1. STATs Shape the Active Enhancer Landscape of T Cell Populations
Golnaz Vahedi, Hayato Takahashi, Shingo Nakayamada, Hong-wei Sun, Vittorio Sartorelli, Yuka Kanno, John J. O’Shea 
Cell 
Volume 151, Issue 5, Pages (November 2012)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Active Enhancer Landscapes in Th1 and Th2 Cells Are Distinct
(A) Chromatin signatures as defined by p300 binding and H3K4me1 map recognized and other putative enhancers in the Il4-Il13 locus. The Il4-Il13 gene track represents 13 p300 binding sites within H3K4me1 domains in Th2 cells, including eight known elements (orange triangles) (Table S1B). “CNS” lane shows conserved noncoding sequences.
(B) Genomic distribution of p300-bound elements in Th1 (total 25,554) and Th2 (total 22,534) cells at promoter (−4 kbp to +500 bp of transcriptional start site [TSS]), intergenic (>4 kbp TSS), and intragenic regions (+500 bp of TSS to transcription end site [TES]).
(C) T helper subsets have thousands of unique p300 binding sites, but almost none are shared among T cells, macrophages, and ES cells. Venn diagram depicts the number and percentages of shared and unique p300 binding sites in each cell type. p300 binding in ES cells and macrophages is from Creyghton et al. (2010) and Ghisletti et al. (2010).
(D–F) In contrast to differentially expressed genes in Th1 and Th2 cells, housekeeping genes have little proximal p300 binding. Box plots show median and quartiles of (D) normalized mRNA expression levels (RPKM) measured by RNA-seq, (E) normalized H3K4me3 (tag per million), and (F) normalized p300 binding (tag per million) for top 100 Th-specific genes versus 100 housekeeping genes selected from Eisenberg and Levanon (2003). The intensity of p300 binding was computed −20 kbp to 20 kbp from the TSS to capture potential enhancers. The intensity of H3K4me3 was computed −4 kbp to 1 kbp from the TSS to capture active promoters (p values for Wilcoxon rank-sum test).
See also Figure S1.
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4. Figure 2 Properties of T-Helper-Specific p300-Bound Elements
(A) T-helper-specific p300 elements are marked by high H3K4me1 and low H3K4me3 in both Th cells but lack p300 binding and H3K4me1 in macrophages and ES cells. Each column depicts p300 binding, H3K4me1, or H3K4me3 within a window centered on the p300-bound sites (indicated as position “0” by red triangle). Three patterns of p300 binding are shown: Th1-specific (9,180), Th1-Th2-common (12,845), and Th2-specific (7,089). Color map corresponds to binding intensities where “black” represents no binding.
(B) H3K4me1 at Th-specific p300 sites shows enrichment in the respective lineage and relative reduction in the opposite lineage. Plots show the normalized distribution of H3K4me1 at Th1 (Th2)-specific p300 elements in Th1 and Th2 cells (±5 kbp) (Kolmogorov-Smirnov test).
(C) Th-specific p300 binding sites are enriched in proximity to genes selectively expressed in T helper cells. Plots depict number of Th-specific p300 binding sites within a given distance to promoters of Th-specific genes (Th1 blue, Th2 black) versus randomly generated sites (red) (Wilcoxon rank-sum test p value < 2.2 × 10−16).
(D) Th-specific genes exhibit enrichment of p300 binding in the corresponding lineage and relative p300 depletion in the opposite lineage. Box plots show median and quartiles of p300 binding in Th1 and Th2 cells around Th1- or Th2-specific genes (±20 kbp from the TSS) (Wilcoxon rank-sum test).
(E) Th-specific p300 elements are enriched for consensus motifs of lineage-appropriate transcription factors. Consensus motifs for T-cell-related transcription factors were computed based on the de novo motif analysis by using ChIP-seq data for each factor. A Gibbs sampling method was used to search for a motif by using the genome as the background (likelihood ratio r > 1,000). Consensus motifs GATA and GAS-4 (STAT6) were preferentially enriched in Th2, whereas T-box, GAS-3 (STAT1,4), and p65 were enriched in Th1-specific p300 elements.
See also Figure S2.
