Remodeling of enhancers in high-grade epithelial dysplasia of gastric mucosa and its effect on expression of proliferation-related gene CD24

Objective‍ ‍To identify the enhancer profile marked by histone H3K27ac modification in high-grade intraepithelial neoplasia (HGIN) in order to reveal the novel regulatory mechanism of HGIN pathogensis. Methods‍ ‍Gastric tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA between June 2022 and June 2023, including 14 normal gastric tissues (Nor group), 31 HGIN tissues (HGIN group) and 17 gastric cancer tissues (GC group). Cleavage under targets and tagmentation (CUT&Tag) technique was employed to capture enhancer regions modified by histone H3K27ac. Multi-omics analysis was performed to identify HGIN-specific active enhancers and their potentially regulated genes. Immunohistochemical profiling was performed to assess differential expression of the gene of interest across clinically stratified specimens, combined with CRISPR-dCas9-mediated ablation of active enhancers to monitor the gene of interest transcriptional dynamics and validate enhancer-mediated regulatory mechanisms. Results‍ ‍Epigenomic sequencing obtained the data with excellent quality, and indicated that obvious remodeling was observed in H3K27ac enhancers in HGIN and GC groups (P<0.05), though no significant difference in the genome-wide distribution of H3K27ac modification among the 3 groups.Combining transcriptome data revealed that enhancer remodeling may up-regulate the expression of the proliferation-related target gene, CD24, in the HGIN tissue; while, inhibiting enhancer activity can notably reduce CD24 expression level (P<0.05). Immunohistochemical assay displayed a positive correlation between the expression levels of CD24 and Ki-67 (P<0.001). Conclusion‍ ‍The remodeling of H3K27ac enhancer represents a significant epigenetic feature of the transformation from normal condition to HGIN. Remodeling of H3K27ac enhancer up-regulates CD24, which may facilitate the abnormal proliferation of gastric epithelial cells.