Remodeling characteristics of H3K27me3-marked silencers in gastric signet-ring cell carcinoma and its transcriptional regulatory function

Objective‍ ‍To draw the genome-wide distribution and remodeling characteristics of H3K27me3 silencers in signet-ring cell carcinoma of the stomach (SRCC) through epigenetic sequencing technology, and to investigate their roles in transcriptional regulation in order to elucidate the regulatory mechanism of SRCC malignant progression. Methods‍ ‍The study was conducted on 35 gastric samples obtained by gastroendoscopic biopsy (15 normal and 20 SRCC tissues) from Department of Gastroenterology of Army Medical Center of PLA between January 2021 and December 2023. Multi-omics analyses, including assay for transposase-accessible chromatin with high-throughput sequencing （ATAC-seq), cleavage under targets and tagmentation （CUT&Tag) and transcriptome sequencing （RNA-seq), were performed to identify chromatin accessibility, H3K27me3 silencer regions, and transcriptional changes, with aid of Illumina NovaSeq 6000. H3K27me3 related differentially expressed genes (|Log2FC|>1, FDR<0.05) were screened using DESeq2. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to analyze the enrichment function, and Homer was employed to identify transcription factor motifs. A regulatory network was constructed using Cytoscape, and then validated using immunohistochemistry to explore its regulatory mechanism. Results H3K27me3 silencers were primarily located in distal intergenic regions (37.06%) in SRCC. Compared with the normal tissues, SRCC showed a significant reduction in H3K27me3 silencer signals (95%CI:1.34~2.30, P=0.007) with 6 257 lost sites (FDR<0.01). Integrating CUT&Tag and RNA-seq revealed 380 up-regulated immune-related genes, particularly in T cell receptor signaling (OR=4.2, 95%CI: 2.8~6.3, P=0.002). Immunohistochemistry confirmed elevated expression of transcription factor EHF (P<0.05). Conclusion‍ ‍There is the remodeling of H3K27me3 silencers in SRCC, and EHF may potentially play a crucial role in the SRCC malignant progression.