Supplementary Figure 1 Transcriptional program of the TE and MP CD8 + T cell subsets. (a) Comparison of gene expression of TE and MP CD8 + T cell subsets by microarray. Genes that are 1.5-fold upregulated in TE or MP CD8 + T cell subsets are highlighted as blue or orange, respectively. Transcripts did not differ significantly in expression following
correction with FDR therefore a fold-change cut-off of 1.5-fold was used for comparisons.(b) Volcano plots of the comparison of total effector and memory CD8 + T cells highlighting TE- or MP-enriched genes. Numbers in bottom corners indicate the number of highlighted genes in that region. (c) The ratio of gene expression of known TFs in TE versus MP subset from microarray. (d) Histograms of protein abundance of key TFs in TE versus MP subset. (e) Comparison of gene expression of TE and MP CD8 + T cell subsets by RNA-seq. Genes that are 2-fold upregulated in TE or MP CD8 + T cell subsets are highlighted as blue or orange, respectively. (f) GSEA plot of total effector versus memory CD8 + T cells with TE- or MP-enriched gene signature generated from (e). (g) The ratio of gene expression of known TFs in TE versus MP subset from RNA-seq. Data are the mean value of gene expression from three independent experiments with pooled spleens from three mice except the TE subset in (a) which is from two independent experiments. Data in (d) are representative of two independent experiments with three mice per group.
Supplementary Figure 2 Experimental design for the characterization of chromatin states and accessibility. (a) Schematic view of sorting strategy for naive (N), terminal-effector (TE), memory-precursor (MP), and memory (M) CD8 + T cell subsets. (b) Schematic view of experimental design for characterization of the global epigenetic landscape and gene expression using ChIP-seq, ATAC-seq and microarray analyses.
Supplementary Figure 3 Dynamic enhancer establishment is associated with gene expression during CD8 + T cell differentiation. (a) Violin plots showing the expression of genes associated with different enhancer clusters generated from Figure 2a in naive (N), total effector (EFF) and memory (M) CD8 + T cells generated from microarray data in Best et al. study 17. (b) Volcano plots of the comparison of TE and MP CD8 + T cells showing expression of enhancer cluster associated genes. Data are representative of three independent experiments with three mice per group (median value). The statistical analysis was performed by a nonparametric Wilcoxon rank-sum test. n.s. * : p value <0.0001.
Supplementary Figure 4 Full list of TF motifs enriched in subset-specific regulatory elements. (a) Schematic view of identification of candidate TFs enriched in subset-specific regulatory elements from ATAC-seq and ChIP-seq. (b) Venn diagram showing the overlap of enhancers between CD8 + T cell subsets. (c) Heatmap showing the p-value of transcription factor motif enrichment at subset-specific promoters (left) or enhancers (right) calculated by binomial test using randomly-picked open chromatin regions as background. Motif enrichment or depletion are indicated as red or blue, respectively.
Supplementary Figure 5 Network construction. Construction of TF regulatory network in CD8 + T cell subsets using ChIP-seq and ATAC-seq as input.
Supplementary Figure 6 Full list of TFs identified by PageRank and comparison of PageRank with TFA. (a) Heatmap showing PageRank fold enrichment of TFs across CD8 + T cell subsets. (b) A list of TFs revealed by PageRank analysis and motif enrichment in Figure 3. Known TFs important for CD8 + T cell differentiation are highlighted in red. (c) Heatmap showing Z
score of PageRank score and TFA score of TFs across CD8 + T cell subsets generated by PageRank and TFA analysis, respectively. (d) Bar graph showing the percentage of known TFs with consistent roles in previous reports for each analysis. (e) Bar graphs showing the fold change of YY1 and Nr3c1 gene expression generated from microarray. Data in (e) are the mean value of gene expression from three independent experiments with pooled spleens from three mice.
Supplementary Figure 7 Ablation of Nr3c1 cofactor Ncor1 and treatment with dexamethasone affect the differentiation of MP CD8 + T cells. (a) Schematic view of experimental design. OT-I CD8 + T cells were activated in vitro and transduced with control shrna or shyy1 retroviral vectors and subsequently co-transferred into recipient mice followed by i.v. infection with Lm-OVA. Splenocytes were isolated and analyzed on day 7 of infection. (b) Flow cytometric analysis of KLRG1 and IL-7R expression for cells transduced with shcd4 and shncor1 in PBL on day 8 of infection. (c) The percentage of TE and MP CD8 + T cells gated on transduced cells on day 8 of infection after knockdown of Ncor1. (d) Flow cytometric analysis of KLRG1 and IL-7R expression for donor cells in mice treated with either vehicle or dexamethasone for 7 days. (e) The percentage of TE and MP CD8 + T cells gated on donor cells on day 8 of Lm-OVA infection after drug treatment. Data are representative from two independent experiments with 5 mice per group. The statistical analysis was performed by two-tailed paired t-test in (c) and two-tailed unpaired t-test in (e). *: p<0.001