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Commensal microbiota contribute to gut homeostasis by inducing transcription of mucosal genes. Analysis of the impact of various microbiota on intestinal tissue provides an important insight into the function of this organ. We used cDNA microarrays to determine the gene expression signature of mucosa isolated from the small intestine and colon of germ-free (GF) mice and animals monoassociated with two E. coli strains. The results were compared to the expression data obtained in conventionally reared (CR) mice. In addition, we analyzed gene expression in colon organoids derived from CR, GF, and monoassociated animals. The analysis revealed that the complete absence of intestinal microbiota mainly affected the mucosal immune system, which was not restored upon monoassociation. The most important expression changes observed in the colon mucosa indicated alterations in adipose tissue and lipid metabolism. In the comparison of differentially expressed genes in the mucosa or organoids obtained from GF and CR mice, only six genes were common for both types of samples. The results show that the increased expression of the angiopoietin-like 4 (Angptl4) gene encoding a secreted regulator of lipid metabolism indicates the GF status.

T-cell factor 4 (TCF4), together with &beta, catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.