FIGURE 7.

EZH2‐mediated H3K27ac enhances METTL3 locus accessibility in GBM cells. (A) Venn diagram illustrates overlap among EZH2, H3K27ac, and H3K27me3 binding sites. ChIP‐seq data were acquired from GSE128275 and GSE112240. (B) METTL3 promoter is occupied by EZH2 and H3K27ac but not by H3K27me3. ChIP‐seq data were acquired from GSE128275 and GSE112240. The y‐axis shows the normalized RPKM (per bin, bin = 25 bp) value. (C) ChIP‐qPCR analysis of EZH2 enrichment at the METTL3 promoter region in U87MG_TMZ_R cells. (D) ChIP‐qPCR analysis of H3K27ac enrichment at the METTL3 promoter region in EZH2 KD (pooled EZH2 shRNAs) or control U87MG_TMZ_R cells. (E) Average intensities of ATAC‐seq signals in EZH2 KD (pooled EZH2 shRNAs) or control U87MG_TMZ_R cells. The ATAC‐seq data of EZH2‐binding genes with H3K27ac modification and EZH2‐binding genes with H3K27me3 modification were analyzed. ATAC‐seq signal around TSSs (shadow) were compared by Student's t‐test. (F) IGV plots of ATAC‐seq peaks at METTL3 mRNAs in U87_TMZ_R cells with or without EZH2 KD (pooled EZH2 shRNAs). The y‐axis shows the normalized RPKM (per bin, bin = 25 bp) value. ATAC‐seq signal around TSS of METTL3 (shadow) was compared by MACS2. (G) mRNA expression of EZH2 and METTL3 in EZH2 KD (pooled EZH2 shRNAs) or control U87MG_TMZ_R cells. (H) Dual‐luciferase reporter assay for the effects of EZH2 KD on the luciferase activity of the METTL3 promoter (–3000 bp‐0 bp) in HEK293T and U87MG_TMZ_R cells. (I) Co‐IP analysis of the interaction between SOX4 and PRC2 complex components in U87MG_TMZ_R cells. (J) Schematic illustration of the working model. *P < 0.05; **P < 0.01; and ***P < 0.001, compared to control (Student's t‐test). All the results were obtained from three independent experiments. Values are presented as mean ± SD. n.s., no significant difference