Chromatin immunoprecipitation (ChIP) using IgG isotype control, anti-Ezh2, and anti-H3K27me3 antibodies.

<p>(<b>A</b>) ChIP-Ezh2-quantitative PCR (qPCR) analysis. The graph shows quantitative PCR on DNA purified from ChIP-Ezh2, using promoter primers for genes associated with selected peaks from the ChIP-Seq data. In NSCs ChIP-Ezh2, there is high enrichment (Ezh2 occupancy) of neuronal, astrocytic and oligodendrocytic lineages determining genes while in pOLs ChIP-Ezh2 only neuronal and astrocytic lineage specific genes show enrichment (occupancy by Ezh2). (<b>B</b>) ChIP-H3K27me3-qPCR analysis. A similar pattern of gene enrichment can be observed in PCR analysis on DNA purified from ChIP-H3K27me3 (trimethylation of H3K27 is a functional mark of Ezh2): in NSCs, there is high enrichment (trimethylation of H3K27) of neuronal, astrocytic and oligodendrocytic lineages determining genes while in pOLs only neuronal and astrocytic lineage specific genes show high enrichment (higher level of trimethylation of H3K27). <i>Rpl32</i> (the gene encoding the 60S ribosomal protein L32, a non-target of Ezh2) was used as a negative control. <i>Acute Ezh2 reduction in NSCs and pOLs mediated by Ezh2-shRNA.</i> (<b>C</b>) Western blot shows the reduction in Ezh2 protein in comparison to control. Lysates were prepared after <i>in vitro</i> transfection with Ezh2-shRNA and non-coding shRNA (nc-shRNA) as control, in both NSCs and pOLs. Reduction in Ezh2 protein resulted in a decrease in the global level of H3K27 tri-methylation, an Ezh2-associated gene repression mark (17 kDa band). (<b>D</b>) Derepression of tested Ezh2 target genes after knocking down Ezh2 protein expression in NSCs and pOLs. The transcript levels were quantified by real-time PCR, normalized to GAPDH (housekeeping gene) and represented as a fold change between nc-shRNA (control) and Ezh2-shRNA in both NSCs and pOLs. Bars represent mean of three independent experiments ± S.D.