Figure 2.

Functional coordination of direct METTL3 targets with epigenetic/transcriptional activation. (A) The abundance of m⁶A-modified transcripts was compared with non-modified transcripts. FPKM (fragments per kilobase of transcript per million mapped reads) of each group was shown as cumulative fraction. m⁶A modified transcripts were high in expression. (B) The cumulative distribution of fold change (shMETTL3-shNT) among methylated transcripts and unmethylated genes was compared. Methylated targets were significantly inhibited by METTL3silencing compared to unmethylated genes (p-value = 0.00001). (C) (Left panel) Gene set enrichment analysis (GSEA) enrichment plot showed a depletion of the GSC-specific transcriptionally activated gene set (ACTIVE_ON_GSC_NSC) compared to Neuronal stem cells (NSC) in METTL3-regulated targets (shMETTL3-shNT). The active gene set contained genes with H3K4me3 (active mark) in their promoters. (Right panel) The normalized read counts of genes (shNT-GSC and shMETTL3-GSC) that were enriched for the ACTIVE_ON_GSC_NSC gene set are depicted as heatmap. Red and green denotes high and low expression, respectively. The majority of genes that are activated by active histone mark in GSCs were downregulated in METTL3-silenced condition. (D) (Left panel) GSEA enrichment plots showing a depletion of the GSC-specific transcriptionally activated gene set (ACTIVE_ON_GSC_DGC) compared to Differentiated glioma cells (DGC) in METTL3-regulated targets (shMETTL3-shNT). The active gene set contained genes with H3K4me3 (active mark) in their promoters. (Right panel) The normalized read counts of genes (shNT-GSC and shMETTL3-GSC) that were enriched for the ACTIVE_ON_GSC_DGC gene set are depicted as heat map. Red and green denotes high and low expression, respectively. The majority of genes that are activated by active histone mark in GSCs were downregulated in METTL3-silenced condition. (E) (Left panel) GSEA enrichment plots showing a depletion of the GSC-specific transcriptionally activated gene set (ACTIVE_ON_ GSC_DGC_H3K27AC) compared to DGC in METTL3-regulated targets (shMETTL3-shNT). The active gene set contained genes with H3K27Ac (active mark) in MGG8 GSCs but not in MGG8 DGCs. (Right panel) The normalized read counts of genes (shNT-GSC and shMETTL3-GSC) that were enriched for ACTIVE_ON_ GSC_DGC_H3K27AC were depicted as heat map. Red and green denotes high and low expression, respectively. The majority of genes that are activated by active histone mark in GSCs were downregulated in METTL3-silenced condition. (F) Heat maps are shown depicting:(a) normalized read counts in shNT RNA-Seq and shMETTL3 RNA-Seq; and(b) the m⁶A enrichment score obtained in shNT RIP-Seq and shMETTL3 RIP-Seq for 19 TFs identified [2] as reprogramming factors in glioma cells. High and low expression values (log₂) are denoted by red and green, respectively. High m⁶A peak enrichment is represented by aqua green and the absence of a peak is depicted by white. (G) The genomic tracks of reprogramming transcription factors (TFs) SOX2, SALL2, POU3F2, and OLIG2 are depicted in shNT and shMETTL3-GSCs. Normalized sequence coverage of m⁶A RIP peaks and RNA-Seq reads is indicated above the gene architecture in UCSC format. Thin black boxes represent the 5′ and 3′UTRs; thick black boxes represent the coding sequences; and thin lines represent introns. Among the four, only SOX2 showed m⁶A RIP peaks in control GSCs, which was depleted in shMETTL3 GSCs. (H) m⁶A enrichment and transcript level was measured for four reprogramming factors by m⁶A-RIP-PCR and RT-qPCR. The enrichment was calculated compared to IgG Ct values. (I) m⁶A enrichment in shNT-GSCs and transcript level changes with METTL3-silencing in MGG8 GSCs was measured by real-time PCR. The enrichment was calculated compared to IgG Ct values. The ddCt value of differential expression was calculated by normalizing with ATP5G.