DNA Methylation Profiling of Human Colonoid Monolayers Under Repeated Submergence Injury

Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells to a stem cell state, but there is a lack of knowledge on how these stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged growth. We used this model to understand how chronic intestinal damage affects the ability of IECs to respond to microbial stimulation, using the TLR5 agonist FliC; and to regenerate and protect the epithelium from further damage. Here, we profiled the effect of repeated rounds of damage on global DNA methylation levels of these human colonid monolayers. We observed a trend towards global demethylation with repeated damage, including in loci associated with genes implicated in DNA repair (NHEJ1) and colon hemostatsis and inflammation regulation (FAM3D). Genome-wide DNA methylation was assessed from 12 cultured human colonoid samples (including 2 replicates) using the Illumina Infinium Human MethylationEPIC BeadChip array. The samples were derived from the same donor but tested over two distinct passages, cultured in air-liquid interface(ALI) for seven days following full confluency, and harvested at 7 days-post culture (differentiated), or after post-submergence injury after differentiation with or without flagellin (at 1 round and 5 rounds of damages).