Deletion of Metal Transporter Zip14 (Slc39a14) Reduces Major Histocompatibility Complex II Expression in Murine Small Intestinal Epithelial Cells

Documented worldwide, impaired immunity is a cardinal signature resulting from loss of dietary zinc, an essential micronutrient. A steady supply of zinc to meet cellular requirements is regulated by an array of zinc transporters. Deletion of the transporter Zip14 (Slc39a14) in mice produced intestinal inflammation. Elevated fecal lipocalin-2, calprotectin, IgG levels and dysbiosis support the inflammatory phenotype. Here we show through RNA-sequencing, using purified intestinal epithelial cells (IECs), that Zip14 deletion produces markedly reduced expression of major histocompatibility complex class II (MHCII) molecules and the master MHCII trans-activator (Ciita). qPCR, western analysis and immunohistochemistry confirmed loss of MHCII. Spectrofluorimetry with zinc probe FluoZin-3 showed reduced labile zinc in IECs from knockout mice. Chromatin immunoprecipitation assays, using Ciita antibody and IEC chromatin, suggest decreased transcription accounts for depressed expression of specific MHCII genes. ATAC-sequencing (ATAC-seq) demonstrated that H2-Aa, H2-Ab1 and other MHCII genes result from chromatin remodeling yielding closed chromatin at regulatory regions of these genes. In agreement, ATAC-seq showed peak density of the chromosomal regulatory region of Ciita is consistent with down regulation of specific MHCII genes in IECs with Zip14 loss. Finally, dietary zinc supplementation of knockout mice and zinc supplementation of intestinal organoids in-vitro with Zip14 deletion restored transcript levels. Taken together, our data suggest that cellular zinc delivery, via Zip14, is necessary for proper chromatin occupancy, required for normal MHCII expression and effective immune functions, and to preclude inflammatory disorders of the small intestine. Overall design: Intestinal epithelial cells were isolated and purified for flow cytometry and cell sorting. The preserved IECs were sent to MedGenome, Foster City, CA, USA). for library preparation and Assay for Transposase-Accessible Chromatin (ATAC) sequencing. Briefly, nuclei were isolated from the IECs and incubated with pre-loaded Tn5 transposase (Cat # FC-121-1030, Illumina, CA, USA) and sequencing adapters. The assembled protein A-Tn5 adapter transposome was activated by Mg2+ to generate adapter-flanked DNA fragments cut near the proteins of interest. The transposed DNA was then extracted, and PCR amplified using NEBNext High-Fidelity PCR Master Mix (Cat # M0541S, New England Biolabs, MA, USA). The library was purified with AMPure XP Magnetic Beads (Cat # A63880, Beckman Coulter, CA, USA), and its quality was assessed using a BioAnalyzer High-Sensitivity DNA Analysis kit (Cat # 5067–4626, Agilent, CA, USA).