Abstract
Aire is a transcription factor that controls T cell tolerance by inducing the expression of a large repertoire of genes specifically in thymic stromal cells. It interacts with scores of protein partners of diverse functional classes. We found that Aire and some of its partners, notably those implicated in the DNA-damage response, preferentially localized to and activated long chromatin stretches that were overloaded with transcriptional regulators, known as super-enhancers. We also identified topoisomerase 1 as a cardinal Aire partner that colocalized on super-enhancers and was required for the interaction of Aire with all of its other associates. We propose a model that entails looping of super-enhancers to efficiently deliver Aire-containing complexes to local and distal transcriptional start sites.
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Acknowledgements
We thank G. Buruzula, K. Rothamel, A. Rhoads, K. Hattori, A. Lopez, G. Gopalan, K. Waraska, M. Thorsen for experimental assistance and C. Laplace for help with manuscript preparation. The NIH Tetramer Core Facility (contract HHSN272201300006C) kindly provided tetramers. This work was supported by NIH grants R01 DK060027 and R01 AI088204. K.B. was supported by American Diabetes Association Mentor-Based Postdoctoral Fellowship #7-12-MN-51 to D.M.
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K.B. and H.Y. performed the experiments. K.B., C.B. and D.M. designed the study, analyzed and interpreted the data, and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Consistency of Aire ChIP-seq in mTECs.
a) Venn diagram displaying number of overlapping Aire peaks identified in two independent experiments. b) Exemplar genome browser views of two independent Aire ChIP-seq experiments in B6.Aire+/+ mTEChi.
Supplementary Figure 2 The mTEC super-enhancer repertoire.
a) Histogram depicting size density of super-enhancers (SEs) across mTEC genome. Dotted yellow line depicts the median length for all super-enhancers. Dotted black line shows a smoothed curve for super-enhancer size density. b) Normalized ChIP-seq profiles at exemplar super-enhancers in B6.Aire+/+ mTEChi. Numbers to the right indicate the ranges of normalized tag densities. SE, super-enhancer. Data are representative of two independent experiments. Data for one of the H3K27ac ChIP-seq experiments in (a, b) came from (ref.22).
Supplementary Figure 3 Super-enhancer repertoire of HEK293T cells.
a) Hockey plot for delineation of super-enhancers in HEK293T cells with H3K27ac ChIP-seq experiment reported in (ref. 24) SE, super-enhancer; CE, conventional enhancer. b) Heatmaps of tag density for the indicated proteins 500kb up- or down-stream of H3K27ac-delimited super-enhancers in HEK293T cells, H3K27ac and H3K4me1 data came from experiments reported in (ref. 24) while Aire, RNA-PolII and IgG data were reported in (ref. 14).
Supplementary Figure 4 shRNA knockdown of Aire-partners to reveal the sequence of Aire-partner associations in HEK293T cells.
Primary data supporting Fig. 3e. IP, immunoprecipitation. Representative immunoblots from two independent experiments.
Supplementary Figure 6 Differential distribution of TOP1 and TOP2A at super-enhancers in mTECs.
a, b) Normalized ChIP-seq profiles of indicated proteins at exemplar super-enhancers (a) or non-super-enhancer regions (b) in B6.Aire+/+ mTEChi to emphasize the lack of enrichment of TOP2A at super-enhancers. Numbers to the right indicate the ranges of normalized tag densities. SE, super-enhancer. Data are representative of two independent experiments. Data for one of the H3K27ac ChIP-seq experiments in (a, b) came from (ref. 22).
Supplementary Figure 7 Lack of thymic stromal cell and thymocyte perturbations after inhibition of topoisomerases.
a, b) Representative cytofluorimetric plots and summary quantitative data for mTEChi (a) and various thymocyte compartments (b) of B6.Aire+/+ mice treated with the indicated topoisomerase inhibitor/s or just vehicle (DMSO) every day for 3 days. Data are representative of three independent experiments with similar results (error bars, mean ± s.e.m. from n=3 measurements pooled from three experiments).
Supplementary Figure 8 Effect of topoisomerase inhibitors on mTEC gene expression and autoimmunity.
a, b) Representative cytofluorimetric plots gated on CD45-Ly51loMHCIIhi mTECs (b) and summary quantitative data (a, b) for mTEC expression of Aire (a) or the topoisomerases (b) in B6.Aire+/+ mice treated with the indicated topoisomerase inhibitor/s or just vehicle (DMSO) every day for 3 days. MFI, mean fluorescence intensity. c) As per Fig. 7a, except total RNA was isolated from mTECs after three weeks of treatment of mice with topoisomerase inhibitors. Gene-expression analyses were performed by quantitative PCR using SYBR Green. d) As per Fig. 6a, except 4-week-old B6.Aire-/- mice were intra-peritoneally injected with topotecan (n=3), etoposide (n=2), both drugs (n=2) or just vehicle (DMSO) (n=3). Orange: transcripts increased >2-fold in vehicle-treated Aire+/+ vs Aire-/- mice; green: transcripts decreased >2-fold. Numbers refer to transcripts up- (right) or down- (left) regulated by the indicated inhibitors (etoposide, P = 1.6 x 10-45; topotecan, P = 4 x 10-58; etoposide + topotecan, P = 9 x 10-101, P values for Aire-induced genes from χ2 test). e) Organ histology scores at 8-12 weeks of age for NOD/LtJ pups (Aire-/-) intra-peritoneally injected with topotecan, etoposide or just vehicle on the 2nd, 4th and 8th day after birth (6-7 mice per group). Scores reflect the scale described in Methods. *P < 0.05 (unpaired Student’s t-test). Data are representative of at least two (d, e) or three independent experiments (a-c) (error bars, mean ± s.e.m. from n = 3 (a-c) or n ≥ 6 (e) measurements).
Supplementary Figure 9 A simplified model of Aire’s mechanism of action.
A detailed scenario is presented in the Discussion section of the text. Not all Aire partners are depicted here, just those highlighted in the text.
1) At the super-enhancer, Aire and TOP1 interact, resulting in stabilization of DNA DSBs, mobilization of the DNA-damage response (e.g. γH2AX, DNA-PKcs, Ku80, PARP-1) and recruitment of general transcription factors (e.g. CBP, BRD4). 2) At the TSS or just downstream of it, additional DSBs occur due to contortion induced by paused RNA-PolII. The super-enhancer delivers its abundant Aire-containing complexes to the TSS, which overcomes pausing and promotes elongation. 3) Effective elongation necessitates relief of torsional stress up- and down-stream of the advancing RNA-PolII as well as eviction of interfering nucleosomes, likely performed by the “histone eviction” complex (including TOP2, DNA-PKcs, Ku80, PARP-1. etc). 4) An as-yet poorly defined Aire-containing complex promotes pre-mRNA processing. Ac = an acetylated residue; me1 = addition of a single methyl group; me3 = a trimethylated site; P = a phosphorylation site; TSS = transcriptional start-site; pTEFb is depicted as having two subunits, CycT1 and CDK9; bold red stretch = super-enhancer; bold black stretch = TSS; bold green = emerging pre-mRNA transcript.
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Efficiency and robustness of various ChIP-seq experiments (PDF 344 kb)
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Bansal, K., Yoshida, H., Benoist, C. et al. The transcriptional regulator Aire binds to and activates super-enhancers. Nat Immunol 18, 263–273 (2017). https://doi.org/10.1038/ni.3675
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DOI: https://doi.org/10.1038/ni.3675
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