Intestinal Zip14 ablation dysregulates tight junction protein expression, inflammatory genes and lncRNAs in intestinal epithelial cells and enteroids.

The mechanisms by which Zip14 ablation mediates mild intestinal inflammation and decreased barrier function are not completely understood. We used RNA sequencing to profile the transcriptome of intestinal epithelial cells (IEC) isolated from Zip14 Cre/flox and control flox/flox mice. RNA-seq analysis identified 1,345 differentially expressed genes in Zip14 Cre/flox mice compared with control mice. In-silico analyses revealed that Zip14ablation promoted dysregulation of specific proinflammatory genes (e.g. IL-6 and Ifn-γ) and genes influencing intestinal barrier homeostasis (specifically, Claudins 1-and-2). Highly dysregulated genes were selected for confirmation by qPCR, and proteins by western analysis, using extracts from isolated IECs from mice of the two genotypes. Highly upregulated genes in the Zip14 Cre/flox mice included Fabp6, Cldn2and Cldn8, while downregulated genes included Cldn1. To explore the possibility that Zip14ablation may alter transcriptional activity, ChIP assays of selected promoters were conducted. Increased binding of Stat3, FXR and NF-kB was observed in Cldn2, FABP6, and Cldn8promoters, respectively, with chromatin from the Zip14 Cre/floxmice compared to control mice. By contrast, Cdx2 binding was decreased for the Cldn1promoter site of the knockout mice. Zip14 ablation also altered expression levels of long noncoding RNAs (lncRNAs). Specifically, the lncRNAs H19, U90926, and Meg3 were highly upregulated, and Gata4 was downregulated as validated by qPCR. Using organoids derived from intestines of mice of both genotypes, it was demonstrated that growth was not impaired with Zip14 ablation. Other biochemical markers of Zip14ablation were recapitulated in organoid cultures. This included indices of enhanced Zn trafficking and differential expression of specific tight junction genes. Decreased HDAC3 activity in IECs from the Zip14 knockout mice suggest that epigenetic modifications account in part for the genomic changes observed. We conclude zinc transported by Zip14 controls intestinal permeability through epigenetic modifications, thus representing a novel health-promoting role for this micronutrient.