DNA demethylating agents can function as cytosine analogues and/or as Dnmt/DNMT inhibitors [56]. Therefore, we investigated Dnmt expression levels in decitabine-treated differentiating NSC by qRT-PCR. In accordance with reduced DNA methylation levels at D2, decitabine treatment caused significant inhibition of transcript levels of all three DNA methyltransferases (Dnmt1, 1.7-fold, P <0.05; Dnmt3a, 1.5-fold, P <0.05 and Dnmt3b, 2.5-fold, P <0.01) (Figure 4E). Even though we anticipated that decitabine withdrawal would restore Dnmt levels, only Dnmt1 levels were elevated (2.2-fold, P <0.05), whereas both Dnmt3a (1.4-fold, P <0.05) and Dnmt3b (1.8-fold, P = 0.06) levels remained inhibited (Figure 4I). In summary, these results indicate that decitabine functions as a DNA demethylating agent in differentiating NSC and globally affects DNA methyl marks. Additionally, our data indicate that decitabine withdrawal would lead to DNA methylation reprogramming in differentiating NSC.
