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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Anderson, G. & Takahama, Y. Thymic epithelial cells: working class heroes for T cell development and repertoire selection. Trends Immunol. 33, 256–263 (2012).
Holländer, G. et al. Cellular and molecular events during early thymus development. Immunol. Rev. 209, 28–46 (2006).
Klein, L., Kyewski, B., Allen, P.M. & Hogquist, K.A. Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see). Nat. Rev. Immunol. 14, 377–391 (2014).
Stritesky, G.L. et al. Murine thymic selection quantified using a unique method to capture deleted T cells. Proc. Natl. Acad. Sci. USA 110, 4679–4684 (2013).
Nehls, M., Pfeifer, D., Schorpp, M., Hedrich, H. & Boehm, T. New member of the winged-helix protein family disrupted in mouse and rat nude mutations. Nature 372, 103–107 (1994).
Nehls, M. et al. Two genetically separable steps in the differentiation of thymic epithelium. Science 272, 886–889 (1996).
Bleul, C.C. et al. Formation of a functional thymus initiated by a postnatal epithelial progenitor cell. Nature 441, 992–996 (2006).
Cheng, L. et al. Postnatal tissue-specific disruption of transcription factor FoxN1 triggers acute thymic atrophy. J. Biol. Chem. 285, 5836–5847 (2010).
Nowell, C.S. et al. Foxn1 regulates lineage progression in cortical and medullary thymic epithelial cells but is dispensable for medullary sublineage divergence. PLoS Genet. 7, e1002348 (2011).
Chen, L., Xiao, S. & Manley, N.R. Foxn1 is required to maintain the postnatal thymic microenvironment in a dosage-sensitive manner. Blood 113, 567–574 (2009).
Zuklys, S. et al. Stabilized β-catenin in thymic epithelial cells blocks thymus development and function. J. Immunol. 182, 2997–3007 (2009).
Zlotoff, D.A. et al. CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus. Blood 115, 1897–1905 (2010).
Ueno, T. et al. CCR7 signals are essential for cortex-medulla migration of developing thymocytes. J. Exp. Med. 200, 493–505 (2004).
Daley, S.R., Hu, D.Y. & Goodnow, C.C. Helios marks strongly autoreactive CD4+ T cells in two major waves of thymic deletion distinguished by induction of PD-1 or NF-κB. J. Exp. Med. 210, 269–285 (2013).
Sansom, S.N. et al. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia. Genome Res. 24, 1918–1931 (2014).
Schlake, T., Schorpp, M., Nehls, M. & Boehm, T. The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system. Proc. Natl. Acad. Sci. USA 94, 3842–3847 (1997).
Ara, T. et al. A role of CXC chemokine ligand 12/stromal cell-derived factor-1/pre-B cell growth stimulating factor and its receptor CXCR4 in fetal and adult T cell development in vivo. J. Immunol. 170, 4649–4655 (2003).
Hozumi, K. et al. Delta-like 4 is indispensable in thymic environment specific for T cell development. J. Exp. Med. 205, 2507–2513 (2008).
Wang, S. et al. Target analysis by integration of transcriptome and ChIP-seq data with BETA. Nat. Protoc. 8, 2502–2515 (2013).
Langfelder, P. & Horvath, S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9, 559 (2008).
Ki, S. et al. Global transcriptional profiling reveals distinct functions of thymic stromal subsets and age-related changes during thymic involution. Cell Rep. 9, 402–415 (2014).
Dennis, G. Jr. et al. DAVID: Database for annotation, visualization, and integrated discovery. Genome Biol. 4, 3 (2003).
Gommeaux, J. et al. Thymus-specific serine protease regulates positive selection of a subset of CD4+ thymocytes. Eur. J. Immunol. 39, 956–964 (2009).
Zambelli, F., Pesole, G. & Pavesi, G. PscanChIP: finding over-represented transcription factor-binding site motifs and their correlations in sequences from ChIP-Seq experiments. Nucleic Acids Res. 41, W535–43 (2013).
Impey, S. et al. Defining the CREB regulon: a genome-wide analysis of transcription factor regulatory regions. Cell 119, 1041–1054 (2004).
Burnley, P. et al. Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging. Cell Death Dis. 4, e932 (2013).
Della Gatta, G. et al. Direct targets of the TRP63 transcription factor revealed by a combination of gene expression profiling and reverse engineering. Genome Res. 18, 939–948 (2008).
Ripen, A.M., Nitta, T., Murata, S., Tanaka, K. & Takahama, Y. Ontogeny of thymic cortical epithelial cells expressing the thymoproteasome subunit β5t. Eur. J. Immunol. 41, 1278–1287 (2011).
Murata, S. et al. Regulation of CD8+ T cell development by thymus-specific proteasomes. Science 316, 1349–1353 (2007).
Fujimoto, Y. et al. CD83 expression influences CD4+ T cell development in the thymus. Cell 108, 755–767 (2002).
Saini, M. et al. Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds. Sci. Signal. 3, ra23 (2010).
Nakagawa, S., Gisselbrecht, S.S., Rogers, J.M., Hartl, D.L. & Bulyk, M.L. DNA-binding specificity changes in the evolution of forkhead transcription factors. Proc. Natl. Acad. Sci. USA 110, 12349–12354 (2013).
Carroll, J.S. et al. Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 122, 33–43 (2005).
Senoo, M., Pinto, F., Crum, C.P. & McKeon, F. p63 Is essential for the proliferative potential of stem cells in stratified epithelia. Cell 129, 523–536 (2007).
Liu, B. et al. Cbx4 regulates the proliferation of thymic epithelial cells and thymus function. Development 140, 780–788 (2013).
Flomerfelt, F.A. et al. Tbata modulates thymic stromal cell proliferation and thymus function. J. Exp. Med. 207, 2521–2532 (2010).
Bredenkamp, N., Nowell, C.S. & Blackburn, C.C. Regeneration of the aged thymus by a single transcription factor. Development 141, 1627–1637 (2014).
Calderón, L. & Boehm, T. Synergistic, context-dependent and hierarchical functions of epithelial components in thymic microenvironments. Cell 149, 159–172 (2012).
Rode, I. et al. Foxn1 protein expression in the developing, aging, and regenerating thymus. J. Immunol. 195, 5678–5687 (2015).
Mayer, C.E. et al. Dynamic spatio-temporal contribution of single β5t+ cortical epithelial precursors to the thymus medulla. Eur. J. Immunol. 46, 846–856 (2016).
Vokes, S.A. et al. Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Development 134, 1977–1989 (2007).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Lunter, G. & Goodson, M. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 21, 936–939 (2011).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).
Buenrostro, J.D., Wu, B., Chang, H.Y. & Greenleaf, W.J. ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 2015, 21.29.1–21.29.9 (2015).
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.).
Author information
Authors and Affiliations
Contributions
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
Ethics declarations
Competing interests
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.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–13 and Supplementary Tables 1–4 (PDF 7935 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.3537
This article is cited by
-
Genetically modified mice as a tool for the study of human diseases
Molecular Biology Reports (2024)
-
Clinical Practice Guidelines for the Immunological Management of Chromosome 22q11.2 Deletion Syndrome and Other Defects in Thymic Development
Journal of Clinical Immunology (2023)
-
Limits to in vivo fate changes of epithelia in thymus and parathyroid by ectopic expression of transcription factors Gcm2 and Foxn1
Scientific Reports (2022)
-
The impact of the gut microbiota on T cell ontogeny in the thymus
Cellular and Molecular Life Sciences (2022)
-
Indispensable epigenetic control of thymic epithelial cell development and function by polycomb repressive complex 2
Nature Communications (2021)