O'Gorman A, Colleran A, Ryan A, Mann J, Egan LJ. Regulation of NF-kappa B responses by epigenetic suppression of I kappa B alpha expression in HCT116 intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 299: G96-G105, 2010. First published April 8, 2010; doi:10.1152/ajpgi.00460.2009.-Intestinal epithelial cells play critical roles in regulating mucosal immunity. Since epigenetic factors such as DNA methylation and histone modifications are implicated in aging, carcinogenesis, and immunity, we set out to assess any role for epigenetic factors in the regulation of intestinal epithelial cell immune responses. Experiments were conducted using the HCT116 cell line, and a subclone was genetically engineered to lack DNA methyltransferases (DNMT). The induction of the chemokine interleukin-8 and the antiapoptotic protein cFLIP by tumor necrosis factor-alpha were markedly less in HCT116 cells lacking DNMT than in parental cells. These effects were accompanied by lower monocyte chemotaxis and higher caspase signaling in HCT116 cells lacking DNMT than parental cells. Tumor necrosis factor-alpha-induced NF-kappa B activation was blocked and I kappa B alpha expression was higher in HCT116 cells lacking DNMT than in parental cells. A CpG island in the I kappa B alpha gene promoter region was found to contain variable levels of methylation in parental HCT116 cells. Chromatin immunoprecipitation analysis of histone proteins bound to the I kappa B alpha gene promoter revealed that higher levels of I kappa B alpha expression in HCT116 cells lacking DNMT compared with parental cells were accompanied by more chromatin marks permissive to gene transcription. These findings show that epigenetic factors influence the NF-kappa B system in intestinal epithelial cells, resulting in a previously unrecognized mechanism of innate immune regulation.