Figure 1.
Genome-wide occupancy of Runx3 and H3K4me1 in resting CD8-TC and NKC.
(A) Venn diagram summarizing the number of Runx3 ChIP-seq peaks (left panel) and corresponding annotated genes (right panel). (B) Runx3-bound and H3K4me1-marked regions in the Cd4, Cd8, Ncr1 and Klrb1a-Klrb1f loci. Brown and blue tracings reflect the Chip-seq tracing wiggle files uploaded to UCSC Genome Browser mm9 genome assembly of Runx3 and H3K4me1, respectively. Brown rectangles mark Runx3 peaks identified by MACS. Positions of the Cd4 silencer and some of the known Cd8 enhancers are marked in green. (C) Pie chart showing the % of Runx3-bound regions relative to the nearest TSS. (D) Overlap of Runx3-bound and H3K4me1-marked regions.
Figure 2.
Enriched motifs within Runx3-bound regions in resting cells.
(A) Overrepresentation of RUNX motif variants among Runx3-bound regions in CD8-TC. (B and C) Results of de novo motif finding analysis spanning Runx3-bound regions in CD8-TC and NKC. The 3 most enriched motifs in Runx3-bound promoter (B) and enhancer (C) regions are shown.
Figure 3.
Runx3-bound regions and their corresponding annotated genes in IL-2-activated compared to resting CD8-TC and NKC.
(A) Overlap of Runx3-bound (upper panels) regions or their corresponding genes (lower panels) in resting and IL-2-activated CD8-TC (left) or NKC (right). The genes corresponding to Runx3-bound regions were derived using GREAT [19]. (B) Recruitment of Runx3 to de novo IL-2-activated regions in Gzme-Gzmc (top) and Serpinb1c-Serpinb1b (bottom) loci, in NKC and CD8-TC, respectively. Brown tracing and rectangles represent Runx3 ChIP-seq wiggle files and the positions of Runx3 peaks, respectively, as in Figure 1. Note that some de novo Runx3-bound regions in Serpin genes in IL-2-activated CD8-TC appear to bind Runx3 in resting CD8-TC, but these regions are not scored by MACS as peaks in resting CD8-TC due to the higher background. (C) Overlap of Runx3-bound regions (upper panel) and their corresponding genes (lower panel) in IL-2-activated CD8-TC and NKC.
Figure 4.
Increased enrichment of RUNX-RUNX, ETS-RUNX and AP1-RUNX modules in uniquely Runx3-bound regions of IL-2-activated compared to resting cells.
Histograms show the degree of enrichment compared to genome of the above modules in Runx3 peaks unique to resting cells, overlapping peaks in resting and IL-2-activated cells and de novo peaks unique to IL-2-activated cells.
Figure 5.
Overlap of Runx3-bound regions in IL-2-activated CD8-TC and NKC with p300 and T-bet-bound regions in Th1 cells.
(A and B) Overlap of Runx3-bound regions in CD8-TC/NKC with p300 and T-bet peaks, respectively, in Th1 cells. The locations of Th1 p300 [24] and T-bet [25] peaks were obtained from processed data in public repository and these papers. (C) De novo found TF motif enrichment of overlapping CD8-TC/NKC Runx3-bound and Th1 T-bet-bound regions. (D) Examples of gene loci with overlap Runx3-bound and H3K4me1-marked regions in resting or IL-2-activated CD8-TC and NKC with p300 and T-bet-bound regions in Th1 cells.
Figure 6.
GSEA analyzed relationship of differential gene expression in WT/Runx3-/- cells and Runx3-bound genes in IL-2-activated CD8-TC and NKC.
(A and B) All microarray genes were pre-ranked according to absolute linear fold changes of WT vs. Runx3-/- and statistical enrichment of Runx3-bound genes within the ranked list was evaluated in CD8-TC (upper panel) and NKC (lower panel). NES, normalized enrichment score.
Figure 7.
Common Runx3-regulated genes in IL-2-activated CD8-TC/NKC and T-bet/p300 bound genes in Th1.
(A) The majority of the 118 common Runx3-regulated genes in IL-2-activated CD8-TC and NKC (R3_CD8_NK_T) harbor overlapping T-bet (R3CD8_Tbet) and p300 (R3_CD8_p300) bound regions in Th1 cells. (B) Transcription factors/regulators that are common Runx3-regulated genes in IL-2-activated CD8-TC and NKC.
Figure 8.
Proliferation-control Runx3-target genes in activated CD8-TC/NKC and common Runx3-regulated genes in resting and IL-2 activated cells.
(A) Runx3-regulated genes that may be involved in the defective proliferation of IL-2-activated Runx3-/- CD8-TC and NKC. (B) Comparisons of common Runx3-regulated genes in resting and IL-2-activated CD8-TC (left panel) or in resting and IL-2-activated NKC (right panel) reveal 72 and 205 common targets, respectively.