Drug-Induced Differentiation Figure 3. Nucleoside drugs affect Polycomb repression in NT2 cells. (A) qRT-PCR expression analysis of HOXA genes (1 to 13 - orthologs 8 and 12 are absent in the HOXA-cluster) after drug treatment for 3 days or knockdown of EZH2 (kd EZH2) for the same time period. Collinear activation of the cluster, similar to RA-treatment (positive control), could only be observed for araC- and DAC-treated NT2 cells. All treatments and measurements were repeated at least five times and internally normalised to the corresponding lamin-b and b-actin expression levels. Y-axis values indicate fold induction compared to the non-treated control. Knockdown of EZH2 was repeated three times in independent experiments (see Figure S3B). Deoxycytidine (dC) was included as a mock treatment control. (B) Chromatin immunoprecipitation (IP) analysis of the HOXA1 promoter region in RA-, drug-treated and control NT2 cells. IP was repeated at least three times with chromatin from biological replicates using antisera specific for H3K27me3 and H3K4me3. Immunoprecipitated DNA was analyzed by real time PCR and a primer specific for the HOXA1 promoter region. Enrichments are shown as percentage of the total input. doi:10.1371/journal.pone.0010726.g003 of SNAP25. Depletion of SAHH, DNMT1, EZH2, NANOG did induce minor changes in SNAP25 expression, but only the knockdown of OCT4 clearly induced neuronal differentiation, and, in parallel, significantly increased expression of SNAP25 (Figure S5A and S5B). Combined knockdown of OCT4 and NANOG or OCT4, NANOG and EZH2, did not enhance the effect. NANOG expression is already strongly reduced in cells depleted for OCT4 (Figure S5B), as OCT4 is a major activator of the NANOG gene [5]. This also explains the lower levels of NANOG transcription upon drug treatment (Figure 5D). Nevertheless, depletion of stem cell factors, in particular OCT4, by RNAi shows similar effects on NT2 cells as treatment with araC and DAC and is sufficient to induce differentiation. To confirm the involvement of proteolytic pathways in drug- induced NANOG and OCT4 degradation, we analysed the expression levels of several key regulatory factors of these pathways in a time course experiment by RT-PCR. As expected, we observed a significant induction of the CASPASE-7 gene by araC and DAC, but also RA (Figure 6). Cytarabine and DAC also triggered moderate upregulation of CASPASE-3 and the c-Jun N- terminal kinase II (JNKII). This was paralleled by the expression of the differentiation marker SNAP25 (Figure 6). We observed no significant changes in gene expression for the apoptotic factor p53, for c-Jun N-terminal kinase I (JNKI), CASPASE-9 and EZH2 (Figure 6). Thus, CASPASE-7, and, to a lesser extent, CASPASE-3, seem to be critical factors in araC- and DAC-induced degradation processes. Drug-induced differentiation is caused by CASPASE-7- dependent degradation of OCT4 In order to further prove the role of caspases in drug-induced differentiation, we combined nucleoside drug treatment with caspase inhibitors (Inhibitor I and II) and a JNKII inhibitor (JNK Inhibitor I). These substances caused a significant reduction of the ability of araC or DAC to activate HOXA1 and SNAP25, whereas RA-mediated induction of these genes and CASPASE-7 expression was not significantly affected (Figure 7A). In addition, all three inhibitors partially rescued OCT4- and EZH2-degradation PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10726