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Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells

Nature Immunology volume 16, pages 933–941 (2015)Cite this article

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Abstract

Expression of tissue-restricted self antigens (TRAs) in medullary thymic epithelial cells (mTECs) is essential for the induction of self-tolerance and prevents autoimmunity, with each TRA being expressed in only a few mTECs. How this process is regulated in single mTECs and is coordinated at the population level, such that the varied single-cell patterns add up to faithfully represent TRAs, is poorly understood. Here we used single-cell RNA sequencing and obtained evidence of numerous recurring TRA–co-expression patterns, each present in only a subset of mTECs. Co-expressed genes clustered in the genome and showed enhanced chromatin accessibility. Our findings characterize TRA expression in mTECs as a coordinated process that might involve local remodeling of chromatin and thus ensures a comprehensive representation of the immunological self.

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Figure 1: Mature mTECs show heterogeneous gene expression at the single-cell level but express a comprehensive set of TRA-encoding genes as a population.
Figure 2: Single mature mTEC transcriptomes reveal numerous low-frequency sets of co-expressed genes.
Figure 3: Confirmation of co-expression in gene sets by independent experimental approaches.
Figure 4: The Tspan8– or Ceacam1–co-expressed gene sets overlap, and corresponding mTECs are organized along a gradient of Tspan8 expression.
Figure 5: Co-expressed genes cluster in the genome.
Figure 6: Promoters of co-regulated genes show increased chromatin accessibility.

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Acknowledgements

We thank K. Hexel and S. Schmitt for single-cell sorting; S. Egle for technical help; C. Sebening and T. Loukanov (University of Heidelberg) for human thymic tissue; The Genomics Core Facility of the European Molecular Biology Laboratory for initial sequencing, and M. Miranda and E. Hopmans for support during subsequent sequencing at the Stanford Genome Technology Center; J. Buenrostro and C. Chabbert for discussions about ATAC-seq experiments and data, respectively; C. Michel and S. Anders for advice and comments on the manuscript; W. Wei and M. Sikora for help with data transfer; and The Central Animal Facility (German Cancer Research Center) for animal care. Supported by the European Union 7th Framework Programme (Health) via Project Radiant (W.H. and A.R.), The Helmholtz Center (K.R.), the Sonderforschungsbereich (DFG 938 to S.P.), the European Research Council (ERC-2012-AdG to B.K.) and the US National Institutes of Health (P01 HG000205 and R01 GM068717 to P.B., M.N. and L.M.S.).

Author information

Author notes

  1. Philip Brennecke, Alejandro Reyes, Sheena Pinto and Kristin Rattay: These authors contributed equally to this work.

  2. Wolfgang Huber, Bruno Kyewski and Lars M Steinmetz: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Genetics, Stanford University, School of Medicine, California, USA

    Philip Brennecke, Michelle Nguyen & Lars M Steinmetz

  2. Stanford Genome Technology Center, Stanford University, California, USA

    Philip Brennecke, Michelle Nguyen & Lars M Steinmetz

  3. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany

    Alejandro Reyes, Wolfgang Huber & Lars M Steinmetz

  4. Division of Developmental Immunology, German Cancer Research Center, Heidelberg, Germany

    Sheena Pinto, Kristin Rattay, Rita Küchler & Bruno Kyewski

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  1. Philip Brennecke
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Contributions

P.B., S.P., B.K. and L.M.S. conceived of the project; P.B., S.P. and K.R. designed experiments; P.B. performed single-cell sequencing experiments, Klk5 single-cell quantitative PCR confirmation experiments and ATAC-seq experiments; S.P. helped with the ATAC-seq experiments; S.P. and K.R. performed experimental mTEC preparations and flow cytometry of single and bulk mTECs; A.R. and W.H. designed analysis strategy and analyzed the data; A.R. prepared the figures; P.B., A.R., S.P., K.R., W.H., B.K. and L.M.S. interpreted the data and wrote the manuscript; M.N. and R.K. provided technical assistance; and L.M.S., B.K. and W.H. supervised the project.

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Correspondence to Wolfgang Huber, Bruno Kyewski or Lars M Steinmetz.

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Integrated supplementary information

Supplementary Figure 1 Histogram of the fraction of genes detected per mTEC that are classified as TRA-encoding genes.

The x-axis shows the fraction of genes detected per cell that are classified as TRA-encoding genes. The y-axis shows the number of cells.

Supplementary Figure 2 Enrichment of TRA-encoding genes among highly variable genes.

