Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells

Abstract

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

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Figure 1: Transgenic rescue of nude phenotype in Foxn1wt*/wt* mice expressing a chimeric Foxn1-Flag protein.
Figure 2: Foxn1 availability in TECs determines T cell developmental defects.
Figure 3: Foxn1 ChIP-seq analysis.
Figure 4: Intersection of Foxn1 ChIP-seq and RNA-seq analyses.
Figure 5: Psmb11 and Cd83 are direct targets of Foxn1.
Figure 6: Comparative analysis of the effect of a loss of Cd83 expression and expression of a hypomorphic Foxn1 allele, respectively, on intrathymic T cell development.

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Acknowledgements

We thank E. Christen, R. Recinos, A. Offinger, D. Nebenius-Oosthulzen and A. Klewe-Nebenius for technical support, M. Gaio and S. Harris for secretarial assistance, and J. Lopez-Rios and M. Osterwalder for help establishing ChIP and provision of plasmids. Supported by the Swiss National Foundation (3100- 68310.02 and 3100-122558 to G.A.H.), The Wellcome Trust (105045/Z/14/Z to G.A.H. and C.P.P.; 100643/Z/12/Z to A.H.) the MRC (C.P.P.) and the European Commission within the Seventh Framework Programme (FP7 project 261387 to G.H.).

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S. Žuklys, S. Zhanybekova, A.H. and G.A.H. designed the experiments. S. Žuklys, C.E.M., S. Zhanybekova, F.G., H.Y.T., K.H., S.M. and M.K. performed the experiments. S. Žuklys, A.H., S. Zhanybekova, F.G., C.E.M., T.B., G.G., C.P.P. and G.A.H. analyzed and/or interpreted the results. G.A.H. wrote the manuscript with contributions from A.H. and S. Žuklys.

Corresponding authors

Correspondence to Saulius Žuklys or Georg A Holländer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Transgenic expression of a Foxn1-3xFlag fusion protein in Foxn1nu/nu mice.

(a) Schematic display of the targeting strategy to achieve homologous recombination of a bacterial artificial chromosome (BAC) to place in exon 2 of the Foxn1 locus a cDNA encoding a Foxn1 fused at its C-terminus to three Flag sequences, designated Foxn1-Flag. FRT: Flipase recombinase target. (b) Western blot (WB) analysis of total thymus tissue lysates and anti-Flag (a-Flag) immunoprecipitates of thymus tissue lysates of both wild type and Foxn1wt*/wt* mice developed using anti-Flag antibodies. (c) Macroscopic analysis of thymic lobes from 4-5 week old mice of the indicated genotype. (d) Hair growth was restored in Foxn1nu/nu mice either heterozygous (designated Foxn1wt*/-) or homozygous (Foxn1wt*/wt*) for the BAC transgene encoding the Foxn1-Flag fusion protein. (e) Gating strategy used in flow cytometric analysis of TEC subpopulations. (f) cTEC and mTEC cellularity in Foxn1+/+, Foxn1wt*/wt* and Foxn1wt*/- mice. The cellularity was calculated based on flow cytometric data presented in Fig. 1e. *p<0.05. (Student’s t-test). Data is representative of two (b-f) independent experiments (mean ±SD) with sample sizes of three (f). Contour plots (e) are representative and the numbers shown in individual gates represent relative frequencies observed in a representative experiment.

Supplementary Figure 2 Foxn1wt*/– mice demonstrate defects in T cell development and increased frequencies of thymic B cells.

Flow cytometric analysis of 5 week old mice with indicated genotype for (a) CD19 and IgM expression on CD4-CD8- thymocytes; (b) CD4 and CD8 expression on thymocytes at sequential developmental stages as defined by the cell surface expression of CD69 and TCR; (c) Foxp3 and CD25 expression CD4+CD8-TCR+CD5+ thymocytes. *p<0.05. (Student’s t-test (a,b)). Data are representative of two (a-c) independent experiments (mean ±SD) with sample sizes of four. Contour plots (a,b) are representative of data in bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a individual experiment.

