Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer

Abstract

Barrett's esophagus (BE) and gastric intestinal metaplasia are related premalignant conditions in which areas of human stomach epithelium express mixed gastric and intestinal features. Intestinal transcription factors (TFs) are expressed in both conditions, with unclear causal roles and cis-regulatory mechanisms. Ectopic CDX2 reprogrammed isogenic mouse stomach organoid lines to a hybrid stomach–intestinal state transcriptionally similar to clinical metaplasia; squamous esophageal organoids resisted this CDX2-mediated effect. Reprogramming was associated with induced activity at thousands of previously inaccessible intestine-restricted enhancers, where CDX2 occupied DNA directly. HNF4A, a TF recently implicated in BE pathogenesis, induced weaker intestinalization by binding a novel shadow Cdx2 enhancer and hence activating Cdx2 expression. CRISPR/Cas9-mediated germline deletion of that cis-element demonstrated its requirement in Cdx2 induction and in the resulting activation of intestinal genes in stomach cells. dCas9-conjugated KRAB repression mapped this activity to the shadow enhancer's HNF4A binding site. Altogether, we show extensive but selective recruitment of intestinal enhancers by CDX2 in gastric cells and that HNF4A-mediated ectopic CDX2 expression in the stomach occurs through a conserved shadow cis-element. These findings identify mechanisms for TF-driven intestinal metaplasia and a likely pathogenic TF hierarchy.

Defining the structure, signals, and cellular elements of the gastric mesenchymal niche

“…Intestinal crypts were plated in Matrigel (Corning 356234) and cultured in complete media (DMEM/F12 supplemented with Glutamax, HEPES, Penicillin, Streptomycin, Neomycin, Primomycin, N2, B27, 1mM N-Acetylcysteine, 50 μg/mL murine rEGF, 100 μg/mL murine rNoggin, 10% Rspo1 conditioned media). Gastric glands were isolated from antrum or corpus separately, incubating the tissue in 10 mM EDTA-PBS at room temperature as previously described (Mahe et al, 2013; Singh et al, 2022). Glands were separated from the stroma and muscularis mucosa by gentle scraping, washed in PBS and cultured in complete media (same as small intestinal organoid complete media with the addition of 10% Afamin/Wnt3a conditioned media, MBL International Corporation, J2-001) or as specified in the text.…”

“…Gastric glands were isolated from the gastric antrum or corpus by incubating the tissue in 10 mM EDTA-PBS at room temperature, as described previously. 46,47 Glands were separated from the stroma and muscularis by gentle scraping, washed in PBS, and cultured in the same medium as SI crypts with addition of 10% Afamin/Wnt3a conditioned medium (MBL International, J2-001) or as specified. Recombinant factors (murine FGF7, Peprotech, 450-60; human BMP3, Peprotech, 120-24B; rat CTGF, R&D Systems, 92-37C-T050) were resuspended in 0.1% BSA/PBS and used at final concentration 100 ng/mL.…”

Helicobacter pylori cancer associated CagA protein drives intestinal metaplastic transition in human gastric organoids

“…A similar phenotypic change has previously been seen in mice as the result of ectopic CDX2 expression in the stomach 28,29 . Recently it was reported that in mouse gastric organoids the ectopic expression of CDX2 results in incomplete reprogramming towards intestinal-like cells 59,74 . Strikingly, in our model CagA led to downregulation of gastric differentiation markers and enrichment of intestine-specific genes and GIM markers, as specified by Companioni et al 48 , indicating that activated CagA leads to trans-differentiation of adult gastric stem cells towards intestinal-like stem cells.…”

Single-cell chromatin profiling of the primitive gut tube reveals regulatory dynamics underlying lineage fate decisions

“…Increasing evidence suggests that pioneer factors mediate embryonic expression signatures in cancer 56 . Interestingly, a recent study has shown that when overexpressed, CDX2 could bind its adult intestinal sites that are inaccessible in the stomach, promoting intestinal metaplasia, a key early process in tumorigenesis 57 . Therefore, it will be important to further investigate pioneer TFs in gut development, stem cell homeostasis, and diseases such as cancer.…”