1. Volume 45, Issue 6, Pages 1341-1354 (December 2016)
CD8+ T Cells Utilize Highly Dynamic Enhancer Repertoires and Regulatory Circuitry in Response to Infections 
Bing He, Shaojun Xing, Changya Chen, Peng Gao, Li Teng, Qiang Shan, Jodi A. Gullicksrud, Matthew D. Martin, Shuyang Yu, John T. Harty, Vladimir P. Badovinac, Kai Tan, Hai-Hui Xue 
Immunity 
Volume 45, Issue 6, Pages (December 2016)
DOI: /j.immuni
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
RNA-Sequencing Identifies Twelve Gene Expression Clusters during CD8+ T Cell Responses
(A) Venn diagram of differentially expressed genes (DEGs) in pairwise comparisons among Tn, Te, and Tcm cells (two biological replicates each). DEGs were identified using the EdgeR algorithm with the criteria of FDR < 0.05 and fold change ≥ 2.
(B) Twelve ExprClusters during CD8+ T cell responses. Transcript clusters were defined using the STEM algorithm. Transcript expression levels were shown as transcript-wise Z score of FPKM values. For each cluster, the numbers of transcripts and corresponding genes are shown in parentheses.
See also Figures S1–S3.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
Epigenomic Profiling Identifies a Dynamic Repertoire of Active Enhancers during CD8+ T Cell Responses
(A) Venn diagram of active enhancers in Tn, Te, and Tcm cells. Numbers in parenthesis indicate enhancer activity profile, e.g., “100” indicates enhancer active in Tn, but inactive in Te and Tcm cells.
(B) Validation of histone mark features at the predicted enhancers using ChIP-qPCR. Tn, Te, and Tcm cells were used for ChIP with anti-H3K4me1, H3K4me3, or H3K27ac antibodies. The relative enrichment of each histone mark was determined by quantitative PCR. Vertical cyan bars highlight the validated enhancer features, i.e., H3K4me1hi, H3K4me3lo, and H3K27achi. Dotted lines denote empirically determined enrichment thresholds for different histone marks. NC, negative control genomic region that is devoid of histone modification signals based on the ChIP-seq data. E2f1 is expressed in all three CD8+ T cell subsets and its transcription start site (TSS) was used as another control for low H3K4me1 signal and high H3K4me3 signal. Data are means ± SD from two independent experiments (n = 4).
(C–F) Genome browser view of predicted enhancers for the Eomes (C), Gzmb (D), Tbx21 (E), and Bcl11b (F) genes. Vertical cyan bars denote the predicted enhancers, and vertical yellow bars denote select enhancers that were validated in (B) or identified experimentally for Bcl11b in a previous study.
See also Figure S4.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
Enhancer-Promoter Interactions Are Highly Dynamic during CD8+ T Cell Responses
(A) Venn diagram of predicted EP interactions in CD8+ T cell subsets.
(B) Expression difference of genes targeted by CD8+ subset-specific enhancers. Genes targeted by enhancers that are specific to a CD8+ subset were identified. Their expression levels (in FPKMs) in all three CD8+ subsets were shown as boxplots. ∗p < 10−10 by Student’s t test.
(C) Cumulative distribution of promoter degree in CD8+ T cell subsets. Promoter degree was defined as the number of enhancers contacting a given promoter. Mean degree value for each subset is shown in parentheses.
(D) Cumulative distribution of enhancer degree in CD8+ T cell subsets. Enhancer degree was defined as the number of promoters contacting a given enhancer. Mean degree value for each subset is shown in parentheses.
(E) Cumulative distribution of the distance between predicted EP pairs in each cell type. Distance was defined as the number of intervening promoters between an EP pair. Dotted horizontal lines indicate the percentage of EP pairs that have no intervening TSS. Inset shows the intersection regions.
(F) Overlap between EP interactions predicted by IM-PET (in orange) and chromatin interactions detected by Hi-C (in cyan).
(G and H) IM-PET-predicted and Hi-C-validated EP interactions at select gene loci in Tn cells. Shown are histone mark ChIP-seq tracks at the Eomes (G) and Myc (H) loci, with IM-PET-predicted interactions shown as green lines ending with bars. Hi-C interactions at 10 kb resolution are shown as gray lines ending with bars, and the shades of gray denote statistical significance of the interactions with darker shades marking more significant interactions. Enhancers highlighted in cyan were also tested using 3C-qPCR in (I) and (L), respectively.
(I–L) 3C-qPCR validation of EP interactions for Eomes (I), Gzmb (J), Tbx21 (K), and Myc (L) genes. Tn, Te, and Tcm cells were subjected to 3C-qPCR analysis. Bacterial artificial chromosome (BAC) clones were used as control template to cover the genomic regions of interest. Relative frequency of 3C product (3C versus BAC template) was plotted along the test loci. Multiple PCR probes were used to determine the relative contact frequency at a target region versus nearby control regions. Each data point represents the average of two biological samples, each measured by qPCR in triplicates (error bars denote SD). Note the signal strength supporting positive interactions with respective promoter should be compared within a cell type, not across cell types.
See also Figures S4 and S5.