Supplementary Figure 3 Foxn1 DNA-binding analysis by ChIP.

(a) ChIP of DNA from two mixed samples (thymoycte:TEC = 5:1, designated TEC +Thymocytes) or sorted thymocytes immunoprecipitated with anti-Flag antibodies (IP) and analyzed by qPCR for enrichment of promoter regions of the FoxN1 candidate genes Psmb11 and Dll4, and Foxp3, as control. (b) Genomic context of Foxn1 ChIP-seq peaks. Enrichment for mm10 RefSeq metagene features and mTEC H3K4me3 ChIP-seq peaks (IDR < 0.01). De novo motif analysis. (c) MEMEChIP-derived Foxn1 binding site motif for all peaks (IDR<0.05; E-value < 10-129). (d) Motif coverage relative to the summit of Foxn1 ChIP-seq peaks for all peaks.

Supplementary Figure 4 RNA-seq differential gene-expression analysis.

Volcano plots of (a) cTEC from Foxn1wt*/- vs. Foxn1wt*/wt* mice, (b) mTEC from Foxn1wt*/- vs. Foxn1wt*/wt* mice, (c) cTEC from iFoxn1Δ7,8 mice vs iFoxn1Δ7,8 mice that lack the Cre-recombinase. Positive fold changes indicate genes with increased expression in the presence of increased transcripts encoding functional Foxn1.

Supplementary Figure 5 Inducible deletion of Foxn1 in cTECs.

(a) Diagram of the targeting strategy to generate mice (designated iFoxn1Δ7,8) with a conditional Foxn1 allele that allows for the deletion of its exons 7 and 8. Germ-line transmitting knock-in mice were crossed to Flp recombinase transgenic mice to remove the PGK-neo cassette. (b) PCR-based analysis of genomic DNA from wild type mice (wt/wt) and mice with either one (wt/lox) or two (lox/lox) targeted alleles using the primers a and b depicted in the panel (a). (c) Strategy to achieve a cTEC-targeted, Dox-inducible deletion of exons 7 and 8 of Foxn1 in these triple-transgenic mice. rtTA is expressed under the transcriptional control of the Psmb11 locus, and TetO is expressed under a minimal CMV promoter.

Supplementary Figure 6 Foxn1 is indispensable for postnatal cTEC function.

(a) Immunofluorescence analysis of thymus tissue from 1 week old iFoxn1Δ7,8 mice injected i.p. with a single dose of Doxycycline (Dox+) or saline (Dox-) and analysed 3 days later. Tissue sections were stained for the expression of Foxn1 (red) and cytokeratin 8 (CK8, a cortical TEC marker; green). Scale bar 100µm. (b) Analysis of total thymic cellularity, and CD4 and CD8 expression on thymocytes of one week old iFoxn1Δ7,8 mice exposed to Doxycycline (Dox+) or saline (Dox-) 3 days earlier. (c) Analysis of total thymic cellularity, and CD4 and CD8 expression on all thymocytes as well as c-kit and CD25 expression on CD4-CD8-Lin- thymocytes isolated from one week old iFoxn1Δ7,8 mice exposed to Doxycycline (Dox+) or saline (Dox-) 4 days earlier. *p<0.0. (student’s t-test (a-c). Data are from two (a-c) independent experiments with sample sizes of three. Data (mean ±SD) is pooled for display in bar graphs (b, c). Contour plots (b,c) are representative of data in corresponding bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a representative experiment.

Supplementary Figure 7 Principal component analysis.

(a) Principal component analysis of RNA-seq datasets from cTEC and mTEC of Foxn1wt*/- and Foxn1wt*/wt* mice. (b) Principal component analysis of RNA-seq datasets from cTEC isolated from Dox-treated iFoxn1Δ7,8 mice and iFoxn1Δ7,8 mice that lack the Cre-recombinase.

