A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.
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We thank the staff at the Wellcome Trust Clinical Research Facility (University of Southampton) where samples were acquired from volunteers; M. North for assisting in patient recruitment, assessment and sample collection; R. Jewel and C. McGuire for providing assistance in the flow cytometry facility (University of Southampton; J. Day for assistance with high-throughput sequencing at the La Jolla Institute for Allergy and Immunology sequencing facility, and A. Moghaddas Gholami at the La Jolla Institute for Allergy and Immunology bioinformatics core for help with the SNP enrichment analysis. L.C. is funded by a Feodor Lynen Research Fellowship from the Alexander von Humboldt Foundation. This work was supported by the Dana Foundation (K.M.A.), GlaxoSmithKline National Clinician Scientist Fellowship Award and Peel Travel Fellowship Award (P.V.), R01 HL114093 (to B.P., A.R. and P.V.) and U19 AI100275 (to B.P., A.R. and P.V.).
The authors declare no competing financial interests.
Integrated supplementary information
Schematic diagram depicts the cell types isolated from peripheral blood of healthy and asthmatic subjects. Sorting strategy for isolating naïve and CCR4– (TH1), CCR4+ (TH2) memory T cells from peripheral blood mononuclear cells (PBMC), and FACS plots pre and post-sorting are shown. The number of samples processed (passing quality control checks) for H3K4me2 ChIP-Seq assay is shown below.
Density plots show pair-wise comparison of sequencing coverage (number of reads) at genome-wide 500 bp windows (MEDIPS v.1.10.0 software, Methods and Supplementary Notes) obtained from 6 independent standard H3K4me2 ChIP-Seq assays performed with 2 × 106 D10 cells. The amount of DNA (post H3K4me2 ChIP; 15ng or 30ng) used for whole genome amplification (see Methods and Supplementary Notes) is shown. Pairwise Spearman correlation values (numbers inside boxes) for genome-wide comparison of H3K4me2 enrichment patterns between replicate samples (labeled as Rep 1-6) are illustrated.
(a) Density plots show pair-wise comparison of sequencing coverage at genome-wide 500bp windows (MEDIPS v.1.10.0 software, Methods and Supplementary Notes) obtained from standard ChIP-Seq (2 × 106 cells) and micro-scaled ChIP-Seq (105, 104 and 103 cell samples) performed with D10 cells. Pairwise Spearman correlation values (numbers inside boxes) for genome-wide comparison of H3K4me2 enrichment patterns between assays performed with different cell numbers are illustrated. (b) Shows density plots and pairwise Spearman correlation between multiple (n = 11) replicate micro-scaled ChIP-Seq assays (105 cell samples) performed in two separate sequencing runs (Run1 and Run2). (c) ROC analysis (detailed methodology is described in Methods and Supplementary Notes) shows the percentage of true and false positives identified by micro-scaled ChIP-Seq assay (105 cell sample) when tested for the top 1% of enriched windows (true positives) identified in the standard ChIP-Seq assay.
ChIP-Seq analysis showing H3K4me2 enrichment patterns from each assay (rows), for the following gene loci: control regions: STIM 1, NUP98, SELP and SELL gene loci; non-expressed gene: SELE locus; TH2 cell-type specific regions: CCR4 and CCR6 locus; TH1 cell-type specific regions: TBX21 (encoding T-BET), performed in peripheral blood naïve, TH1 and TH2 memory T cells from all study subjects. The significant cell-specific H3K4me2 enrichment across (enhancers and promoters) in these loci is highlighted in the red dashed line boxes.
ChIP-Seq analysis showing cell-specific H3K4me2 enrichment patterns, for the following gene loci: HNRPLL, ADAM19, miR155 and miR221-222, in naïve, TH1 and TH2 cells. For each specific cell-type, data was merged from all donors including assay duplicates. H3K4me2 enrichment values for specific 500 bp windows (highlighted in red boxes) are shown in the graphs below. Each dot represents data from a single assay-donor; error bars indicate mean ± (s.e.m.).
Diagnostic plots examining different characteristics of the ChIP-Seq data based on raw counts (left) and quantile normalized counts (right) resulting from the three pairwise cell type comparisons. The MA plots (top) contrast log2 fold changes (y-axis) against mean sequencing coverage (x-axis) for all genome wide 500 bp windows. Genomic windows with a Bonferroni adjusted P-value <0.05 are indicated in red. The density plots (middle) show the relative distribution of read counts (x-axis) at genome wide 500 bp windows for windows with read counts ≤10. The boxplots (bottom) show read counts at genome wide 500 bp windows for individual assays.
(a) As examples, cell-type specific enhancer DER tracks (pre- and post-normalization (norm)) along with UCSC tracks are shown for the following gene loci: TBX21, IFNG, CXCR3, CXCR6, STAT1 and STAT4, where additional DERs were detected following quantile normalization of the ChIP-Seq data (Supplementary Fig. 7). (b) The number and size distribution of DERs after merging consecutive DERs (see Methods and Supplementary Notes).
