Figure 5. Durability of IFNγ-induced de... | Rockefeller University Press

Figure 5.

$Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D). Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.$

Durability of IFNγ-induced de novo enhancers is dependent on continued JAK/STAT signaling by IFNγ. (A) Schematic of experimental design: Human macrophages were stimulated with IFNγ (100 ng/ml) for 8 h. Cells were subsequently washed and cultured for an additional 88 h in standard media or media supplemented with 1 µM ruxolitinib or 8 µg/ml anti-IFNγ neutralizing antibody. H3K4me1 CUT&Tag was performed at each time point. (B) Boxplot quantifying log2 fold changes of reads within IFNγ-induced H3K4me1 CUT&Tag peaks after 8 h of IFNγ stimulation and after washout for each condition. (C) Barplot showing fraction of IFNγ-induced H3K4me1 peaks at 8 h that persist 4 days after washout in each condition. Persistence was defined as L2FC ≥ 0, FDR < 0.01. (D). Heatmap of Z-scored reads within H3K4me1 peaks induced IFNγ (L2FC > 2, FDR < 0.01) after 8 h of stimulation and 4 days after washout for each condition. Clusters were generated by unsupervised k-means clustering. Each column represents a biological replicate from the same human donor. (E) Representative genome browser tracks of peaks from each cluster in D. All box/whisker plots indicate interquartile range and 1.5× interquartile range. Statistical tests were determined by paired Wilcoxon test. ***P < 0.001; ****P < 0.0001.