Barplot showing the fraction of TRA-encoding genes (y-axis) in two subsets of genes (x-axis), those that were detected to be highly variable in Fig 1c and the rest of the genes that were not detected to be highly variable (See Methods for details). The difference in the fraction of genes overlapping with TRA-encoding genes was detected to be significantly different between the two gene subsets (p-value < 2.2 x 10-16, Fisher’s exact test).

Supplementary Figure 3 Enrichment of genes in the Tspan8–co-expressed gene set among genes from cluster B resulting from the k-medoids clustering.

Barplot showing the fraction of genes from the Tspan8 co-expressed gene set (y-axis) in two subsets of genes (x-axis), genes belonging to cluster B resulting from the k-medoids clustering (Fig. 2a) and genes grouped to the rest of the k-medoids clusters (See Methods for details). The difference in the fraction of genes overlapping with the Tspan8 co-expressed gene set was detected to be significantly different between the two gene subsets (p-value < 2.2 x 10-16, Fisher’s exact test).

Supplementary Figure 4 Confirmation of co-expressed gene sets by independent experimental approaches.

Each point depicts one gene. The y-axis shows the logarithmic fold change (base 2) of the unselected mature mTECs where TRA expression was detected by the scRNA-seq assay with respect to the unselected mature mTECs where the scRNA-seq assay did not detect the expression of the TRA. The x-axis shows the logarithmic fold change (base 2) between the mature mTECs selected for the expression of the TRA (by either qPCR or flow cytometry) and the unselected mature mTECs where the scRNA-seq assay did not detect the expression of the TRA. Each column panel shows the data for one TRA. The row panels split the genes according to whether they were detected to be co-expressed with the specific TRA or not.

Supplementary Figure 5 Enrichment of genes in the Klk5–co-expressed gene set among genes from cluster D resulting from the k-medoids clustering.

Barplot showing the fraction of genes from the Klk5 co-expressed gene set (y-axis) in two subsets of genes (x-axis), genes belonging to cluster D resulting from the k-medoids clustering and genes grouped to the rest of the k-medoids clusters (See Methods for details). The difference in the fraction of genes overlapping with the Klk5 co-expressed gene set was detected to be significantly different between the two gene subsets (p-value < 9.6 x 10-4, Fisher’s exact test).

Supplementary Figure 6 Expected versus observed genomic proximity of the 11 groups of co-expressed genes resulting from the k-medoids clustering.

Each panel shows the data for one of the 11 clusters from Fig. 2b. The histograms show the expected distribution of the median genomic distance between pairs of genes in the genome according to the size of each co-expressed gene set. This expected distribution was estimated by sampling random genes of the same size of the co-expressed gene set for 1,000 times. The observed median distance for each cluster is depicted with vertical solid lines.

Supplementary Figure 7 Karyogram of the genomic position of the 11 groups of co-expressed genes resulting from the k-medoids clustering.

Each panel corresponds to one gene cluster. The colors correspond to the colors from Fig. 2. The colored vertical lines in the chromosomes mark the genomic position of genes.

Supplementary Figure 8 Examples of co-expressed genomic loci and their locations in the genome.

(a) The genomic locus of the BPI fold-containing B gene family is shown. The x-axis represents the genomic positions from chromosome 2 of the mouse genome. Protein coding genes are shown in boxes. Genes colored in purple belong to cluster “D”. (+) = plus strand, (-) = minus strand. (b) The genomic locus of the Glutathione S-transferase Mu gene family is shown. The x-axis represents the genomic positions from chromosome 3 of the mouse genome. Protein coding genes are shown in boxes. Genes colored in purple belong to cluster “D”. (+) = plus strand, (-) = minus strand. (c) Example of co-expressed genes from unrelated gene families that are clustered in the genome. The x-axis represents the genomic positions from chromosome 6 of the mouse genome. Protein coding genes are shown in boxes. Genes colored in purple belong to cluster “D”. (+) = plus strand, (-) = minus strand.

Supplementary Figure 9 Fraction of cells with detection of expression of genes encoding proteases of the kallikrein family.

The y-axis shows the fraction of cells for which the scRNA-seq assay detected gene expression. The x-axis shows genes ordered by genomic location. The upper panel shows the data for the qPCR-selected Klk5+ cells. The lower panel shows the data for the unselected mature mTECs for which Klk5 expression was not detected by the scRNA-seq assay.

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Brennecke, P., Reyes, A., Pinto, S. et al. Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells. Nat Immunol 16, 933–941 (2015). https://doi.org/10.1038/ni.3246

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