Supplementary Figure 9 Genomic distribution of differentially enriched cis-regulatory regions (DERs).
Pie chart (left) shows the distribution of DERs in different genomic regions, and compared to the reference annotation (right)
Supplementary Figure 10 Concordant changes in gene expression and H3K4me2 enrichment patterns at promoter and distal cis-regulatory elements (enhancers).
(a) Heat map shows the comparison of gene expression (RNA-Seq) and H3K4me2 enrichment patterns (across an extended genomic region) for transcripts having a DER in their promoter region when comparing naïve to TH2 memory CD4+ T cells (each row represents one transcript). Upstream = -20 kb from transcription start site (TSS); Promoter = +/-1 kb around TSS; Transcript body = region between TSS and transcript end site (TES); Downstream = +20 kb from TES; RNA = RNA-Seq data. The heat map indicates concordant gain (red) or loss (blue) of H3K4me2 enrichment in TH2 memory compared to naïve CD4+ T cells, at the promoter and at enhancers located in or close to that transcript (see Methods and Supplementary Notes). Similarly, the last column indicates concordant up- (red) or down- (blue) regulation of gene expression in TH2 memory cells compared to naïve CD4+ T cells for the corresponding transcripts. (b) As examples, H3K4me2 enrichment and RNA-Seq tracks for naïve and TH2 memory T cells (data merged from all donors including duplicate assays) are shown for the following gene loci: CCL20 and CCR8 (TH2 gain); S100B and LY86 (TH1 gain), along with UCSC gene tracks (top row) and cell-type specific enhancer DERs.
Supplementary Figure 11 Concordant changes in gene expression and H3K4me2 enrichment patterns at promoter and distal cis-regulatory elements.
(a) Heat map shows the comparison of gene expression (RNA-Seq) and H3K4me2 enrichment patterns (across an extended genomic region) for genes that are differentially expressed in any of the three pairwise comparisons of naïve, TH2 and TH1 cells (each row represents one gene). First column of the heat map (labeled RNA-Seq) displays log2 fold change in gene expression values. Next to the RNA-Seq data, fold change in H3K4me2 enrichment pattern across each extended gene locus (transcription start site (TSS) +/-50 kb) is displayed (see Methods and Supplementary Notes). The heat map indicates concordant changes in (up-red or down-blue) gene expression and H3K4me2 enrichment at the promoter and distal enhancer regions. Bottom panel shows genes with no significant changes in expression levels, but display a promoter-localized differentially enriched H3K4me2 region (DER) in any of the three pairwise comparisons of naïve, TH2 and TH1 cells. (b) As examples, RNA-Seq and H3K4me2 enrichment tracks for naïve and memory T cells (data merged from all donors including duplicate assays) are shown for the following gene loci: CCR6, CCR8 and FAM129a (TH2 active genes); CCL5 and CX3CR1 (TH1 active genes); IL4 and IL21 (TH2 poised genes), along with UCSC gene tracks (top row), cell-type specific DERs and differentially expressed genes (DEX). Red dashed line boxes highlight cell-specific DERs. (c) Shows MA plots (vertically displayed) for the pairwise comparisons of naïve, TH2 and TH1 cells, and red dots indicate differentially expressed genes (false discovery rate of 1%). (d-e Shows principal component analysis (PCA) for RNA-Seq data from each sample, and the genes contributing to the PCA.
Supplementary Figures 1–11, Supplementary Note (PDF 3029 kb)
The detailed description of 120 ChIP-seq assays. (XLSX 47 kb)
List of differentially enriched cis-regulatory regions (DERs) for cell types comparison. (XLSX 21830 kb)
Classification of the DERs into subgroups. (XLSX 10 kb)
List of all RefSeq promoters covered by DERs. (XLSX 135 kb)
Biological process-enrichment analysis. (XLSX 251 kb)
Genomic coordinates of enhancer DERs and linked genes. (XLSX 2697 kb)
Biological process and pathway-enrichment analysis of target genes linked to enhancer DERs. (XLSX 6051 kb)
Differential gene expression. (XLSX 3779 kb)
Transcription factors motif enrichment in DERs enhancers. (XLSX 15 kb)
Transcription factor binding site enrichment analysis. (XLSX 55 kb)
GWAS SNPs enrichment analysis. (XLSX 5147 kb)
Genomic coordinates of disease-specific enhancer DERs and linked genes. (XLSX 68 kb)
Transcription factor binding site enrichment analysis for disease-specific DERs. (XLSX 20 kb)
Biological process and pathway-enrichment analysis. (XLSX 12 kb)
Details of study subjects. (XLSX 9 kb)
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Seumois, G., Chavez, L., Gerasimova, A. et al. Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility. Nat Immunol 15, 777–788 (2014). https://doi.org/10.1038/ni.2937
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