Abstract
Genetic variations underlying susceptibility to complex autoimmune and allergic diseases are concentrated within noncoding regulatory elements termed enhancers1. The functions of a large majority of disease-associated enhancers are unknown, in part owing to their distance from the genes they regulate, a lack of understanding of the cell types in which they operate, and our inability to recapitulate the biology of immune diseases in vitro. Here, using shared synteny to guide loss-of-function analysis of homologues of human enhancers in mice, we show that the prominent autoimmune and allergic disease risk locus at chromosome 11q13.52,3,4,5,6,7 contains a distal enhancer that is functional in CD4+ regulatory T (Treg) cells and required for Treg-mediated suppression of colitis. The enhancer recruits the transcription factors STAT5 and NF-κB to mediate signal-driven expression of Lrrc32, which encodes the protein glycoprotein A repetitions predominant (GARP). Whereas disruption of the Lrrc32 gene results in early lethality, mice lacking the enhancer are viable but lack GARP expression in Foxp3+ Treg cells, which are unable to control colitis in a cell-transfer model of the disease. In human Treg cells, the enhancer forms conformational interactions with the promoter of LRRC32 and enhancer risk variants are associated with reduced histone acetylation and GARP expression. Finally, functional fine-mapping of 11q13.5 using CRISPR-activation (CRISPRa) identifies a CRISPRa-responsive element in the vicinity of risk variant rs11236797 capable of driving GARP expression. These findings provide a mechanistic basis for association of the 11q13.5 risk locus with immune-mediated diseases and identify GARP as a potential target in their therapy.
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Data availability
RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE128198. Sequencing data for H3K27ac hQTL and mRNA eQTL analyses are deposited under the European Genome-phenome Archive (EGA; study accession EGAS00001003516, datasets EGAD00001004828 and EGAD00001004830).
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Acknowledgements
The research was supported by Wellcome Trust–Royal Society Fellowship 105663/Z/14/Z, Wellcome Trust grant WT206194, Biotechnology and Biological Sciences Research Council grants BB/N007794/1, BBS/E/B/000C0427 and BBS/E/B/000C0428, Cancer Research UK grant C52623/A22597, Medical Research Council grants MR/N014995/1, MR/S024468/1 and MC_UU_00014/5, Wellcome Trust Major Award 208363/Z/17/Z, Associazione Italiana per la Ricerca sul Cancro (AIRC) grant IG 20607, and US National Institutes of Health (NIH) grants RM1-HG007735, U19-AI142733 and R01-AI121920. We thank members of the Babraham Institute Biological Services Unit, flow cytometry facility and sequencing facility, and Wellcome Sanger Institute flow cytometry, sequencing, IT and data access facilities for data generation and processing. We thank all participating blood donors, and Cambridge and Oxford NHS Blood and Transplant, New York Blood Center and Policlinico San Matteo Pavia Fondazione for the recruitment of study participants. We thank F. De Paoli (Humanitas) for processing human PBMC samples and M. Turner, K. Okkenhaug, E. Shevach, M. Linterman and G. Butcher for support and discussion.
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R.N., C.J.I., F.M.G., M.D., L.P., L.K, P.K., F.S., S.K.W., A.C., P.V., C.E.W., T.L., T.F., H.F., E.L., D.U., S.M. and R.R. performed experiments. L.B.-C., D.G. and G.T. performed and analysed hQTL and eQTL analyses of human T cells. R.N., C.J.I., F.S., M.R.M., H.F., J.Y., A.L., S.A., G.T. and R.R. analysed data. R.N., C.J.I, L.B.-C., G.T. and R.R. wrote the manuscript. H.Y.C., D.U., G.T. and R.R. provided overall supervision of the work.
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Extended data figures and tables
Extended Data Fig. 1 Highly linked genetic polymorphisms at a distal intergenic region of 11q13.5 are associated with risk of multiple immune-mediated diseases.
a–d, Graph showing association of polymorphisms within the 11q13.5 locus with risk of indicated immune-mediated disorders (−log10 (P); left axis). Each point represents an individual SNP. Point colours depict R2 values reflecting the level of linkage disequilibrium between each polymorphism and the indicated lead GWAS variant (diamond symbol). Chromosomal position (GRCh38) is represented on the x-axis. GWAS summary statistics and replicate information from ref. 45 (a), ref. 4 (b) ref. 6 (c) and ref. 2 (d).
Extended Data Fig. 2 Alignments showing distribution of H3K27ac at the indicated genomic region in primary human lymphocytes.
Alignment of H3K27ac enrichment at the indicated locus within the indicated lymphocyte lineages; sample information and replicate statistics are from the Roadmap Epigenomics Project8. Grey shaded area marks the risk locus containing the single nucleotide polymorphism (SNP) rs11236797 (position indicated by red triangle).
