Figure 5. The Rroid locus promotes open chromatin and STAT5 deposition at the Id2 promoter.
A. Histone modifications at the Id2 locus. Gene browser depiction of Id2 locus and ChIP-qPCR primer locations (above). ChIP-qPCR showing H3K27ac and H3K36me3 enrichment within the Id2 promoter and gene body (below). NK cells were expanded in vivo with IL-2/αIL-2 complexes and sorted from spleen (n=3 groups of 2 pooled mice per group). Results were normalized to enrichment at the Gapdh locus. Data are representative of two independent experiments.
B. Representative gene browser tracks of ATAC-seq reads at the Id2 and Rroid loci in Rroid+/+ and Rroid−/− NK cells.
C. Representative gene browser tracks of ATAC-seq reads at the Id2 locus in Rroid+/+ and Rroid−/− NK cells.
D. Density plot of log2 fold change distribution for ATAC-seq peaks in Rroid+/+ and Rroid−/− NK cells. Fold changes were estimated as the ratio of the trimmed mean of M-values (TMM)-normalized read counts in consensus peak regions via the DiffBind R package. Vertical dashed lines show the 5th and 95th percentiles. The green arrowhead shows the Id2 promoter.
E. Relative chromatin accessibility at the Id2 locus in Rroid+/+ and Rroid−/− NK cells. Chromatin accessibility is depicted as an average of TMM-normalized read counts across replicates. Statistics were obtained using the DiffBind R package (n=3 mice per group).
F. Id2 (left) and Rroid (right) expression in in vitro expanded NK cells stimulated with IL-15. Splenic NK cells were sorted and expanded 7d ex vivo. Prior to the experiment, cells were rested in culture media without cytokines media for 5h. Either media alone (0ng/mL), 0.5ng/mL, or 5ng/mL IL-15/IL-15Rα complex was added and cultured for an additional 3h. Gene expression was analyzed by qPCR. Results are normalized to Hprt (n=3 biological replicates per group).
G. qPCR analysis of Id2 expression in Rroid+/+and Rroid−/− NK cells. Sorted splenic NK cells were expanded as in F, rested 4h, and stimulated 3h with IL-15/IL-15Rα complex (n=3 biological replicates per group).
H. Representative flow cytometry plots of splenic NK cell maturation in Stat5a+/+ Stat5b+/+ and Stat5a−/− Stat5b+/− mice. Data are representative of two independent experiments.
I. Fold change of Id2 expression in splenic control (Stat5a+/+ Stat5b+/+) or Stat5a−/− Stat5b+/− NK cells (n=4 Stat5a+/+ Stat5b+/+ and 5 Stat5a−/− Stat5b+/− mice pooled from two independent experiments). Normalized to Hprt expression and shown as fold change relative to controls.
J. STAT5 occupancy at the Id2 promoter. Splenic NK cells were sorted and expanded in vitro for 7d. Cells were rested for 2h in unsupplemented media, then stimulated with 20ng/mL IL-15/IL-15Rα complex for 1h. Fixed nuclei were then isolated for chromatin immunoprecipitation and qPCR (n=3 biological replicates per group).
*p≤0.05, **p≤0.01, ***p≤0.001. Two-tailed t-test; F and G were analyzed by one way ANOVA with Tukey’s post hoc test. All error bars represent SEM. See also Figure S5.