Resistance to 5-aza-2'-deoxycytidine in genic regions compared to non-genic repetitive sequences

Abstract

The DNA methyltransferase (Dnmt) inhibitor and demethylating agent 5-aza-2'-deoxycytidine (5azadC) has been used to induce cellular differentiation and gene activation. It has been approved for treating several kinds of malignancies due to its ability to reactivate silenced tumor suppressor genes. Considering the potential effect of 5azadC on non-targeted genomic regions in normal cells, we investigated its effect on repetitive sequences and selected gene loci, Oct-4, Sall3, Per1, Clu, Dpep1 and Igf2r, including tissue-dependent and differentially methylated regions, by treating mouse NIH/3T3 fibroblast cells with concentrations of 5azadC ranging from 0.001 to 5 μM. Demethylation of minor satellite repeats and endogenous viruses was concentration dependent, and the demethylation was strong at 1 and 5 μM. In genic regions, the methylation level decreased only at 0.1 μM, but was minimally altered at concentrations lower or higher, regardless of the abundance of CpG sites. Thus, repeats are strongly demethylated, but genic regions are only demethylated at effective doses. Genes were activated by 5azadC treatment and were accompanied by a unique combination of histone modifications in genic regions, including an increased level of H3K9me3 and a decreased level of AcH3. Increase of H3K9me3 in genic regions was not observed in Dnmt knock out cells. We identified differential effects of 5azadC on repetitive sequences and genic regions and revealed the importance of choosing appropriate 5azadC doses to achieve targeted gene recovery. © 2010 by the Society for Reproduction and Development.