DNA Methylation Represses IFN-γ-Induced and Signal Transducer and Activator of Transcription 1-Mediated IFN Regulatory Factor 8 Activation in Colon Carcinoma Cells

IFN regulatory factor 8 (IRF8) is both constitutively expressed and IFN-gamma inducible in hematopoietic and nonhematopoietic cells. We have shown that IRF8 expression is silenced by DNA methylation in human colon carcinoma cells, but the molecular mechanism underlying methylation-dependent IRF8 silencing remains elusive. In this study, we observed that IRF8 protein level is inversely correlated with the methylation status of the IRF8 promoter and the metastatic phenotype in human colorectal carcinoma specimens in vivo. Demethylation treatment or knocking down DNMT1 and DNMT3b expression rendered the tumor cells responsive to IFN-gamma to activate IRF8 transcription in vitro. Bisulfite genomic DNA sequencing revealed that the entire CpG island of the IRF8 promoter is methylated. Electrophoresis mobility shift assay revealed that DNA methylation does not directly inhibit IFN-gamma-activated phosphorylated signal transducer and activator of transcription 1 (pSTAT1) binding to the IFN-gamma activation site element in the IRF8 promoter in vitro. Chromatin immunoprecipitation assay revealed that pSTAT1 is associated with the IFN-gamma activation site element of the IRF8 promoter in vivo regardless of the methylation status of the IRF8 promoter. However, DNA methylation results in preferential association of PIAS1, a potent inhibitor of pSTAT1, with pSTAT1 in the methylated IRF8 promoter region. Silencing methyl-CpG binding domain protein 1 (MBD1) expression resulted in IRFS activation by IFN-gamma in human colon carcinoma cells with methylated IRF8 promoter. Our data thus suggest that human colon carcinoma cells silence IFN-gamma-activated IRF8 expression through MBD1-dependent and PIAS1-mediated inhibition of pSTAT1 function at the methylated IRF8 promoter. (Mol Cancer Res 2008;6(12):1841-51)