DNA Methylome Mapping Identifies Epigenetic Abnormalities in Intestinal Lymphocyte Regulation in Human Necrotizing Enterocolitis

Abstract

Background: Epigenetic changes occur in response to environmental factors during the pathogenesis of necrotizing enterocolitis (NEC) in animal models, but the DNA methylation signature in human patients with NEC has not been examined.

Aim: To illustrate the signature and function of DNA methylation in the intestine of human NEC.

Methods: DNA methyltransferases (DNMTs) were compared between intestinal tissue with NEC and control. Genome-wide DNA methylation was analyzed by reduced representation bisulfite sequencing (RRBS). The biological functions of the potential methylation regulated genes were analyzed by Gene Ontology. Gene methylation and expression were confirmed by bisulfite genomic sequencing (BGS) and RT-qPCR.

Results: By screening the expression of DNMTs, we identified a marked reduction in DNMT3A at both the mRNA and protein levels in NEC. Genome-wide variation of DNA methylation was detected in NEC lesions. The CG methylation level in almost all unique regions except CpG islands (CGIs) was lower in NEC compared with control. A total of 287 differentially methylated regions (DMRs) were identified across the whole genome in NEC, 123 of them are located on the CGI in the promoter. The DMR-associated genes were linked to intestinal epithelial permeability, platelet aggregation, and lymphocyte proliferation. Four genes (ZNF335, MPL, RASAL3, and KDM6A) with roles in the regulation of lymphocytes that may predispose the intestine to imbalanced immune processes were further confirmed to be hypermethylated and transcriptionally downregulated.

Conclusions: These findings underscore the novel relationship between epigenetic changes and lymphocyte regulation in human NEC, which may have potential diagnostic and therapeutic relevance for NEC.

Keywords: CpG island; DNA methylation; Epigenetic gene regulation; Human necrotizing enterocolitis; Lymphocyte regulation.