Abstract
Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.
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Acknowledgements
We thank M. Anderson (University of California at San Francisco) for Aire-Igrp-GFP mice; D. Graf (University of Zurich) for B6.Foxn1-Cre mice (obtained with the consent of N. Manley (University of Georgia)); H. Clevers (Hubrecht Institute) for Tcf7−/− mice; and Y. Peleg, G. Yona and V. Krupalnik for experimental expertise and help. Supported by the Israel Science Foundation (1825/10 and 1376/13), the Sy Syms Foundation, the Dr. Celia Zwillenberg-Fridman and Dr. Lutz Fridman Career Development Chair (J.A.), the Weizmann-German Cancer Research Center PhD fellowship program (Y.H., M.D., J.A., M.F.), the German Cancer Research Center–Israeli Ministry of Science and Technology foundation for German-Israeli co-operation (2431 to J.A. and M.F.), the Agence Nationale de Recherche (2011-CHEX-001-R12004KK to M.Gi.) and the European Federation of Immunological Societies fellowship program (M.R.B.).
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Y.H. and J.A. designed the study and wrote the manuscript; Y.H. performed most of the experimental work; S.N. performed several experiments, including ChIP, protein immunoprecipitation and Aire intracellular staining; C.B. performed the 'indexing-first' ChIP and ATAC-Seq experiments; M.R.B set up and conducted luciferase-based assays and analyzed the Tbx21-mutant mice; L.W. and S.V. constructed the Tet1fl/flTet2fl/flTet3fl/fl mice; M.D. assisted in performing the DNA bisulfite experiments; and S.B.-H., A.S., M.E.-B., Y.G., B.L., E.D., S.B.-D., M.G., J.H.H., A.B., F.L., I.A., M.F. and J.A. helped in performing, analyzing and/or designing some of the experiments.
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Integrated supplementary information
Supplementary Figure 1 Aire expression in various immune-cell populations.
(a) Representative flow cytometry analyses of the expression of Aire.GFP (x-axis) and MHC class II (MHCII) (y-axis) in cell populations obtained from (n=3) 6-7week-old Aire.GFP mice, gated on thymic CD45+ EpCAM- CD11c- (T cells); CD45+ EpCAM- CD11c+ (Dendritic Cells; DCs); CD45- EpCAM+ Ly51+ (cortical thymic epithelial cells; cTECs); CD45- EpCAM+ Ly51- (medullary thymic epithelial cells; mTECs) or CD45+ EpCAM- CD19+ (B cells) populations. Numbers in outlined areas indicate %GFP+ cells, gated on an age-matched WT mouse. Data are representative of two independent experiments with similar results. (b) Quantitative PCR analysis of mean Aire expression in various sorted cell types obtained from same mice as indicated in (a). Results are normalized to the expression of Hprt and are presented relative to the expression in mature mTECs. (c) Genomic sequence of the predicted CpG islands. Color coded are the CG pairs. In light green are mTEC-specific differentially demethylated and in yellow are immature (MHCIIlo) mTEC-specific differentially demethylated cytosine residues.
Supplementary Figure 2 Quantitative PCR analysis verifies the mTEChi-cell-specific transcriptional regulator signature.
Clustered heat map depicting relative mRNA expression of indicated transcription regulators in sorted EpCAM+ Ly51neg-lo mature (MHCIIhi) vis-à-vis immature (MHCIllo) medullary or cortical (Ly51hi) thymic epithelial cells obtained from 6-7 weeks old mice (n=3). Shown are relative signal values normalized to expression of Hprt and relative to cTECs. Colors represent high (yellow) or low (blue) expression levels. Data are representative of at least three independent experiments with similar results.
Supplementary Figure 3 Aire reporter assays highlight potential mTEC-specific Aire regulators.
(a) Outline of the Aire promoter-based luciferase and RFP screening systems. (b) Representative flow cytometry analysis showing Aire.RFP reporter activity (x-axis) in HEK 293FT cells co-transfected with the Aire.RFP reporter vector alongside either an empty vector or expression vectors encoding all ~50 candidate mTEChi–specific transcription regulator genes for 48h. Numbers outlined present %RFPhi cells. Data are representative of at least three independent experiments with similar results. (c) Clustered heat map depicting mRNA expression profiles of candidate Aire-regulators that are predominantly expressed in antigen presenting cells or T cells and are predicted to activate the Aire promoter by either the Luciferase or the RFP reporter assays (Fig. 3e). Shown are normalized relative expression values of indicated immune cell populations. Colors represent high (yellow) or low (blue) expression levels. DCs, Dentiritic cells; T γδ, gamma delta T cells; T DP, Double positive (CD4+ CD8+) T cells; NK, Natural Killer cells.
