To identify epigenetically-regulated genes in breast cancer, MCF-7 cells were exposed to 250 nM 5-aza or 5-aza + 50 nM TSA for three weeks followed by a five week recovery period after treatment withdrawal and gene expression patterns were examined by microarray analysis. We identified 20 genes that are associated with a > or = 2-fold increase in expression in response to the demethylating treatment but returned to control levels after treatment withdrawal. RT-PCR verified that the genes identified were expressed at low or undetectable levels in control MCF-7 cells, but increased expression in treated cells. Most of these putative epigentically-regulated genes in MCF-7 cells do not contain CpG islands. In fact, these genes could be classified based upon their promoter CpG features, including genes with: (1) typical CpG features (CpG islands), (2) intermediate CpG features (weak CpG islands), and (3) atypical CpG features (no CpG islands). Prototype genes from each class (including CpG-deficient genes) were shown to be methylation-sensitive (subject to CpG methylation and responsive to demethylating agents), suggesting that not all gene targets of DNA methylation in breast cancer will contain a CpG island. Based upon the results of the current study and observations from the literature, we propose expansion of the current model for methylation-dependent regulation of gene expression to include genes lacking typical CpG islands. The expanded model we propose recognizes that all promoter CpG dinucleotides represent legitimate targets for DNA methylation and that the methylation of specific CpG dinucleotides in critical domains of regulatory regions can result in gene silencing.