Fig 2: BATF directly regulates Ezh2 expression to drive effector CTL differentiation(A) Representative CUT&RUN signal tracks of WT and Batf−/− effector P14 cells. See also Figure 3.(B) Schematic for designing a set of sgRNA targeting BATF-dependent enhancer region of Ezh2 gene (Ezh2-BDH). Genome tracks represent chromatin accessibility at the Ezh2 gene in WT and Batf−/− P14 cells.(C) WT recipient mice were adoptively transferred with congenically distinct WT P14 cells treated with either control sgRNA (sgCtrl) or sgRNA targeting Ezh2-BDH mixed at a 1:1 ratio (500 each cell), followed by infection with LCMV Arm. (Top) Flow cytometric analysis of splenocytes of the recipient mice at transfer and day 8 post-infection. Plots are gated on P14 cells. (Bottom) The numbers of each P14 cell in the spleen of recipient mice (per 106 splenocytes).(D) Expression of Ezh2, BATF, and IRF4 at day 8 post-infection in the recipient mice. Graphs represent the MFI of indicated markers in each P14 cell.(C and D) Data from two independent experiments were merged and analyzed. ***p < 0.001 and ****p < 0.0001 (paired t test).

Fig 3: IRF4 is indispensable for chromatin remodeling, but not for BATF binding, during effector differentiation of CTLsWT recipient mice were adoptively transferred with 1 × 106 of CD4Cre and IRF4cKO P14 cells, followed by infection with LCMV Arm. Effector P14 cells were collected at day 3 post-infection and subjected to ATAC-seq and BATF CUT&RUN (n = 2).(A) Volcano plot represents differences in chromatin accessibility between CD4Cre and IRF4cKO effector P14 cells.(B) Scatterplots showing relationship between differences in chromatin accessibilities between WT and Batf−/− effector P14 cells (horizontal axis) in Figure 2 and between CD4Cre and IRF4cKO effector P14 cells (vertical axis). OCRs that were differentially accessible between either WT and Batf−/− P14 cells or CD4Cre and IRF4cKO P14 cells were analyzed.(C) Representative ATAC-seq signal tracks for WT, Batf−/−, CD4Cre, and IRF4cKO effector P14 cells.(D) PCA of ATAC-seq data of WT and Batf−/− effector P14 cells in Figure 2 and CD4Cre and IRF4cKO effector P14 cells.(E) Volcano plot showing differences in chromatin accessibilities between Batf−/− and IRF4cKO effector P14 cells.(F) Volcano plot represents differences in BATF binding between CD4Cre and IRF4cKO effector P14 cells.(G) BATF CUT&RUN signal coverages at OCRs differentially accessible or shared between CD4Cre and IRF4cKO effector P14 cells.(H) Scatterplots showing relationship between differences in BATF bindings (horizontal axis) and those in chromatin accessibilities (vertical axis) between CD4Cre and IRF4cKO effector P14 cells.