Figure 1.
Figure 1. Induction of DNA demethylation by RUNX1 overexpression. (A) Confirmation of RUNX1 overexpression by western blotting. (B) Scatter plot showing DMCs caused by RUNX1 overexpression in 293T cells. x- and y-axes show M-values for 293T-mock and RUNX1-overexpressing 293T (293T-RUNX1) cells, respectively. Dashed lines represent ΔM borders of >2. Green, purple, and gray dots represent significantly methylated, demethylated, and insignificant probes, respectively. Numbers of DMCs are shown in the upper left (methylated) and bottom right (demethylated). Larger purple and gray dots represent targets and controls of qMSP analysis (Figures 2B and 4G; supplemental Figure 2). An enlarged view of the area surrounded by dashed red lined is shown in the left, and probe identifications of the targets and controls of qMSP analysis are labeled. (C) Distribution of enrichment scores for RUNX1-binding motifs within ±5000 bp of demethylated CpGs in RUNX1-overexpressing 293T cells. x- and y-axes indicate distance from probe CpG position and enrichment score, respectively. (D) ZENBU browser screenshots showing typical relationships between RUNX1 binding and the demethylated CpGs at cg07236781 (left) and cg03333149 (right) regions in RUNX1-overexpressing 293T cells. Demethylated CpG tracks show positions of demethylated CpG. ChIP-seq peaks and ChIP-seq tracks show the peak positions and tag per million (tpm), respectively. (E) A histogram showing distribution of RUNX1 ChIP-seq peaks in RUNX1-overexpressing 293T cells around demethylated CpGs (pink) and randomly selected probes (blue). Overlapped regions are shown as purple. x- and y-axes show distance from CpG (bp) and frequency of ChIP-seq peak, respectively. (F) DNA methylation patterns of PTPN22 and RUNX3 regions were determined using bisulfite sequencing in mock vector overexpressing 293T cells (293T-mock) and RUNX1-overexpressing 293T cells (293T-RUNX1). Horizontal lines show sequencing results for each subclone. Arrows represent positions of demethylated probes that were identified by methylation arrays. Circles represent cytosines of CpGs: black, methylated; white, unmethylated. Significant demethylation: *P < .05; **P < .01. Percentages and P values are shown at the bottom right. Percentages of methylated cytosines among all cytosines in the target region of all measured subclones were compared using Fisher’s exact test.

Induction of DNA demethylation by RUNX1 overexpression. (A) Confirmation of RUNX1 overexpression by western blotting. (B) Scatter plot showing DMCs caused by RUNX1 overexpression in 293T cells. x- and y-axes show M-values for 293T-mock and RUNX1-overexpressing 293T (293T-RUNX1) cells, respectively. Dashed lines represent ΔM borders of >2. Green, purple, and gray dots represent significantly methylated, demethylated, and insignificant probes, respectively. Numbers of DMCs are shown in the upper left (methylated) and bottom right (demethylated). Larger purple and gray dots represent targets and controls of qMSP analysis (Figures 2B and 4G; supplemental Figure 2). An enlarged view of the area surrounded by dashed red lined is shown in the left, and probe identifications of the targets and controls of qMSP analysis are labeled. (C) Distribution of enrichment scores for RUNX1-binding motifs within ±5000 bp of demethylated CpGs in RUNX1-overexpressing 293T cells. x- and y-axes indicate distance from probe CpG position and enrichment score, respectively. (D) ZENBU browser screenshots showing typical relationships between RUNX1 binding and the demethylated CpGs at cg07236781 (left) and cg03333149 (right) regions in RUNX1-overexpressing 293T cells. Demethylated CpG tracks show positions of demethylated CpG. ChIP-seq peaks and ChIP-seq tracks show the peak positions and tag per million (tpm), respectively. (E) A histogram showing distribution of RUNX1 ChIP-seq peaks in RUNX1-overexpressing 293T cells around demethylated CpGs (pink) and randomly selected probes (blue). Overlapped regions are shown as purple. x- and y-axes show distance from CpG (bp) and frequency of ChIP-seq peak, respectively. (F) DNA methylation patterns of PTPN22 and RUNX3 regions were determined using bisulfite sequencing in mock vector overexpressing 293T cells (293T-mock) and RUNX1-overexpressing 293T cells (293T-RUNX1). Horizontal lines show sequencing results for each subclone. Arrows represent positions of demethylated probes that were identified by methylation arrays. Circles represent cytosines of CpGs: black, methylated; white, unmethylated. Significant demethylation: *P < .05; **P < .01. Percentages and P values are shown at the bottom right. Percentages of methylated cytosines among all cytosines in the target region of all measured subclones were compared using Fisher’s exact test.

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