Abstract
The transcription factor Aire controls immunological tolerance by inducing the ectopic thymic expression of many tissue-specific genes, acting broadly by removing stops on the transcriptional machinery. To better understand Aire's specificity, we performed single-cell RNA-seq and DNA-methylation analysis of Aire-sufficient and Aire-deficient medullary epithelial cells (mTECs). Each of Aire's target genes was induced in only a minority of mTECs, independently of DNA-methylation patterns, as small inter-chromosomal gene clusters activated in concert in a proportion of mTECs. These microclusters differed between individual mice. Thus, our results suggest an organization of the DNA or of the epigenome that results from stochastic determinism but is 'bookmarked' and stable through mTEC divisions, which ensures more effective presentation of self antigens and favors diversity of self-tolerance between individuals.
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Acknowledgements
We thank S. Mostafavi for advice on computational analysis; M. Anderson (University of California, San Francisco) for the Aire-GFP line; and K. Hattori, G. Buruzula, and K. Waraska for help with mice, sorting and sequencing. Supported by the US National Institutes of Health (DK060027; and a Children's Hospital in Pediatric Gastroenterology training grant for M.M.) and Boehringer Ingelheim Fonds (D.Z.).
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M.M., data collection, data analysis and manuscript writing; D.Z., data analysis and manuscript writing; D.M., manuscript writing; C.B. data analysis and manuscript writing.
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Integrated supplementary information
Supplementary Figure 1 Less increase in frequency of expression of Aire-activated genes than predicted from sampling statistics.
We modeled the change in frequency of cells expressing a given gene resulting from the increase in its per-cell expression level. For each Aire-induced gene Gi we randomly picked, from the general distribution of Aire-neutral genes, 50 genes whose mean expression levels in positive cells matched the levels of Gi in Aire KO and WT cells, respectively, and we calculated the average pairwise difference in expression frequency. The distribution of these “simulated Aire-induction” changes (grey dots) matched the general distribution of intensity vs frequency of Fig. 4D but was very different from the corresponding changes for Aire-induced genes in the presence or absence of Aire (red dots), where equal changes in intensity led to far smaller increases in frequency (KS test p<10-15).
Supplementary Figure 2 Absence of co-expressed microclusters among Aire-neutral genes.
A) Gene-gene correlations were computed for a set of Aire-neutral genes (expression-matched with Aire-induced genes in Fig. 5A) and clustered by affinity propagation as in Fig. 5A. B) Direct comparison of the size and mean intra-cluster correlation for Aire-WT MEC microclusters for Aire-induced genes (red dots) and Aire-neutral genes (from S2A; black dots).
Supplementary Figure 3 Locally clustered Aire-induced genes are co-expressed in individual mTECs.
Representative raw read pileups for a few cells at locally clustered gene families in the A) Sprr locus on chromosome 3 and B) Mup locus on chromosome 4. Only one exon per gene was detected because the SCS technique only tags polyA-proximal sequences.
Supplementary Figure 4 MBD1 motifs are not over-methylated or over-represented at Aire-induced loci.
A) Distribution of methylation frequencies at CpGs in TCGCA (MBD1 binding site) motifs and non-TCGCA sites in Aire-induced and Aire-neutral promoters. Most CpGs in all four groups were unmethylated (<10%), and three exceptions of Aire-induced promoters containing a methylated TCGCA motif are indicated. B) Overall profiles of expression level and fold change in Aire WT/KO of genes with promoters that contain a TCGCA motif (regardless of methylation status) and those that do not. Table at bottom shows counts and proportions of TCGCA motifs in Aire-induced and neutral loci. C) Volcano plot showing MBD1’s effect on MEC transcriptome (all genes in black) overlaid with Aire-induced genes (red). Numbers show how many genes from each group fall in the two sectors of the plot.
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Meredith, M., Zemmour, D., Mathis, D. et al. Aire controls gene expression in the thymic epithelium with ordered stochasticity. Nat Immunol 16, 942–949 (2015). https://doi.org/10.1038/ni.3247
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DOI: https://doi.org/10.1038/ni.3247
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