A Novel Epigenetic Mechanism of Intestinal NHE3 Gene Regulation by Histone Acetylation

NHE3 is an essential Na+ transporter in the intestine, contributing to diarrhea associated with inflammatory bowel disease (IBD) or intestinal infections. Nevertheless, the influence of histone acetylation on intestinal NHE3 gene expression has not yet been investigated. Various methods were employed, including human intestinal epithelial cells (Caco2 cells), ex vivo mouse and human ileal enteroids, and in vivo mouse models, along with techniques such as real-time qPCR, Western blot, immunofluorescence staining, ChIP, and H3K27Ac HiChIP sequencing analyses. Our findings indicated that valproic acid (VPA, a class I HDAC inhibitor) significantly elevated both NHE3 mRNA and protein levels in IECs. ChIP and Chromatin accessibility assays revealed an enhanced enrichment of acetylated histones (H3/H4) and a more open chromatin state at the p-1329/p-541 region of the NHE3 promoter in Caco2 cells treated with VPA. HDAC2/3 inhibition by MI192 in Caco2 cells and mouse/human ileal organoids or siRNA knockdown of HDAC2 in Caco2 cells significantly increased NHE3 expression. Activation or ectopic overexpression of histone acetylase p300 in Caco2 cells increased NHE3 mRNA expression and enhanced accumulation of acetylated histone (H3) within the p-1329 to -161 bp region of the NHE3 promoter. Additionally, in vivo experiments revealed that VPA significantly increased NHE3 mRNA levels in the ileum and proximal colon of mice, along with increased NHE3 immunostaining. H3K27Ac HiChIP sequencing analysis of isolated IECs from the mouse ileum indicated that VPA increased H3K27Ac acetylation and facilitated chromatin looping between distal enhancers and the NHE3 promoter, thereby stimulating NHE3 transcription. In conclusion, HDAC2 inhibition and histone acetylation play pivotal roles in upregulating NHE3 expression, with therapeutic implications for inflammation-associated diarrhea linked to reduced NHE3 expression/function.