Extended Data Fig. 3 Steady-state immune phenotype of Enh-KO mice.
a, b, Body mass at indicated age (a) and Kaplan–Meier plot showing survival (b) of mice of indicated genotypes (n = 8 and 7, WT and Enh-KO from 8 independent breedings). c, Representative flow cytometry (left) and replicate measurements (right) of the frequency of the indicated thymocyte subsets in the thymi of mice of the indicated genotypes at 8–10 weeks of age (n = 6 and 5; WT and Enh-KO). d, Representative flow cytometry (left) and replicate measurements of the frequency and absolute number (right) of Foxp3+ Treg cells in the spleens of mice of the indicated genotypes at 8–10 weeks of age (n = 5 mice per group). Representative of two independent experiments (c, d). e, Representative flow cytometry (left) and replicate measurements of the frequency of naive and helper CD4+Foxp3− Tconv cells in the spleens of mice of the indicated genotypes at 8–10 weeks of age (n = 9 mice per group, pooled from two independent experiments). f, Frequency of cells expressing IFN-γ (left) and TNF (right) upon intracellular cytokine staining analysis of splenic CD4+ and CD8+ T cells from mice of the indicated genotypes at 8–10 weeks of age (n = 10 mice per group pooled from two independent experiments). Unpaired two-tailed Student’s t test (a, c-f), Mantel–Cox test (b). Data are mean ± s.e.m.
Extended Data Fig. 4 Increased susceptibility of Enh-KO mice to colitis induced by DSS.
a, b, Replicate length measurements (a) and photographs (b) of large intestine from WT and Enh-KO mice treated with DSS or vehicle control (vehicle). n = 4 (WT vehicle), 4 (Enh-KO vehicle), 10 (WT DSS) and 8 (Enh-KO DSS). c, Histopathological scores of sections of large intestine from WT and Enh-KO treated for 16 days with DSS or vehicle. The following scoring criteria were used: extent of inflammation: 0, none; 1, mucosa; 2, mucosa and submucosa; 3, transmural. Crypt damage: 0, none; 1, basal one-thirds; 2, basal two-thirds; 3, only surface epithelium intact; 4, loss of entire crypt and surface epithelium. Tissue involvement: 1, 0 to 25%; 2, 26 to 50%; 3, 51 to 75%; 4, 76 to 100%. Severity of inflammation: 0, none; 1, mild; 2, moderate; 3, severe. Data are representative of two independently repeated experiments with 10 and 4 mice per DSS- and vehicle-treated group. Unpaired two-tailed Student’s t test (a); Wilcoxon-Mann–Whitney Test (c). Data are mean ± s.e.m.
Extended Data Fig. 5 Analysis of cytokine expression in WT and Enh-KO mice treated with DSS.
a, Replicate measurements of IFN-γ (left) and Foxp3 (right) in CD4+ T cells from large intestinal lamina propria (n = 8 mice per group). Data representative of two independent experiments. b–e, Concentration of the indicated cytokines in the serum of WT and Enh-KO mice treated with vehicle or DSS for 16 days. Data pooled from two independent experiments with n = 8, 8, 20 and 17 for WT (vehicle), Enh-KO (vehicle), WT (DSS), Enh-KO (DSS) groups. Data analysed using unpaired two-tailed Student’s t test. Data are mean ± s.e.m.
Extended Data Fig. 6 Loss of LRRC32 and GARP expression in Foxp3+ Treg cells from Enh-KO mice.
a, Scatter plot showing global differences in chromatin accessibility at called peaks using ATAC-Seq analysis of WT and Enh-KO CD4+ Foxp3GFP+ Treg cells. Red dots show significantly differentially accessible peaks (FDR < 0.05). Mean log2 reads per million (RPM) values for each called peak (represented as points) from three independent biological replicates are shown, with samples isolated on different days. Two-tailed Wald test with Benjamini–Hochberg correction. b, Representative alignment of gene expression (top) and chromatin accessibility (bottom) within the indicated cell types sorted by FACS from WT and Enh-KO Foxp3eGFP reporter mice. Expected loss of ATAC-seq reads mapping to the deleted region (Enh; highlighted in grey) in Enh-KO cells is observed. Data representative of three independent biological replicates with samples isolated on different days. c, Representative flow cytometry analysis of GARP and Foxp3 expression by CD4+ T cells of mice of the indicated genotypes from mesenteric lymph nodes. d, GARP expression from mice of indicated genotypes in spleen (n = 5 per genotype), thymus (n = 6 and 5 for WT and Enh-KO groups), and mesenteric lymph node (n = 3 and 4 for WT and Enh-KO groups). Data are representative of three and two independent experiments (c, d). Unpaired two-tailed Student’s t test (d). Data are mean ± s.e.m.
Extended Data Fig. 7 Lrrc32 +70k is not required for induction of GARP on the surface of CD4+ Tconv cells following stimulation in vitro.
a, Representative flow cytometry showing gating strategy and representative GARP expression on resting (CD44lowCD62L+) and activated (CD44highCD62L−) Treg cells (top) and naive (CD44lowCD62L+) and memory (CD44highCD62L−) Tconv cells (bottom). Data are representative of two independent experiments. b, Representative flow cytometry (top) and replicate measurements (bottom) of CD25 and GARP expression in CD4+Foxp3− Tconv cells following stimulation under the indicated conditions for 16 h in vitro. Data representative of three independently repeated experiments with five independent biological replicates per group. c, Representative alignments of known H3K27ac ChIP-seq data from the indicated cell types. Sample information and replicate statistics are in ref. 20. Grey bar shows position of the enhancer. Unpaired two-tailed Student’s t test (b). Data are mean ± s.e.m.
