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. 2022 Jan 1;36(1-2):38–52. doi: 10.1101/gad.348983.121

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© 2022 Singh et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 6. — Tightly correlated HNF4A and CDX2 expression in gastric organoids and tissue. (A) Forced HNF4A expression in gastric organoids induces Cdx2, as shown by normalized RNA read counts. Protein levels (see Fig. 1A) also match this induction. (B, left) HNF4A expression in human endoderm-derived tissues (highest in the liver, lowest in the stomach) taken from the genotype-tissue expression (GTEX) consortium (Melé et al. 2015). (Right) HNF4A is up-regulated in human BE compared with normal stomach (data from Owen et al. 2018). (C,D) Sequential tissue sections from primary human BE (C) and GIM (D) reveal perfect concordance of HNF4A up-regulation with CDX2 expression. Nonmetaplastic stomach epithelium (dotted area in C; asterisk in D) shows low basal HNF4A and no CDX2, whereas areas of IM with goblet cells (arrowheads; see also the corresponding hematoxylin and eosin-stained sections in Supplemental Fig. S5A) show high HNF4A and CDX2. Additional examples are in Supplemental Figure S5. (E, top) ATAC-seq data tracks on isogenic organoid lines showing increased ATAC activity at the Cdx2 promoter in HNF4A⁺ compared with control GFP⁺ gastric organoids. HNF4A binding, assessed by CUT&RUN (CnR), is not detected at the promoter but at a previously accessible area immediately downstream from the 3′ untranslated region (blue box); GFP⁺ organoids lack HNF4A binding at this candidate cis-element. (Bottom) ATAC-seq tracks from primary mouse gastric and intestinal Lgr5⁺ stem cells and H3K27ac ChIP-seq tracks from mouse stomach and intestinal epithelium. The 3′ Cdx2 enhancer is accessible (ATAC+) but inactive (H3K27ac−) in the stomach and encompassed within a superenhancer in the intestine. (F) The novel 3′ Cdx2 shadow enhancer is homologous in mice and humans; a representative 42-bp region within the HNF4A CnR peak (red) shows one base mismatch and shared HNF4A motifs. Pooled (pseudo-bulk) scATAC-seq data from all epithelial cells in an intestinal and two independent human stomach samples (Singh et al. 2021) show chromatin accessibility in both tissues, centered on the HNF4A motif (asterisk).