Drug-Induced Differentiation Figure 1. Nucleoside drugs araC and DAC induce neuronal differentiation of NT2 EC cells. (A) Microscopic images (106 magnification) of NT2 control cells (cont.) and NT2 cells treated with DZNep, araC, DAC or RA for 3 days. Neuron-like morphology appears most pronounced for araC and DAC. (B) qRT-PCR expression analysis of neuronal differentiation markers NEFL and SNAP25 in drug-treated, RA- treated and control cells after 3 and 6 days of treatment. NEFL and SNAP25 are prominently induced by araC and DAC. All qRT-PCR measurements were repeated at least three times and internally normalised to the corresponding lamin-b and b-actin expression levels. Deoxycytidine (dC) was included as a mock treatment control. Y-axis shows fold expression changes compared to the untreated control (cont.). P-values (Student’s t-test) for the expression differences between control and treated cells for highly significant cases (p#0.01) are shown. Asterisks indicate expression values that are significantly different from controls. doi:10.1371/journal.pone.0010726.g001 knockdown of EZH2, which only resulted in a very weak activation of the HOXA cluster (Figure 3A and S3B), indicating that EZH2 degradation is not sufficient for cellular differentiation. Reactivation of HOXA genes by RA is accompanied by epigenetic changes in their histone modification patterns [8,9]. Cytarabine and DAC have not been reported to affect global histone modifications. In contrast, DZNep has been shown to cause genome-wide loss of H3K27me3 [14] and additional repressive and active histone modifications [16]. We therefore analysed the effect of drug treatment on the histone methylation pattern of the HOXA1 promoter by chromatin immunoprecipita- tion (ChIP). In control cells this region is marked by H3K27me3 and lacks the activating mark H3K4me3 (trimethylation of lysine 4 of histone H3), consistent with its silent state (Figure 3B). When HOXA1 was induced by RA, the promoter was marked mainly by H3K4me3. DZNep did not cause any significant changes, whereas the HOXA1-activating drugs araC and DAC caused a substantial increase in H3K4me3, in parallel with a reduction in H3K27me3 levels (Figure 3B). This indicates that drug-induced activation of HOXA1 is accompanied by histone modification changes, reflecting the loss of PcG repression at this target gene. Analysis of drug-dependent DNA methylation changes To further elucidate the pathways mediating drug-induced differentiation we also investigated DNA methylation. Previous studies have linked altered HOX gene expression patterns to changes in the DNA methylation status [29–31]. In addition, it has been suggested that DZNep might cause concomitant DNA methylation changes [14]. Lastly, DAC is an established inhibitor of DNA methylation and inhibition of DNA methylation has also been associated with cellular differentiation [13]. We therefore compared DZNep, araC, DAC and RA in their ability to alter HOXA methylation in NT2 cells. Methylation analysis by COBRA indicated dense DNA methylation of several HOXA CpG islands, some of which were demethylated upon incubation with DZNep or DAC, but not with araC or RA (Figure 4A). Similar results were also obtained using capillary electrophoresis to measure global cytosine methylation. Untreated NT2 cells showed a methylation level of 4.3%, which was not significantly altered after treatment with araC or RA (Figure 4B). However, treatment with DAC or DZNep significantly reduced global DNA methylation levels to 2.4% and 3.2%, respectively (Figure 4B). These results provide important confirmation for the DNA methylation inhibiting activity of DZNep [14,16]. However, DZNep did not cause degradation of DNA methyltransferase 1 (DNMT1) (Figure 4C), which represents the main pathway for DAC-induced DNA demethylation [32]. Interestingly, depletion of SAHH by RNA interference (RNAi) also led to significant demethylation of HOXA CpG islands (Figure 4D and Figure S3C), which suggests that the demethylating activity of DZNep is a consequence of the PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10726