Figure 3.
Stochastic expression of Aire-induced transcripts revealed by deep single-cell analysis. (A) t-SNE visualization of 1,980 TECs, color-coded by cluster assignment. (B) t-SNE plot color-coded for the mean expressions of 669 out of the 683 Aire-DEGs predominantly up-regulated in scMulti-seq/c3 (see also Fig. 2). (C) Volcano plot comparing RamDA-seq WT/c2 and Aire-KO/c3, with Bonferroni-adjusted P values from the Wilcoxon rank-sum test. Aire-DEGs defined by scMulti-seq in Fig. 2 A are highlighted in red. (D) Heatmap of 2,717 Aire-DEG expression across mTEChigh clusters, represented by standardized expressions. Genes were grouped as shown in Fig. 2. Genes expressed in fewer than five cells were excluded. (E) Expression of Aire-DEGs shown in D by individual cells. Expression levels are normalized to the 97.5th quantile and represented by color coding. Cell identities are indicated at the bottom. (F) Fractions of WT cells expressing Aire-DEGs predominantly up-regulated in each cluster. The median proportions of expressing cells are shown at the top and represented by red dashed lines in histograms. (G) Pearson’s correlation coefficient among Aire-enhanced genes (812 Aire-DEGs up-regulated in scMulti-seq-c4, scMulti-seq-c10, scMulti-seq-c11, scMulti-seq-c15-17, and scMulti-seq-c19 calculated with RamDA-c7, RamDA-c8, RamDA-c11, RamDA-c13, and RamDA-c14, top) and Aire-driven genes (588 Aire-DEGs predominantly up-regulated in scMulti-seq/c3 calculated with RamDA-c2 and RamDA-c3, bottom). Left: Gene-to-gene correlations after clustering by affinity propagation. Right: Circos plots indicating genomic positions for highly correlated genes (Pearson’s r > 0.6), with 1,157 and 24 connections by red lines for Aire-enhanced genes and Aire-driven genes, respectively (see also Fig. S2 H). (H) Histograms representing the distance between highly correlated genes within two Aire-DEG groups. Top: The relative frequency for the intra- and interchromosomal correlations (4.2% and 93.2% interchromosomal correlations for Aire-driven genes and Aire-enhanced genes, respectively; P = 7.7 × 10−51 by the chi-square test). Bottom: The frequency within the same chromosomes (total 23 connections for Aire-driven genes and 79 for Aire-enhanced genes; P = 2.4 × 10−11 by the Wilcoxon rank-sum test). (I) Illustration of two types of Aire-DEGs: Aire-driven genes and Aire-enhanced genes. Both types are directly induced by Aire’s transcriptional activity within Aire-expressing mTECs in a stochastic manner. Aire-enhanced genes are additionally expressed in mimetic cells in a coordinated manner.

Stochastic expression of Aire-induced transcripts revealed by deep single-cell analysis. (A) t-SNE visualization of 1,980 TECs, color-coded by cluster assignment. (B) t-SNE plot color-coded for the mean expressions of 669 out of the 683 Aire-DEGs predominantly up-regulated in scMulti-seq/c3 (see also Fig. 2). (C) Volcano plot comparing RamDA-seq WT/c2 and Aire-KO/c3, with Bonferroni-adjusted P values from the Wilcoxon rank-sum test. Aire-DEGs defined by scMulti-seq in Fig. 2 A are highlighted in red. (D) Heatmap of 2,717 Aire-DEG expression across mTEChigh clusters, represented by standardized expressions. Genes were grouped as shown in Fig. 2. Genes expressed in fewer than five cells were excluded. (E) Expression of Aire-DEGs shown in D by individual cells. Expression levels are normalized to the 97.5th quantile and represented by color coding. Cell identities are indicated at the bottom. (F) Fractions of WT cells expressing Aire-DEGs predominantly up-regulated in each cluster. The median proportions of expressing cells are shown at the top and represented by red dashed lines in histograms. (G) Pearson’s correlation coefficient among Aire-enhanced genes (812 Aire-DEGs up-regulated in scMulti-seq-c4, scMulti-seq-c10, scMulti-seq-c11, scMulti-seq-c15-17, and scMulti-seq-c19 calculated with RamDA-c7, RamDA-c8, RamDA-c11, RamDA-c13, and RamDA-c14, top) and Aire-driven genes (588 Aire-DEGs predominantly up-regulated in scMulti-seq/c3 calculated with RamDA-c2 and RamDA-c3, bottom). Left: Gene-to-gene correlations after clustering by affinity propagation. Right: Circos plots indicating genomic positions for highly correlated genes (Pearson’s r > 0.6), with 1,157 and 24 connections by red lines for Aire-enhanced genes and Aire-driven genes, respectively (see also Fig. S2 H). (H) Histograms representing the distance between highly correlated genes within two Aire-DEG groups. Top: The relative frequency for the intra- and interchromosomal correlations (4.2% and 93.2% interchromosomal correlations for Aire-driven genes and Aire-enhanced genes, respectively; P = 7.7 × 10−51 by the chi-square test). Bottom: The frequency within the same chromosomes (total 23 connections for Aire-driven genes and 79 for Aire-enhanced genes; P = 2.4 × 10−11 by the Wilcoxon rank-sum test). (I) Illustration of two types of Aire-DEGs: Aire-driven genes and Aire-enhanced genes. Both types are directly induced by Aire’s transcriptional activity within Aire-expressing mTECs in a stochastic manner. Aire-enhanced genes are additionally expressed in mimetic cells in a coordinated manner.

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