EZH2 mediates an EMX2-to-HOXB9 transcriptional switch upon neoplastic transformation A.Figure 3Schematic representation of EMX2 and HOXB9 expression in the embryonic nervous system. ChIP-seq signal for EZH2 and H3K27me3 (left) at EMX2 and HOXB9 loci. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. mRNA expression of EMX2 and HOXB9 (right) as detected by RNA-seq. Expression values represent mean ± SEM from three biological replicates. UT: untransformed, PN: pre-neoplastic, TR: transformed. TPM: transcripts per million. qRT-PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days. Values represent mean ± SEM from three biological replicates. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). UT: untransformed, TR: transformed. Expression levels of EMX2 and HOXB9 in primary human neural cells, as detected by RNA-seq. Data sourced from Brainseq2. Expression values represent mean ± SEM from four, twelve, one and five biological replicates of fetal astrocytes, mature astrocytes, neurons and oligodendrocytes, respectively. FPKM: fragments per kilobase of transcript per million. Expression levels of EMX2 and HOXB9 in 62 glioma cell lines. Data sourced from CCLE. Every dot represents a cell line. RPKM: reads per kilobase of transcript per million. In the boxplot, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. Quantification of EZH2 binding at EMX2, HOXB9 and GAPDH (negative control) promoters by ChIP-qPCR in M059K GBM cells. Values from two biological replicates are shown. Three asterisks indicate p-value < 0.001 of EZH2 samples relative to the negative control GAPDH (two-way ANOVA followed by pairwise comparisons using Holm-Sidak method). Ns: non-significant. Quantification of EMX2 levels by qRT-PCR in five different GBM cell lines upon treatment with 1μM, 3μM or 10μM of EZH2i for eight days, or a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Two asterisks indicate p-value <0.01 comparing EZH2i- and DMSO-treated cells (Two-way ANOVA). Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines. Values for EMX2 upregulation are the fold-change in mRNA expression induced upon treatment with 10μM EZH2i (see: panel G), and are expressed relative to a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines. All data sourced from the CCLE. The significance of the anti-correlation between mRNA levels and DNA methylation levels across cell lines is indicated (Spearman rank correlation). Heatmap visualizing the fold change in EMX2 expression as detected by qRT-PCR after treatment of the indicated PDX-derived (blue) and established cancer cell lines (black) with EZH2i for eight days. Values are from three technical replicates.