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5. Figure 3
STAT6 Has a Major Role in Generating Active Enhancers of Th2 Cells
(A) STAT6 is critical for the global chromatin signature of Th2-specific enhancers. Globally, p300 binding and H3K4me1 at 77% of Th2-specific p300 sites (5,451) were STAT6 dependent. The plot in each column represents the pattern of p300 binding and H3K4me1 in wild-type or Stat6−/− cells centered on the Th2-specific p300-bound sites (as indicated by position “0”). Color map corresponds to binding intensities where “black” represents no binding.
(B) H3K4me1 at Th2-specific p300 sites is STAT6 dependent. Plot shows the normalized distribution of H3K4me1 at 5,451 STAT6-dependent p300 elements (Kolmogorov-Smirnov test).
(C) STAT6 positively regulated genes are enriched with STAT6-dependent p300 binding sites. By using RNA-seq data from wild-type Th2 and STAT6-deficient cells, we identified positively regulated genes by STAT6 (>2-fold change). Accumulation of p300 binding at these genes in wild-type and STAT6-deficient cells was computed (±20 kbp from the TSS). Box plots show median and quartiles of gene expression levels in RPKM (left) and p300 binding in tag per million (right) at STAT6-dependent genes in wild-type and STAT6-deficient cells (Wilcoxon rank-sum test).
See also Figure S3.
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6. Figure 4
STAT4 and STAT1, but Not T-bet, Are Critical for Active Enhancers of Th1 Cells
(A) STAT1 and STAT4, but not T-bet, play major roles in generating the active enhancer landscape of Th1 cells. Globally, 60% of Th1-specific enhancers were STAT dependent, whereas 17% were T-bet dependent. Each column represents the pattern of p300 binding in wild-type, Stat4−/−, Stat1−/−, or T-bet−/− cells centered on the Th1-specific p300-bound sites. Color map corresponds to binding intensities where “black” represents no binding.
(B) Genes positively regulated by STAT4 are enriched with STAT4-dependent p300 binding sites. Using RNA-seq data in wild-type Th1 and STAT4-deficient cells, we identified genes that were positively regulated by STAT4 (>2-fold change). Accumulation of p300 binding at these genes in wild-type and STAT4-deficient cells was computed (±20 kbp). Box plots show normalized gene expression levels in RPKM (left) and p300 binding in tag per million (right) at STAT4-dependent genes in wild-type and STAT4-deficient cells (Wilcoxon rank-sum test).
(C) p300 binding at the extended loci of genes positively regulated by T-bet is not T-bet dependent. By using RNA-seq data in wild-type Th1 and T-bet-deficient cells, we selected positively regulated genes by T-bet (>2-fold change). Box plots show normalized gene expression levels in RPKM (left) and p300 binding in tag per million (right) at T-bet-dependent genes in wild-type and T-bet-deficient cells.
See also Figure S4.
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7. Figure 5
Quantification of Direct Contribution of STATs to p300 Binding
(A) Global binding of STAT6 leads to both gain and loss of cognate p300 binding. Two-dimensional histogram depicts STAT6 binding, resulting in a change in p300 recruitment in wild-type versus Stat6−/− cells. Percentages of STAT6-bound sites with positive or negative effect on p300 are represented in the marked area (>4-fold change). The x axis corresponds to intensity of STAT6 binding (log2). The y axis measures the fold change of p300 binding in wild-type versus Stat6−/− cells (log2). Color map corresponds to the number of binding events. Examples of genes with proximal STAT6 binding include Nfil3, Il24, and Gata3 (for positive effect) and Ifng, Xcl1, and Il18r1 (for negative effect).
(B) STAT6 has direct negative effects on Ifng enhancers in Th2 cells. Gene track shows that STAT6 binding (dotted box) in Th2 cells leads to loss of p300 binding and H3K4me1 at Ifng enhancers. RNA-seq lanes depict the expression of Ifng gene increased in the absence of STAT6 (14–135 RPKM).
(C) STAT4 binding correlates with gain and loss of p300 binding. Examples of genes with proximal STAT4 binding include Ifng, Nfatc2, and Il18r1 (for positive effect) and Il2 and Il4ra (for negative effect).
(D) Contrasting effect of T-bet on p300 binding. T-bet has a dominant role as a repressor rather than an activator based on p300 binding. Examples of genes with proximal T-bet binding include Ifng and Xcl1 (for positive effect) and Eomes and Il4-Il13 (for negative effect).
See also Figure S5.
