STATs shape the active enhancer landscape of T cell populations

Abstract

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells.

Figure 1. Active Enhancer Landscapes in Th1 and Th2 Cells Are Distinct

(A) Chromatin signatures as defined by p300 binding and H3K4me1 identify recognized and new potential enhancers in the Il4-Il13 locus. The Il4-Il13 gene track represents 13 p300 binding sites within H3K4me1 domains in Th2 cells including 8 known elements (orange triangles) (Table S1B). “CNS” lane shows conserved non-coding sequences. (B) Genomic distribution of p300-bound elements in Th1 (total 25,554) and Th2 (total 22,534) cells at promoter (−4kbp to +500bp of TSS), intergenic (>4kbp TSS), and intragenic regions (+500bp of TSS to 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; Ghisletti et al., 2010). (D–F) In contrast to differentially expressed genes in Th1 and Th2 cells, housekeeping genes have little proximal p300 binding. Boxplots show median and quartiles of (D) normalized mRNA expression levels (RPKM: reads per kilobase exon model per million reads) 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 −20kbp to 20kbp from the TSS to capture potential enhancers. The intensity of H3K4me3 was computed −4kbp to 1kbp from the TSS to capture active promoters. (p-values wilcoxon rank-sum test).

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 (+/− 5kbp) (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 <2.2e-16). (D) Th-specific genes exhibit enrichment of p300 binding in the corresponding lineage and relative p300 depletion in the opposite lineage. Boxplots show median and quartiles of p300 binding in Th1 and Th2 cells around Th1- or Th2-specific genes (+/− 20kbp 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 using ChIP-seq data for each factor. A Gibbs sampling method was used to search for a motif using the genome as the background (likelihood ratio r>1000). 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.

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. Using RNA-seq data from wildtype Th2 and STAT6-deficient cells, we identified positively regulated genes by STAT6 (>2 fold-change). Accumulation of p300 binding at these genes in wildtype and STAT6-deficient cells was computed (+/− 20kbp from the TSS). Boxplots show median and quartiles of gene expression levels in RPKM (left) and p300 binding in tag-permillion (right) at STAT6-dependent genes in wildtype and STAT6-deficient cells (wilcoxon rank-sum test).

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) STAT4-positively regulated genes are enriched with STAT4-dependent p300 binding sites. Using RNA-seq data in wildtype Th1 and Stat4-deficient cells, positively regulated genes by STAT4 were identified (>2-fold change). Accumulation of p300 binding at these genes in wildtype and STAT4-deficient cells was computed (+/− 20kbp). Boxplots show normalized gene expression levels in RPKM (left) and p300 binding in tag-per-million (right) at STAT4-dependent genes in wildtype and STAT4-deficient cells (wilcoxon rank-sum test). (C) p300 binding at the extended loci of positively regulated genes by T-bet is not T-bet dependent. Using RNA-seq data in wildtype Th1 and T-bet-deficient cells, we selected positively regulated genes by T-bet (>2 fold-change). Boxplots show normalized gene expression levels in RPKM (left) and p300 binding in tag-per-million (right) at T-bet-dependent genes in wildtype and T-bet-deficient cells.

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 to 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 data. Examples of genes with proximal T-bet binding include Ifng, and Xcl1 (for positive effect) and Eomes and Il4-Il13 (for negative effect).

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, 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. Boxplots 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. Boxplots 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.