Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

Moringa (Moringa oleifera Lam.) seed extract (MSE) has anti-inflammatory and antioxidant activities. We investigated the effects of MSE enriched in moringa isothiocyanate-1 (MIC-1), its putative bioactive, on ulcerative colitis (UC) and its anti-inflammatory/antioxidant mechanism likely mediated through Nrf2-signaling pathway. Dextran sulfate sodium (DSS)-induced acute (n = 8/group; 3% DSS for 5 d) and chronic (n = 6/group; cyclic rotations of 2.5% DSS/water for 30 d) UC was induced in mice that were assigned to 4 experimental groups: healthy control (water/vehicle), disease control (DSS/vehicle), MSE treatment (DSS/MSE), or 5-aminosalicyic acid (5-ASA) treatment (positive control; DSS/5-ASA). Following UC induction, water (vehicle), 150 mg/kg MSE, or 50 mg/kg 5-ASA were orally administered for 1 or 2 wks. Disease activity index (DAI), spleen/colon sizes, and colonic histopathology were measured. From colon and/or fecal samples, pro-inflammatory biomarkers, tight-junction proteins, and Nrf2-mediated enzymes were analyzed at protein and/or gene expression levels. Compared to disease control, MSE decreased DAI scores, and showed an increase in colon lengths and decrease in colon weight/length ratios in both UC models. MSE also reduced colonic inflammation/damage and histopathological scores (modestly) in acute UC. MSE decreased colonic secretions of pro-inflammatory keratinocyte-derived cytokine (KC), tumor necrosis factor (TNF)-α, nitric oxide (NO), and myeloperoxidase (MPO) in acute and chronic UC; reduced fecal lipocalin-2 in acute UC; downregulated gene expression of pro-inflammatory interleukin (IL)-1, IL-6, TNF-α, and inducible nitric oxide synthase (iNOS) in acute UC; upregulated expression of claudin-1 and ZO-1 in acute and chronic UC; and upregulated GSTP1, an Nrf2-mediated phase II detoxifying enzyme, in chronic UC. MSE was effective in mitigating UC symptoms and reducing UC-induced colonic pathologies, likely by suppressing pro-inflammatory biomarkers and increasing tight-junction proteins. This effect is consistent with Nrf2-mediated anti-inflammatory/antioxidant signaling pathway documented for other isothiocyanates similar to MIC-1. Therefore, MSE, enriched with MIC-1, may be useful in prevention and treatment of UC.

Plasticity among cell lineages is a fundamental, but poorly understood, property of regenerative tissues. In the gut tube, the small intestine absorbs nutrients, whereas the colon absorbs electrolytes. In a striking display of inherent plasticity, adult colonic mucosa lacking the chromatin factor SATB2 is converted to small intestine. Using proteomics and CRISPR-Cas9 screening, we identify MTA2 as a crucial component of the molecular machinery that, together with SATB2, restrains colonic plasticity. MTA2 loss in the adult mouse colon activated lipid absorptive genes and functional lipid uptake. Mechanistically, MTA2 co-occupies DNA with HNF4A, an activating pan-intestinal transcription factor (TF), on colonic chromatin. MTA2 loss leads to HNF4A release from colonic chromatin, and accumulation on small intestinal chromatin. SATB2 similarly restrains colonic plasticity through an HNF4A-dependent mechanism. Our study provides a generalizable model of lineage plasticity in which broadly-expressed TFs are retained on tissue-specific enhancers to maintain cell identity and prevent activation of alternative lineages, and their release unleashes plasticity.

Enterocytes modulate the extent of postprandial lipemia by storing dietary fats in cytoplasmic lipid droplets (cLDs). We have previously shown that the integrin ligand MFGE8 links absorption of dietary fats with activation of triglyceride (TG) hydrolases that catabolize cLDs for chylomicron production. Here, we identify CES1D as the key hydrolase downstream of the MFGE8-αvβ5 integrin pathway that regulates catabolism of diet-derived cLDs. Mfge8 knockout (KO) enterocytes have reduced CES1D transcript and protein levels and reduced protein levels of the transcription factor HNF4γ. Both Ces1d and Hnf4γ KO mice have decreased enterocyte TG hydrolase activity coupled with retention of TG in cLDs. Mechanistically, MFGE8-dependent fatty acid uptake through CD36 stabilizes HNF4γ protein level; HNF4γ then increases Ces1d transcription. Our work identifies a regulatory network that regulates the severity of postprandial lipemia by linking dietary fat absorption with protein stabilization of a transcription factor that increases expression of hydrolases responsible for catabolizing diet-derived cLDs.

Autophagy defects are a risk factor for inflammatory bowel diseases (IBDs) through unknown mechanisms. Whole-body conditional deletion of autophagy-related gene (Atg) Atg7 in adult mice (Atg7Δ/Δ) causes tissue damage and death within 3 mo due to neurodegeneration without substantial effect on intestine. In contrast, we report here that whole-body conditional deletion of other essential Atg genes Atg5 or Fip200/Atg17 in adult mice (Atg5Δ/Δ or Fip200Δ/Δ) caused death within 5 d due to rapid autophagy inhibition, elimination of ileum stem cells, and loss of barrier function. Atg5Δ/Δ mice lost PDGFRα+ mesenchymal cells (PMCs) and Wnt signaling essential for stem cell renewal, which were partially rescued by exogenous Wnt. Matrix-assisted laser desorption ionization coupled to mass spectrometry imaging (MALDI-MSI) of Atg5Δ/Δ ileum revealed depletion of aspartate and nucleotides, consistent with metabolic insufficiency underlying PMC loss. The difference in the autophagy gene knockout phenotypes is likely due to distinct kinetics of autophagy loss, as deletion of Atg5 more gradually extended lifespan phenocopying deletion of Atg7 or Atg12. Thus, autophagy is required for PMC metabolism and ileum stem cell and mammalian survival. Failure to maintain PMCs through autophagy may therefore contribute to IBD.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.