Immunity  , DOI: ( /j.immuni )
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5. Figure 4
Epigenetic States of Enhancers Correlate with Their Activities and Regulatory Output during CD8+ T Cell Responses
(A) Heatmap of epigenetic clusters associated with enhancers (EnhEpi clusters). Histone modification profiles of 7,387 enhancers targeting 2,310 DEGs were clustered using the Consensus Clustering algorithm. Overlap between EnhEpi clusters and ExprClusters was determined using the hypergeometric distribution with a p value cutoff of Size of each EnhEpi cluster and its overlapping ExprCluster(s) are shown on the right of the heatmap.
(B) Graphical summary of histone modification changes at enhancers between adjacent CD8+ T cell subsets. Blue square, transition from Tn to Te; Green square, transition from Te to Tcm; G, gain of histone mark; L, loss of histone mark; white square, no significant change in a histone mark during the transition.
(C–F) Genome browser view of enhancer epigenetic modification and expression profiles during CD8+ T cell responses at the Notch1 (C), Pdcd1 (D), Fos (E), and Socs3 (F) genes. Vertical cyan bars denote the enhancers of interest. Cluster memberships are indicated in parentheses.
Immunity  , DOI: ( /j.immuni )
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6. Figure 5
Epigenomic Profiling Identifies a Dynamic Repertoire of Super Enhancers during CD8+ T Cell Responses
(A) Venn diagram of super enhancers in Tn, Te, and Tcm cells.
(B) Super enhancers are transcribed at higher levels than typical enhancers. Typical enhancers or super enhancers were separated into two groups: those that are specific to Tn, Te, or Tcm stage, and those that are common to two or all three stages. The enhancer transcripts, expressed in FPKM values, were determined for both types of enhancers. p values determined by Student’s t test are also shown.
(C) Target genes of super enhancers are expressed at higher levels than those of typical enhancers. The target genes of typical enhancers or super enhancers were predicted using the IM-PET. The relative expression of a gene was computed as Expr_predicted/Expr_all where “Expr_predicted” was the expression of a gene in a specific CD8+ subset where it was predicted to be an enhancer target, and “Expr_all” was the average expression of the gene in all three CD8+ subsets.
(D–E) Genome browser view of super enhancers targeting the Ccr7 (D) and Tcf7 (E) genes. Super enhancers are highlighted in cyan. EP interactions predicted by IM-PET are shown in green. Chromatin interactions detected by Hi-C are shown in gray. Multiple enhancers in the super enhancers contact the respective promoters.
See also Figure S6.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6
Network Analysis Reveals Extensive Re-wiring of Regulatory Circuitry during the Tn to Te Transition
(A) The numbers of target genes of TFs whose motifs were enriched at Tn and/or Te enhancers.
(B) Changes in the core TF network during the Tn-to-Te transition. Node color represents expression fold change of each TF during the transition. Red, upregulation in Te cells; Green, downregulation in Te cells. Node size is proportional to the fraction of TF target genes showing expression changes during the transition. Arrows represent regulatory interactions among TFs. Red arrows, interactions acquired in Te cells (Te-specific); Green, interactions lost in Te cells (Tn-specific); grey, interactions that existed in both Tn and Te cells.
(C) Tn-specific regulatory interaction at a Tcf7 enhancer. A –24 kb enhancer (highlighted in cyan) regulating the Tcf7 gene was active in Tn but lost in Te cells. The EP interaction predicted by IM-PET is shown in green, and the supporting Hi-C data is shown in gray. The enhancer sequence contains the binding motifs for three TFs, FOXJ3, ERRα (ESSRA), and Tcf1 (TCF7). The sequence logo and p value of motif match are shown for each TF. The track of Tcf1 ChIP-seq data in Tn cells is included for comparison.
(D) Expression levels of predicted Tcf1 targets and non-Tcf1 targets in Tcf1/Lef1-deficient Tn cells. Tcf7−/−Lef1−/− or control Tn cells (two biological replicates each) were sorted and analyzed by RNA-seq. The expression of predicted Tcf1 targets and non-Tcf1 targets (defined as all other predicted enhancer target genes in Tn cells) was compared, with p values shown.
(E) Te-specific regulatory interaction at a Tbx21 enhancer. A –19 kb enhancer regulating the Tbx21 gene was inactive in Tn but was activated in Te cells. The enhancer contains an ERRα (ESSRA) binding motif.
See also Figure S7.
Immunity  , DOI: ( /j.immuni )
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8. Figure 7
Network Analysis Reveals Extensive Re-wiring of Regulatory Circuitry during the Te to Tcm Transition
(A) The numbers of target genes of TFs whose motifs were enriched at Te and/or Tcm enhancers.
(B) Changes in the core TF network during the Te-to-Tcm transition. Node color represents expression fold change of each TF during the transition. Red, upregulation in Tcm cells; Green, downregulation in Tcm cells. Node size is proportional to the fraction of TF target genes showing expression changes during the transition. Arrows represent regulatory interactions among TFs. Red arrows, interactions acquired in Tcm cells (Tcm-specific); Green arrows, interactions lost in Tcm (Te-specific); grey, interactions that existed in both Te and Tcm cells.
(C and D) Tcm-specific regulatory interactions at the Thra (C) and Foxo1 (D) enhancers. A +18 kb enhancer regulating the Thra gene (C) and a +110 kb enhancer regulating the Foxo1 gene (D), both marked in cyan, were inactive in Te but were activated in Tcm cells. RREB1 and SP1 motifs were found in Thra and Foxo1 enhancers, respectively, and their sequence logos and p values are shown.
See also Figure S7.
Immunity  , DOI: ( /j.immuni )
Copyright © 2016 Elsevier Inc. Terms and Conditions