Demethylation by 5-aza-2'-deoxycytidine in colorectal cancer cells targets genomic DNA whilst promoter CpG island methylation persists

Colorectal cancer cell lines were treated with 5-aza-dC, with and without TSA, to analyse global methylation decreases by High Performance Liquid Chromatography (HPLC). Re-methylation was observed with removal of drug treatments. Expression arrays identified silenced genes with differing patterns of expression after treatment, such as short term reactivation or long term reactivation. Sodium bisulfite sequencing was performed on the CpG island associated with these genes and expression was verified with real time PCR.Treatment with 5-aza-dC was found to affect genomic methylation and to a lesser extent gene specific methylation. Reactivated genes which remained expressed 10 days post 5-aza-dC treatment featured hypomethylated CpG sites adjacent to the transcription start site (TSS). In contrast, genes with uniformly hypermethylated CpG islands were only temporarily reactivated.These results imply that 5-aza-dC induces strong de-methylation of the genome and initiates reactivation of transcriptionally inactive genes, but this does not require gene associated CpG island de-methylation to occur. In addition, for three of our selected genes, hypomethylation at the TSS of an epigenetically silenced gene is associated with the long term reversion of gene expression level brought about by alterations in the epigenetic status following 5-aza-dC treatment.DNA methylation is an epigenetic modification that occurs on cytosine residues in the sequence context 5'-CG-3'. It is well established that DNA methylation acts as a transcriptional repressor of gene expression via recruitment of repressive proteins. These include the Methyl-CpG Binding Protein 1 (MeCP1) and proteins with a methyl-binding domain, such as MBD1, MBD2, MBD3, MBD4 and MeCP2. These proteins hinder transcription through the recruitment of other factors such as nucleosome remodelling complex [1]. In the case of MeCP2, the protein is capable of binding to a single symmetrically methylated cytosine and contributing