of high quality. This study significantly contributes to the research fields of Wnt signaling, tissue-specific enhancers, and intestinal homeostasis.

Weaknesses:

One weakness of this manuscript is an insufficient discussion on the Ctnnb1 enhancers for different tissues. For example, do specific DNA motifs and transcriptional factors contribute to the tissue-specificity of the neocortical and gastrointestinal enhancers? It is also worth discussing the potential molecular mechanisms controlling the gastrointestinal expression of Ctnnb1 in different species since the identified human and mouse enhancers don't seem to share significant similarities in primary sequences.

We agree with the reviewer that the manuscript lacks sufficient discussions on how enhancers control cell-type-specific expressions of target genes, which is one of the most important questions in the field of transcription regulation. Equally important are the common and species-specific features of this regulation. In general, motif composition, location, order, and affinity with trans-factors within enhancers are four key elements. We will elaborate the point in follow-up revision.

Reviewer #2 (Public Review):

Wnt signaling is the name given to a cell-communication mechanism that cells employ to inform on each other's position and identity during development. In cells that receive the Wnt signal from the extracellular environment, intracellular changes are triggered that cause the stabilization and nuclear translocation of β-catenin, a protein that can turn on groups of genes referred to as Wnt targets. Typically these are genes involved in cell proliferation. Genetic mutations that affect Wnt signaling components can therefore affect tissue expansion. Loss of function of APC is a drastic example: APC is part of the β-catenin destruction complex, and in its absence, β-catenin protein is not degraded and constitutively turns on proliferation genes, causing cancers in the colon and rectum. And here lies the importance of the finding: β-catenin has for long been considered to be regulated almost exclusively by tuning its protein turnover. In this article, a new aspect is revealed: Ctnnb1, the gene encoding for β-catenin, possesses tissue-specific regulation with transcriptional enhancers in its vicinity that drive its upregulation in intestinal stem cells. The observation that there is more active β-catenin in colorectal tumors not only because the broken APC cannot degrade it, but also because transcription of the Ctnnb1 gene occurs at higher rates, is novel and potentially game-changing. As genomic regulatory regions can be targeted, one could now envision that mutational approaches aimed at dampening Ctnnb1 transcription could be a viable additional strategy to treat Wnt-driven tumors.

We appreciate the reviewer for acknowledging the potential significance represented by the manuscript. We also recognize that targeting genomic regulatory regions to dampen Ctnnb1 transcription could be a promising strategy for treating Wnt-driven tumors, including many colorectal carcinomas. However, we would like to point out that three are significant technical challenges associated with AAV delivery to the GI epithelium, including the hostile environment, immune response, and low delivery efficiency.

Reviewer #3 (Public Review):

The authors of this paper identify an enhancer upstream of the Ctnnb1 gene that selectively enhances expression in intestinal cells. This enhancer sequence drives expression of a reporter gene in the intestine and knockout of this enhancer attenuates Ctnnb1 expression in the intestine while protecting mice from intestinal cancers. The human counterpart of this enhancer sequence is functional and involved in tumorigenesis. Overall, this is an excellent example of how to fully characterize a cell-specific enhancer. The strength of the study is the thorough nature of the analysis and the relevance of the data to the development of intestinal tumors in both mice and humans. A minor weakness is that the loss of this enhancer does not completely compromise the expression of the Ctnnb1 gene in the intestine, suggesting that other elements are likely involved. Adding some discussion on that point would be helpful.

We are quite encouraged by the reviewer’s positive comments. We agree with the reviewer that other cis-regulatory elements may be involved in the transcription of Ctnnb1 within the GI epithelium. It is also possible that the basal transcription of Ctnnb1 within the GI epithelium is relatively high, and that enhancers can only boost transcription within a certain range. We will discuss these possibilities in the revision.