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8. Figure 6
Overexpression of T-bet or GATA3 in STAT-Deficient Cells Fails to Reconstitute STAT-Dependent Active Repertoires
(A) GATA3 expression recovers half of STAT6-dependent elements in Th2 cells. Of 5,041 Th2-specific-STAT6-dependent p300 sites, 2,639 (50%) regulatory elements are recovered in STAT6-deficient cells in which GATA3 was reconstituted. Overall, 36% of Th2-specific enhancers are STAT6 dependent and GATA3 independent. Each column represents p300 binding in wild-type (Stat6−/−) cells transduced with control or GATA3-expressing retrovirus centered on the Th2-specific p300 sites. Color map corresponds to binding intensities where “black” represents no binding.
(B) GATA3 recovers p300 binding at genes whose expression levels are recovered by GATA3. Box plots show median and quartiles of expression levels in RPKM (left) and normalized p300 binding in tag per million (right) in wild-type (Stat6−/−) cells transduced with control or GATA3-expressing retrovirus at genes recovered by GATA3.
(C) GATA3 has no effect on p300 binding at genes whose expression levels are not affected by GATA3.
(D) T-bet overexpression fails to recover the chromatin signature of STAT4-dependent enhancers. Of 6,820 Th1-specific STAT4-dependent sites, 1,614 (23%) regulatory elements are recovered in STAT4-deficient T-bet-expressing cells. Each column represents p300 binding in wild-type (Stat4−/−) cells infected with control or T-bet-expressing retrovirus centered on the Th1-specific p300 sites.
(E) T-bet fails to recover p300 binding at genes whose expression levels are recovered by T-bet. Box plots show median and quartiles of gene expression levels in RPKM (left) and normalized p300 binding in tag per million (right) in wild-type (Stat4−/−) cells transduced with control or T-bet-expressing retrovirus.
(F) T-bet has no effect on p300 binding at genes whose expression levels are not recovered by T-bet.
See also Figure S6 and Table S3.
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9. Figure S1
Enhancer Landscapes Are Highly Cell-Type-Specific, Related to Figure 1
(A) Global comparisons of p300 ChIP-seq experiments between biological duplicates (after log2 transformation on tag-per-million intensity) for Th1 (left) and Th2 (right) cell populations reveal reproducibility of p300 data. The Spearman’s correlation coefficient for each comparison is indicated in the insert.
(B–D) Chromatin signatures p300 binding and H3K4me1 identify previously recognized and new potential enhancers in T helper cells. Gene tracks represent p300 binding and H3K4me1 modification in Th1 and Th2 cells for (B) Cd4, CD8a, Ifng, (C) Tbx21, Gata3, Foxp3, (D) Hlx, Bcl6, Prdm1. Y-scale is normalized tag intensity (tag-per-million). CNS lane represents elements with more than 50% conservation in human. Previously known enhancers are marked by an orange triangle.
(E) Genomic distribution of p300 elements at promoters (−4kbp to +500bp of TSS), intergenic (>4kbp TSS), and intragenic regions (+500bp of TSS to TES) for macrophages (total 12,237 peaks) and ES (total 12,033 peaks) cells.
(F–H) Quantification of gene-expression levels in constitutively expressed housekeeping genes and differentially expressed genes in Th1 and Th2 cells. Boxplots show median and quartiles of normalized mRNA expression levels (RPKM: reads per kilobase exon model per million reads) measured by RNA-seq for (F) top 100 Th1-specfic, (G) top 100 Th2-specfic, and (H) 100 housekeeping genes in Th1 and Th2 cells (Wilcoxon rank-sum test).
(I) H3K4me1 modification is not significantly different between housekeeping genes and T helper specific genes for Th2 cells. Boxplots show median and quartiles of H3K4me1 in Th1 (Th2) cells for the top 100 Th1 (Th2)-specific genes compare to 100 housekeeping genes (−20kbp to 20kbp of TSS). (Wilcoxon rank-sum test).
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10. Figure S2
Organizational Properties of T-Helper-Specific Enhancers, Related to Figure 2
(A) Th-specific p300 elements are devoid of repressive chromatin mark, H3K27me3. Plots show distribution of H3K27me3 chromatin modification in Th1 and Th2 cells at Th1-specific and Th2-specific p300-bound elements (y axis).
