Figure 6.
Pik3cdE1020Kreshapes the epigenetic landscape of CD4+T cells. (A and B) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq (n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cdE1020K/+-specific, and common peaks. (B) WT and Pik3cdE1020K/+-specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cdE1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cdE1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cdE1020K/+) and non-DEGs from bulk RNAseq data (Fig. 3 C) were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cdE1020K/+). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cdE1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cdE1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cdE1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cdE1020K/+) and DEGs showing repressed CTCF peaks in Pik3cdE1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; Xie et al., 2021) of TFTs (ChEA; Lachmann et al., 2010) was performed using these two categories of DEGs. Adjusted P values (−log10) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cdE1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments (n = 3), quantified in Fig. S4 C. (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cdE1020K/+ cells. Data are representative of three independent experiments (n = 3), Fig. S4 D. NC, negative control. Source data are available for this figure: SourceData F6.

Pik3cd E1020K reshapes the epigenetic landscape of CD4 + T cells. (A and B) Naïve CD4 T cells from WT and Pik3cdE1020K/+ mice were polarized under Th2 conditions and evaluated by ATACseq (n = 3). A total of 71,040 peaks were detected. (A) Venn diagram of WT-specific, Pik3cdE1020K/+-specific, and common peaks. (B) WT and Pik3cdE1020K/+-specific peaks examined by motif enrichment analysis. Enrichment P values were plotted for both groups. Red: motifs specifically enriched in WT peaks; blue: motifs specifically enriched in Pik3cdE1020K/+ peaks; orange: motifs enriched in both groups. (C) Peak heatmap of CTCF CUT&Tag peaks from WT and Pik3cdE1020K/+ naïve, Th1, and Th2 cells organized into six clusters (1–6) specific to each indicated population, as described. (D) CTCF motif enrichment P value (top) and fold enrichment (bottom) in clusters 1–6. (E) DEGs (WT vs Pik3cdE1020K/+) and non-DEGs from bulk RNAseq data (Fig. 3 C) were compared in the indicated populations for percentages of genes showing differential CTCF peaks (WT versus Pik3cdE1020K/+). (F) Frequencies of CTCF peaks repressed, induced, or both induced and repressed in Pik3cdE1020K/+ Th2 cells (versus WT Th2) near DEGs (WT Th2 versus Pik3cdE1020K/+ Th2). (G) Th2 DEGs (WT Th2 versus Pik3cdE1020K/+ Th2) were organized into two categories: DEGs showing no change in CTCF (WT versus Pik3cdE1020K/+) and DEGs showing repressed CTCF peaks in Pik3cdE1020K/+ relative to WT. Pathway enrichment analysis (Enrichr; Xie et al., 2021) of TFTs (ChEA; Lachmann et al., 2010) was performed using these two categories of DEGs. Adjusted P values (−log10) of the top 25 enriched TF signatures in each category plotted against each other. (H) Western blot evaluating CTCF and Zap70 in lysates from WT and Pik3cdE1020K/+ Th2-polarized cells, cultured in the presence or absence of Cal101 (10 nM) or rapamycin (200 nM). Data are representative of three independent experiments (n = 3), quantified in Fig. S4 C. (I) Western blot evaluating CTCF and Zap70 in lysates from Th2-polarized NC and Foxo1 gRNA-Cas9–nucleofected WT CD4 T cells, compared with Pik3cdE1020K/+ cells. Data are representative of three independent experiments (n = 3), Fig. S4 D. NC, negative control. Source data are available for this figure: SourceData F6.

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