Figure 3: Zfp423 is downregulated in PT upon H3K27 trimethylation and its binding motif is overrepresented among DEG promoters.

(a) Left: heatmap representing the gene expression levels (red: increased expression, blue: reduced expression) between AstroEGFR* and PT of the most significant, overrepresented TFs identified with Pscan (Bonferroni P-value<0.05). The second heatmap represents H3K27me3 status of the promoter of the same genes (white: unmethylated, green: methylated). Right: Zfp423-binding motif. (b) Left: IGV visualization of the RNAseq tracks for Zfp423. Right: analysis of transcript levels measured by qRT–PCR in different cell populations. Data are represented as dCt (log₂ scale) relative to Tbp. Each symbol represents a different sample for the specified category; black bars within the symbols represent the median. (*P<0.05 and **P<0.01) (P-values were calculated using the Mann–Whitney test). (c) Top: IGV visualization of the H3K27me3 ChIPseq tracks for the Zfp423 gene. Bottom: ChIP–qPCR validation of the H3K27me3 enrichment. Data represent the fold increase of the K27me3-positive region of the promoter normalized on the total H3 (K27⁺) over a K27me3-negative region in close proximity, again normalized on the total H3 (K27⁻). Each symbol represents a different sample in each category; black bars within the symbols represent the median (*P<0.05 and **P<0.01) (P-values were calculated using the Mann–Whitney test). (d) Top: schematic representation of a different glioma mouse model in which Zfp423 downregulation was confirmed. Neural precursors (NPs) were isolated from telencefalic vesicles of embryos at 13.5 dpc. Cells were infected with the mouse Pdgf-β and transplanted stereotaxically into recipient mice. GPCs were derived from PTs. Bottom: Zfp423 messenger RNA levels were measured in the different cell populations by qRT–PCR. Data are represented as dCt (log₂ scale) relative to Tbp; error bars represent the s.d. of the technical replicates.