Figure 6. Ezh2 Is Required in Activated Treg Cells to Stabilize the Foxp3-Driven Treg Program.

(A) Principal-component analysis of the transcriptomes of sorted CD62L^hi and CD62L^lo YFP⁺ Treg cells from lymph nodes and spleens of female Foxp3^YFP-cre/ Foxp3^WT;Ezh2^fl/+ (Ezh2^Δ/+) and Foxp3^YFP-cre/Foxp3^WT;Ezh2^fl/fl (Ezh2^Δ/Δ) mice. The contribution of each principal component to the total variance in the data is shown on each axes (%). Each point represents the combined average of three biological samples except for CD62L^hi Ezh2^Δ/+, which is from two samples.

(B) Comparison of all differentially expressed genes (DEGs = 3097, FDR < 0.05) between CD62L^hi and CD62L^lo Treg cells in the absence of Ezh2 (Ezh2^Δ/Δ, y axis) or presence of Ezh2 (Ezh2^Δ/+, x axis). Dashed line represents the trend of all genes differentially regulated between the Ezh2^Δ/Δ and Ezh2^Δ/+ datasets. Data points are colored by significant differences in gene expression between the CD62L^lo populations of Ezh2^Δ/Δ versus Ezh2^Δ/+ Treg cells: red, increased expression; blue, reduced expression. Differentially expressed “Treg cell signature” genes are labeled and colored with respect to expression change.

(C) Cumulative distribution of fold changes in all DEGs (black dotted line, 3097 genes) or DEGs with increased H3K27me3 marks in activated Treg cells (green line, 465 genes) between CD62L^lo Ezh2^Δ/Δ and Ezh2^Δ/+ Treg cells, p < 7.8 × 10⁻⁸ using the two-sample Kolmogorov-Smirnov (K-S) statistical test.

(D) Venn diagram of DEGs shared between Foxp3^D Treg cells (Foxp3^gfpkoTreg versus WT Treg: 1,678 genes) and activated Ezh2^Δ/Δ Treg cells (CD62L^lo Ezh2^Δ/Δ versus CD62L^lo Ezh2^Δ/+ Treg cells: 831 genes).

(E) Clustering of all genes up or down (341 DEGs, FDR < 0.05) between Foxp3^D and Ezh2^Δ/Δ data sets (left) and expression of Foxp3-bound genes that are up- or downregulated ≥0.5 log₂ fold change (right). *, genes also identified by GSEA of Ezh2^Δ/Δ Treg cells compared to Treg versus Tconv immunological signatures.

(F) Left to right: Venn diagram depicting Foxp3-bound DEGs in-common between Foxp3^D and Ezh2^Δ/Δ Treg cells (left); overlap in Foxp3-bound DEGs either up-(top) or downregulated (bottom) in both datasets (middle); percentage of shared up- (top) or downregulated Foxp3-bound DEGs (bottom) in Foxp3^D and Ezh2^Δ/Δ that are associated with increased H3K27me3 marks in activated versus resting WT Treg cells (right).

See also Figure S5.