Extended Data Fig. 8 Specific loss of GARP expression on Treg cells from Enh-KO mice.
a, Representative flow cytometry of GARP expression on CD45−CD31+ endothelial cells from lungs of WT and Enh-KO mice. b, Frequency of GARP+ cells among WT and Enh-KO B220+CD19+GL7+ cells, stimulated with bacterial LPS or vehicle control (Veh) for 48 h (n = 6 and 8 for WT and Enh-KO groups). c, Percentage of GARP+ cells among indicated cell types from WT and Enh-KO mice (n = 6 per genotype; unpaired two-tailed Student’s t test). d, Representative histograms showing GARP expression in the cell types shown in c. e, Representative flow cytometry indicating gating strategy for cells shown in c. Unpaired two-tailed Student’s t test (b). Data are representative of two independent experiments. Data are mean ± s.e.m.
Extended Data Fig. 9 Molecular and functional characterization of mouse and human enhancer homologues.
a, Evolutionarily conserved STAT5 and NF-κB binding motifs within Lrrc32 +70k. Genomic sequence alignments of reference genome sequences of indicated mammals are shown. The position of conserved STAT5 and NF-κB binding motifs (V$STAT5A_03 and V$NFKAPPAB_01, respectively) are highlighted in grey. The position of rs11236797 is shown. b, Alignment of previously determined STAT5 ChIP-seq binding at the indicated locus in human Treg and Tconv cells. Sample information and replicate statistics are in ref. 21. The identified distal enhancer is shown indicated by the grey shaded area. c, GARP expression in CD4+Foxp3+ Treg cells following stimulation under the indicated conditions for 16 h in vitro. n = 4 technical replicates per condition; data are representative of three independent experiments. d, Representative flow cytometry showing gating (left) and replicate measurements (right) of CD45.2+ (transferred Treg) and CD45.2− (transferred Tconv) cells within the spleen and mesenteric lymph node of cell-transfer recipients. e, Replicate measurements of GARP expression on the indicated cell types from the spleen. f, Replicate measurements of expression of indicated cytokines by CD45.2− Tconv cells from indicated tissues following brief restimulation ex vivo. n = 5 and 6, WT and Enh-KO Treg recipients (d–f). Data representative of two independent experiments. Unpaired two-tailed Student’s t test (c–f). Data are mean ± s.e.m.
Extended Data Fig. 10 Conformational topography and eQTL analysis of human 11q13.5.
a, Visualization of intrachromosomal interactions at human 11q13.5 within the B lymphoid line GM1287829. A sub-topologically associated domain containing the identified enhancer and the promoter of LRRC32 is indicated. Sample information and replicate statistics are in ref. 29. b, Analysis of intrachromosomal H3K27ac-enriched HiChIP interactions in human CD4+ naive (CD45RA+CD25−CD127high), Treg (CD25+CD127low), and TH17 cells (CD45RA−CD25−CD127highCCR6+CXCR5−) isolated directly from human peripheral blood. Sample information and replicate statistics are in ref. 28. c, Expression quantitative trait locus (eQTL) analysis of the association between genetic polymorphisms at the indicated SNP with LRRC32 expression in human CD4+CD127−CD25+ Treg cells isolated by FACS from the blood of 123 healthy human donors. Point colours reflect linkage disequilibrium (R2) relative to rs11236797. Nominal P value and FDR of the most significantly associated SNP are shown; linear regression, two-sided.
Supplementary information
Supplementary Table 1
Genomic sequence alignment of Human Chr11:76588236-76592844 (GRCh38) with the reverse complement of mouse Chr7:105711382-105713753 (mm9).
Supplementary Table 2
Analysis of global gene expression differences between WT and Enh-KO CD4+ Foxp3GFP+ Treg cells and Foxp3GFP– Tconv cells.
Supplementary Table 3
Normalised H3K27Ac enrichment at Chr11:76586431-76600121 in CD4+ CD127- CD25+ Treg cells freshly isolated from 91 genotyped healthy donors.
Supplementary Table 4
Normalised H3K27Ac enrichment at Chr11:76586431-76600121 in CD4+ Naive Tconv cells isolated from 142 genotyped healthy donors.
Supplementary Table 5
Association of distinct polymorphic variants within the 11q13.5 risk locus with IBD risk and H3K27ac enrichment at chr11:76586431-76600121.
Supplementary Table 6
sgRNA sequences used in CRISPRa screen.
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Nasrallah, R., Imianowski, C.J., Bossini-Castillo, L. et al. A distal enhancer at risk locus 11q13.5 promotes suppression of colitis by Treg cells. Nature 583, 447–452 (2020). https://doi.org/10.1038/s41586-020-2296-7
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DOI: https://doi.org/10.1038/s41586-020-2296-7
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