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

Abstract

Background: DNA methylation and histone acetylation are epigenetic modifications that act as regulators of gene expression. Aberrant epigenetic gene silencing in tumours is a frequent event, yet the factors which dictate which genes are targeted for inactivation are unknown. DNA methylation and histone acetylation can be modified with the chemical agents 5-aza-2'-deoxycytidine (5-aza-dC) and Trichostatin A (TSA) respectively. The aim of this study was to analyse de-methylation and re-methylation and its affect on gene expression in colorectal cancer cell lines treated with 5-aza-dC alone and in combination with TSA. We also sought to identify methylation patterns associated with long term reactivation of previously silenced genes.

Method: 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.

Results: 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.

Conclusion: 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.

Figure 1

Global re-methylation in cell lines. All cells exhibited demethylation after 5-aza-dC treatment, and re-methylation occurred over the 10 days of drug free growth.

Figure 2

Selection of genes for bisulphite sequencing analysis. Shown in the heatmap are the genes reactivated in the SW480 cell line. Filters were applied to select silenced genes that were reactivated upon 5-aza-dC treatment. Genes demonstrating differential expression between cell lines were chosen for further analysis.

Figure 3

MAGEA3 and ZFP3 CGI methylation and expression with 5-aza-dC treatment. Figure 3A illustrates hypomethylated cytosines at the TSS in otherwise hypermethylated CpG island and the corresponding expression (B). Figure C and D shows CGI hyper-methylation and expression of the temporarily reactivated ZFP3 gene in HT29 cells.