Figure 3.
Transcriptome analysis revealed high correlation between demethylation of marker regions and expression of the associated genes. (A) Heatmap showing the top 50 most variable genes in an unbiased hierarchical clustering of ILC1-3 and LTi cells, as revealed by DESeq2 expression analysis (B) Expression values (reads per kilobase maximum transcript length per million mapped reads) of marker-associated genes were translated into a heatmap and ranked according to the respective ILC population. (C) Correlation analyses visualize the relation between methylation status of marker regions (methylation difference, x axis) and associated gene expression (log2 fold-change [FC], y axis). Analysis of ILC1 vs. ILC2, ILC3 vs. ILC2, or LTi vs. ILC2 revealed R = −0.82 (P < 0.001), R = −0.67 (P < 0.001), or R = −0.86 (P < 0.001), respectively. Plots show the comparison between ILC2 (red dots) and ILC1 (blue dots), ILC3 (cyan dots), or LTi (magenta dots) including the linear regression line. (D) Methylation profile of the Nmur1 locus for ILC1-3 and LTi cells, including smoothed linear display of CpG motifs (bar code), methylation values of the marker region (light gray box), and the surrounding gene body (exons in dark gray boxes, TSS indicated by arrow). (E) Representative flow cytometry plot showing Gata3 expression in ILC2 following 7 d of expansion in the presence of IL-2, IL-7, and IL-33. (F) sgRNA recognizing Nmur1-associated marker region (crNmur1) or negative control sgRNA (crNeg) were electroporated into in vitro–cultured ILC2 by electroporation. Nonelectroporated ILC2 and in vitro–differentiated Th2 cells served as additional controls. Graph depicts expression of Nmur1 determined 3 d after electroporation, shown as relative expression to Actb. RNA-seq was performed in n = 3 independent experiments and shown as triplicates (A) or mean values (B). Methylation data for the correlation analysis (C and D) was derived from the initial WGBS. In vitro targeting of ILC and Th2 cells (E and F) was performed in n = 3 independent experiments. Statistical significance was analyzed using unpaired two-tailed Student’s t test with ***, P ≤ 0.001.

Transcriptome analysis revealed high correlation between demethylation of marker regions and expression of the associated genes. (A) Heatmap showing the top 50 most variable genes in an unbiased hierarchical clustering of ILC1-3 and LTi cells, as revealed by DESeq2 expression analysis (B) Expression values (reads per kilobase maximum transcript length per million mapped reads) of marker-associated genes were translated into a heatmap and ranked according to the respective ILC population. (C) Correlation analyses visualize the relation between methylation status of marker regions (methylation difference, x axis) and associated gene expression (log2 fold-change [FC], y axis). Analysis of ILC1 vs. ILC2, ILC3 vs. ILC2, or LTi vs. ILC2 revealed R = −0.82 (P < 0.001), R = −0.67 (P < 0.001), or R = −0.86 (P < 0.001), respectively. Plots show the comparison between ILC2 (red dots) and ILC1 (blue dots), ILC3 (cyan dots), or LTi (magenta dots) including the linear regression line. (D) Methylation profile of the Nmur1 locus for ILC1-3 and LTi cells, including smoothed linear display of CpG motifs (bar code), methylation values of the marker region (light gray box), and the surrounding gene body (exons in dark gray boxes, TSS indicated by arrow). (E) Representative flow cytometry plot showing Gata3 expression in ILC2 following 7 d of expansion in the presence of IL-2, IL-7, and IL-33. (F) sgRNA recognizing Nmur1-associated marker region (crNmur1) or negative control sgRNA (crNeg) were electroporated into in vitro–cultured ILC2 by electroporation. Nonelectroporated ILC2 and in vitro–differentiated Th2 cells served as additional controls. Graph depicts expression of Nmur1 determined 3 d after electroporation, shown as relative expression to Actb. RNA-seq was performed in n = 3 independent experiments and shown as triplicates (A) or mean values (B). Methylation data for the correlation analysis (C and D) was derived from the initial WGBS. In vitro targeting of ILC and Th2 cells (E and F) was performed in n = 3 independent experiments. Statistical significance was analyzed using unpaired two-tailed Student’s t test with ***, P ≤ 0.001.

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