(B) Cell-type-specific enhancers are highly conserved among mammals. Enrichment of evolutionary conserved elements based on phastCons score around Th1-specific and Th2-specific p300 elements (red) in contrast to random regions of the genome (blue) (+/− 1kbp the center of p300 binding) (Kolmogorov-Smirnov test p-value < 2e-300).
(C) Cell-type-specific enhancers are enriched for CpG islands. CpG content around p300-elements proximal or distal to promoters compared to random elements (Kolmogorov-Smirnov test p-value < 1e-200).
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11. Figure S3
STAT6-Dependent p300 Binding Is Enriched at Genes Upregulated by STAT6, Related to Figure 3
(A) Global comparisons of p300 ChIP-seq experiments between duplicates (after log2 transformation on tag-per-million intensity) for STAT6-deficient cells reveal the reproducibility of p300 data. The Spearman’s correlation coefficient for the comparison is indicated in the insert.
(B) p300 binding at Il4-Il13 loci is STAT6-dependent. Gene track represents STAT6 binding along with p300 and H3K4me1 profiles in wild-type and Stat6−/− cells in Il4-Il13 loci.
(C) Th2-specific-STAT6-dependent p300 binding sites are enriched in proximity to genes positively regulated by STAT6. Using RNA-seq data in wild-type Th2 and STAT6-deficient cells, we selected positively regulated genes by STAT6 (>2-fold change). Plots depict number of Th2-specific-STAT6-dependent p300 binding sites within a given distance to promoters of genes upregulated by STAT6 (green) versus randomly generated sites (red) (Wilcoxon rank sum test p-value < 2.2e-16).
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12. Figure S4
Role of STAT4, T-bet, and STAT1 in Generating Th1-Specific Enhancers, Related to Figure 4
(A) Global comparisons of p300 ChIP-seq experiments between duplicates (after log2 transformation on tag-per-million intensity) for STAT1, STAT4, and T-bet-deficient cells reveal the reproducibility of p300 data. The Spearman’s correlation coefficient for each comparison is indicated in the insert.
(B and C) STAT4, STAT1 and T-bet are important for enhancers of the Ifng gene. Gene track (B) shows p300 binding and H3K4me1 in wild-type Th1, Stat4−/−, Stat1−/−, and T-bet−/− cells for the Ifng locus. Heatmap (C) quantitates intensity of p300 binding (log2) in (B) where “black” represents no binding.
(D) STAT4, STAT1 and T-bet colocalize at p300-bound regions in Ifng locus. Heatmap shows the quantification of (log2) binding of p300, STAT4, T-bet, and STAT1 across all p300 peaks in the locus.
(E–G) STAT4, T-bet, and STAT1 recruit p300 at selected enhancers of Ifng gene. Quantification of direct role of STAT1, STAT4, or T-bet on p300 binding in the Ifng locus reveals that 13, 7, and 3 p300-bound regions are directly dependent on STAT4, T-bet and STAT1, respectively.
(H) H3K4me1 at Th1-specific p300 elements is largely independent of STAT1, STAT4, or T-bet. Plots show distribution of H3K4me1 in wild-type, Stat4−/−, Stat1−/− and T-bet−/− cells at Th1-specific p300 elements (y axis).
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13. Figure S5
Direct Effects of Transcription Factors on the Chromatin Signature of Enhancers, Related to Figure 5
(A) One-third of STATs or T-bet-dependent p300 binding shows the binding of cognate factor. Number of STAT1, STAT4, T-bet and STAT6-dependent p300 binding that had the binding of cognate factor based on ChIP-seq data. Percentages refer to the portion of STAT or T-bet-dependent p300 sites with the transcription factor binding. ChIP-seq data were analyzed using normal rabbit serum as negative control (Ji et al., 2008). Furthermore, in STAT4, T-bet and STAT6 cases, ChIP-seq data in STAT4, T-bet, and STAT6-deficient cells were used to exclude non-specific binding.
(B) Binding sites of STAT4, STAT6, and T-bet with an effect on p300 are proximal to genes differentially regulated by each factor. T-bet binding at p300 correlates more with genes negatively regulated by T-bet. Using RNA-seq data in wild-type and STAT-deficient cells, differentially regulated genes by STATs were identified (>2-fold between wild-type and knockout cells). Similarly, differentially regulated genes by T-bet were identified (>1.5-fold change). Binding of each factor leading to p300 gain or loss were determined in (Figure 5) based on 4-fold change in p300 level between wild-type and knockout cells. Box plots show expression levels and number of genes within 20kbp of these p300 elements that are also regulated by each factor.
