Correlation between the single-site CpG mutationwithin the gene promoter and the expression silencingof XIAP-Associated Factor 1 (XAF1) in human coloncancer cell lines

Abstract

Background and Aims XIAP-Associated Factor 1 (XAF1) is anovel inhibitor of X-linked inhibitor of apoptosis protein (XIAP).TheXAF1 mRNA is expressed at low or undetectable levels in variouscancer cell lines, suggesting that the silencing expression of XAF1 incancer cells is important for malignant cell survival.The mechanismsin regulating XAF1 expression has not been clearly determined. Inthis study, we examined the potential epigenetic gene silencing mechanisms of XAF1 in colon cancer cell lines.Methods (1) We cloned 3 different fragments around TSS (Transcription Start Site) of XAF1 gene and examined their putativepromoter activities by Luciferase Report Assay. (2) We mutated theCpG sites in fragment F291 by Site-Directed Mutagenesis techniqueand detected the change of promoter activity of this fragment byLuciferase Report Assay. (3) Genomic DNAs were isolated from 6colon cancer cell lines and normal human blood cells and the methy-lation status was determined by bisulfite DNA sequencing analysis of8 CpG sites located around TSS (nucleotides from -59 to +194).Results By bisulfite DNA sequencing method, we showed thatnormal blood cells do not display XAF1 methylation around TSS. Incontrast, all cancer cell lines except Colo 205 showed different levelsof hypermethylation which can be removed by 5¢-AZA treatment.Thefragment F291 that contains 8 sporatic CpG sites around TSS showedthe highest promoter activity, which could be downregulated by muta-tion of some CpG sites in this fragment especially the mutation at -2 CpG site, which suppressed promoter activity by over 50%. In con-trast, single-site mutation at -1 CpG site suppressed activity by only20%.Conclusions Results from our study showed that the hypermethy-lation status of sporatic CpG sites around TSS of XAF1 coincidenceswith the gene expression suggesting that the aberrant methylationcontributes at least in part to the epigenetic silencing of XAF1 gene,which may be important for malignant cell survival. In those CpGsites in XAF1 gene promoter, -2 CpG dinucleotides appears to befunctionally more important than others associated with transcrip-tional inactivation of the XAF1 gene.