Supplementary Figure 8 Integration of ChIP-seq and RNA-seq datasets.

BETA analysis of Foxn1 function by integration of ChIP-seq data sets and data sets from cTEC of Dox-treated iFoxn1Δ7,8 mice with the TetO-Cre transgene (designated here TetO+) vs. iFoxn1Δ7,8 mice that lack the TetO-Cre transgene (designated TetO-). Cumulative proportion of genes either upregulated (red), downregulated (blue) or unchanged (black) by increased levels of active Foxn1 against different regulatory score cut-offs.

Supplementary Figure 9 Linear relationship between relative expression of Foxn1-binding exons and Foxn1 target-gene expression.

(a) All high confidence Foxn1 gene targets. Overall mean gene expression is shown in dark red (r2=0.92, p<0.0001). (b) Ccl25 (r2=0.95, p<0.0001). (c) Dll4 (r2=0.90, p<0.0001). (d) Cd83 (r2=0.92, p<0.0001). Relative Foxn1 DNA binding exon expression was calculated as the mean number of reads aligned to the Foxn1 DNA binding exons 7 and 8 normalized by RNA-seq library size and overall Foxn1 expression.

Supplementary Figure 10 Comparative analysis of differential gene expression in cTECs.

Scatter plot of log2 fold change in gene expression in cTEC data sets from Foxn1wt*/- vs. Foxn1wt*/wt* mice and from Dox-treated iFoxn1Δ7,8 mice with TetO-Cre transgene (designated here TetO+) vs. iFoxn1Δ7,8 mice that lack the TetO-Cre transgene (designated TetO-). Genes significantly changed in both models at FDR < 0.05 are highlighted in red. Positive fold change indicates higher expression in Foxn1wt*/wt* mice than Foxn1wt*/- or TetO-iFoxn1Δ7,8 than TetO+ iFoxn1Δ7,8, respectively.

Supplementary Figure 11 Weighted correlation network analysis (WGCNA).

(a) Network permutation analysis showing median bidirectional weighted correlation coefficients for the high confidence Foxn1 gene targets and randomly selected genes matched by expression decile. This co-expression network includes only TEC data (cTEC, mTEClo and mTEChi). The vertical red line indicates real data. (b) Soft threshold r2 for different threshold powers. (c) Cluster dendrogram depicting WGCNA gene modules.

Supplementary Figure 12 Network analysis and assessment of putative binding partners.

(a) A direct seed co-expression network for cTEC generated from TEC-specific microarray data23. Interactions are shown for r2 > 0.7. The local network surrounding Foxn1 (yellow) is shown in the magnified graphic view. This network includes only cTEC data. (b) Likely binding partners of Foxn1 detected by PScan ChIP local motif enrichment. Specific motif IDs are CREB1_MA0018.1, TP63_MA0525.1, Mycn_MA0104.2, Bach1::Mafk_MA0591.1, NRF1_MA0506.1, HIF1A::ARNT_MA0259.1, REST_MA0138.2, E2F1_MA0024.2 and Mafb_MA0117.1. (c) Motif enrichment for binding motifs for several PScan predicted binding partners near Foxn1 ChIP-seq peaks. Enrichment is shown relative to the mean coverage near Foxn1 ChIP-seq peaks.

Supplementary Figure 13 Reduced Psmb11 expression in Foxn1wt*/– mice.

Immunofluorescence analysis for Psmb11(green) and CD4 (red) expression in thymus tissue sections of 1 week old mice with indicated genotype. Scale bar 50µm. The data is representative of two independent experiments analyzing 2 mice per group.

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Žuklys, S., Handel, A., Zhanybekova, S. et al. Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells. Nat Immunol 17, 1206–1215 (2016). https://doi.org/10.1038/ni.3537

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