(C) STAT1 binding demonstrates a direct effect on p300 deposition. Two-dimensional histograms relating STAT1 binding to the fold-change in p300 binding in wild-type versus STAT1-deficient cells. x axis corresponds to the (log2) intensity of protein-DNA binding while y axis measures the (log2) fold-change of p300 in wild-type versus knockout cells. Color-map corresponds to the number of events.
(D) STAT6, but not STAT4, STAT1, or T-bet, has an impact on H3K4me1 deposition. At 1,152 (7%) of STAT6 binding sites, the absence of STAT6 resulted in more than 4-fold reduction in H3K4me1 modification. Two-dimensional histograms relating STAT4, T-bet, STAT1, or STAT6 binding to the fold-change in H3K4me1 modifications in wild-type versus STAT4, T-bet, STAT1, or STAT6-deficient cells, respectively.
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14. Figure S6
Recovery of p300 and Expression by T-bet and GATA3, Related to Figure 6
(A) Overexpression of GATA3 in STAT6-deficient cells is sufficient to restore IL-4 production. Naive CD4+ T cells from wild-type or Stat6−/− mice were infected with either control (blue) or GATA3-expressing (red) retrovirus and were cultured in Th2 condition. Transduced cells were isolated and levels of intracellular GATA3 and IL-4 proteins were measured by flow cytometry as shown in histograms. Values in parenthesis represent the percentages and MFI of the indicated markers. Staining with an isotype control antibody (gray) was used to set gates (dotted lines).
(B) Overexpression of GATA3 recovers p300 binding at STAT6-dependent enhancers of Il4-Il13 except the most conserved element (dotted box). Gene track shows p300 binding in Il4-Il13 locus for wild-type or STAT6-deficient cells when cells were treated with either the control or Gata3-expressing retrovirus under Th2 culture condition. Heatmap quantitates (log2) p300 binding for all peaks in wild-type (control vector) Th2 cells where “black” represents no binding. Overall, 8 STAT6-dependent peaks were recovered by GATA3 while 3 binding sites were still missing including the most conserved element upstream of Il4. The previously known elements along with the CNS lane were provided.
(C) Th-specific p300 elements in Th1 and Th2 cells with control retrovirus confirm the extent of cell-type-specific p300 elements in two cell populations. Venn diagram represents the number of common and unique p300 elements in Th1 and Th2 cells with control vector.
(D) GATA3 overexpression in STAT6-deficient cells could recover the expression of 41% of STAT6-dependent genes. Using RNA-seq data in wild-type and Stat6−/− cells transduced with control retrovirus, genes positively regulated by STAT6 were identified based on a 2-fold change difference between wild-type and knockout cells (239 genes). Using the RNA-seq data in wild-type and Stat6−/− cells transduced with GATA3 retrovirus, we identified GATA3 recovered (99, 41%) or not recovered genes (140, 58%) based on 1.5-fold change between wild-type and Stat6−/− cells.
(E) Overexpression of T-bet in STAT4-deficient cells is sufficient to restore IFNγ production. Active T cells were infected, differentiated and analyzed as in (A) using control (blue) or T-bet expressing (red) retrovirus under Th1 condition.
(F) Overexpression of T-bet recovers p300 binding at the promoter and selected enhancers of Ifng while many elements still lack p300 binding. Gene track shows p300 binding in Ifng locus for wild-type or STAT4-deficient cells when cells were treated with either the control or T-bet-expressing retrovirus. Heatmap quantitates (log2) p300 binding for all peaks in wild-type (control vector) Th1 cells where “black” represents no binding. Overall, 7 STAT4-dependent peaks recovered by T-bet including the promoter of Ifng while 23 binding sites were STAT4-dependent, and T-bet-independent. Previously characterized CNS elements along with the previously known elements were provided.
(G) T-bet overexpression in STAT4-deficient cells recovers the expression of 11% of STAT4-dependent genes. Using RNA-seq data in wild-type and Stat4−/− cells transduced with control retrovirus, genes positively regulated by STAT4 were identified based on a 2-fold change difference between wild-type and knockout cells (366 genes). Among these genes, T-bet recovered expression of (37, 11%) genes while (329, 89%) genes did not restore their expression (>1.5-fold change between wild-type and Stat4−/− cells).
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