Supplementary Figure 4 AIRE and AIRE-dependent genes are induced following expression of various transcriptional regulators.
(a+b) AIRE mRNA expression in HEK 293FT cells, treated with or without 5-Aza, encoding highlighted transcriptional regulators (a) or their combinations (b), and tested 48h later. (c-e) qPCR analyses showing mRNA expression of the AIRE-dependent genes ALOX12 (c) and KRT14 (d) or AIRE independent gene CAND1 (e) in 5-Aza treated cells transfected with the indicated factors and tested 48h later. (f-i) qPCR analyses showing AIRE mRNA levels in 5-Aza treated HEK 293FT cells transfected with expression vectors of Myb (f), Tbx21 (g), Tcf7 (h) or Tox4 (i) alongside short-listed candidates and measured 48h later; Results are normalized to the expression of HPRT and are presented relative to the expression in untreated cells transfected with an empty expression vector. Data in all experiments are representative of at least two independent experiments with similar results (mean and S.E.M of n = 2 biological replicates).
Supplementary Figure 5 mTECs with 50% Aire expression show normal TRA expression.
Quantitative PCR analysis of expression of Aire, several Aire-responsive TRAs (Ins2, Csn a, Mup4, Pcp4 and Spt1), and several Aire-neutral TRAs (Csn b and Gad67) in FACS-sorted mTEChi cells obtained from 6-8 week old Aire+/+, Aire+/– or Aire−/− mice (n=3). Results are normalized to the expression of Hprt and are presented relative to the expression values in Aire+/+ mTECs. Data are representative of two independent experiments with similar results (mean and S.E.M).
Supplementary Figure 6 Most candidate regulators bind the AIRE promoter independently of DNA-methylation status.
Chromatin immunoprecipitation followed by quantitative PCR assessing relative enrichment of indicated transfected candidates (vs. IgG control) along the AIRE TSS (corresponding to fragment 3 in Fig. 6b), in HEK 293FT cells treated 48h or untreated with 5-Aza. Results are normalized to values obtained from 5-Aza untreated cells. Data are representative of at least two independent experiments with similar results (mean and S.E.M of n = 2 biological replicates).
Supplementary Figure 7 Binding dynamics and endogenous expression of candidate transcriptional regulators.
(a-b) Chromatin immunoprecipitation followed by quantitative PCR assessing relative enrichment of CTCF following expression of Irf4/Irf8/Tbx21/Tcf7 (a) or of HA-tagged Irf4/Irf8/Tbx21/Tcf7 following expression of Ctcfl (b) in 5-Aza treated HEK 293FT cells transfected for 48h (n=3); (c-g) Quantitative PCR analyses showing endogenous expression of CTCF (c) HNF4G (d), IRF4 (e), IRF8 (f), TBX21 (g) and TCF7 (h) in 5-Aza treated HEK 293FT cells (pooled n=4) transfected with an siRNA set targeting expression of CTCF, and tested 48h later. Results are normalized to the expression of HPRT and are presented relative to the expression in the same cells transfected with a non-targeting siRNA set; Data are representative of at least two independent experiments with similar results. n.s. not significant, *P < 0.05, **P < 0.01 and ***P < 0.001 (Student's t-test, S.E.M).
Supplementary Figure 8 The molecular mechanisms that control the expression of Aire.
Aire is not expressed in the vast majority of cells in the body, except for a few cell types, primarily mature mTECs. To achieve such regulation, the Aire expression is regulated at multiple levels: First, the Aire locus is physically inaccessible and hypermethylated at specific CpG residues upstream and downstream to the TSS in cells and tissues that do not express it. Second, Aire locus is insulated by a global chromatin organizer - CTCF, which specifically binds at Aire’s TSS and downstream of the last Aire exon; Third, in mTECs, Aire expression is facilitated by concurrent eviction of CTCF, specific DNA demethylation and coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which act on mTEC-specific accessible regions in the Aire locus.
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Herzig, Y., Nevo, S., Bornstein, C. et al. Transcriptional programs that control expression of the autoimmune regulator gene Aire. Nat Immunol 18, 161–172 (2017). https://doi.org/10.1038/ni.3638
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DOI: https://doi.org/10.1038/ni.3638
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