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.

Figure 1

Transgenic rescue of nude phenotype in Foxn1^wt*/wt* mice expressing a chimeric Foxn1-Flag protein. (a) Absolute thymus cellularity of mice with indicated genotype and age. (b, c) Immunofluorescence analysis of thymus tissue from 4 week old mice with indicated genotype (b) for the expression of cytokeratin 8 (CK8; a cortical TEC marker, blue), CK5 (a medullary TEC marker, green), and ERTR7 (endothelial cells and fibroblasts; red), and (c) for the expression of Foxn1 (red) and the Autoimmune Regulator (Aire, identifying a subpopulation of mature mTEC; blue). Scale bar 50µm (c) and 100µm (b). (d) RT-qPCR analysis of functional Foxn1 transcripts in cTEC from Foxn1^wt*/wt* and Foxn1^wt*/- mice using oligonucleotides specific for transgene specific FLAG tag sequence. Values are normalized to EpCAM expression. (e) Flow cytometric analysis of TEC subpopulations isolated from 4 week old mice with the indicated genotype (left panels). The gating strategy is detailed in Supplementary Fig. 1e. Frequencies of cTEC and mTEC in middle and right panels, respectively. (f,g) Flow cytometric analysis of mTEC isolated from 4 week old mice mice with the indicated genotype for the expression of MHCII and (f) CD80 or (g) CD86. Frequencies of double positive cells displayed on the right. (h) Flow cytometric analysis of MHCII expression on cTEC and mTEC isolated from 4 week old mice with the indicated genotypes. Bar graph displays frequencies of MHCII^hi cells as defined by the gate in the histograms. *p<0.05. (Student’s t-test (a,d-h)). Data in bar graphs are pooled from two (a,e,h) independent experiments (n=7, mean ±SD ) or display one experiment (b,c,f,g) representative of two independent experiments with four replicates each. Contour plots (e-g) and histograms (h) are representative of data in bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a representative experiment. ND: not detected

Figure 2

Foxn1 availability in TEC determines T cell developmental defects. Flow cytometric analysis of 4 week old mice with indicated genotype for (a) CD44⁺c-kit⁺ thymocytes. Bar graph display frequencies of CD44⁺c-kit⁺CD25^- thymocytes. (b) CD44 and CD25 expression on CD4^-CD8^-Lin^- thymocytes, (c) CD4 and CD8 expression on total thymocytes; (d) CD69 and TCR β-chain expression on total thymocytes; (e) Helios and PD1 expression on Foxp3^-CCR7^- thymocytes; (f) CD24 and CCR7 expression on CD4⁺CD8^-TCR⁺CD5⁺Foxp3^- thymocytes; (g) CD24 and CCR7 expression on CD4^-CD8⁺TCR⁺CD5⁺ thymocytes; and (h) Foxp3 and CD25 expression by CD4⁺CD8^-TCR⁺CD5⁺ thymocytes. Representative contour plots for (h) are displayed in Supplementary Fig. 2c. *p<0.05. (Student’s t-test (a-h). Data in bar graphs are from one experiment representative of two independent experiments with four replicates each. Contour plots (a-g) are representative of data in bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a representative experiment.

Figure 3

Foxn1 ChIP-seq analysis. (a) Proportion of Foxn1 ChIP-seq peaks falling within different RefSeq meta-gene regions. (b) Left: Enrichment of Foxn1 ChIP-seq signal across a meta-gene profile comprising all RefSeq mouse genes relative to control. TSS = transcriptional start site; TES = transcriptional end site. Right: Relative coverage of cTEC ATAC-seq transposon insertions relative to the summit of Foxn1 ChIP-seq peaks. (c) MEMEChIP-derived Foxn1 binding site motif for TSS-associated peaks (-5kb before and 100bp after TSS, E-value < 10^-80). (d) Motif coverage relative to the summit of Foxn1 ChIP-seq peaks for TSS-associated peaks.

Figure 4

Intersection of Foxn1 ChIP-seq and RNA-seq analyses. (a) Thymus cellularity and expression of c-kit and CD25 on CD4^-CD8^-Lin^- thymocytes in one week old iFoxn1^Δ7,8 mice 72hrs after i.p. exposure to Doxycycline (Dox+) or saline (Dox-). (b) Left: Foxn1 ChIP-seq binding profiles for Ccl25, Cxcl12 and Dll4. Scale bars represent 5kb (top) and 2kb (middle and bottom). Right: RT-qPCR analysis for Cxcl12, Dll4, Ccl25 and Foxn1 transcripts in cTECs isolated at the indicated times after treatment of one week old iFoxn1^∆7,8 mice with a single dose of Dox relative to untreated iFoxn1^∆7,8 mice. (c) Number of with decreased Foxn1 activity downregulated genes filtered from all protein coding Ensembl genes by sequential use of indicated data sets. ChIP-seq targets were defined as genes with a Foxn1 peak (IDR < 0.05) 5kb upstream or 100 bases downstream of the TSS. Genes with FDR < 0.05 were called as differentially expressed. (d) Distribution of Foxn1 ChIP-seq peaks around the transcriptional start site (TSS) of genes categorized by the direction of change in TetO^-iFoxn1^∆7,8 vs TetO⁺iFoxn1^∆7,8 mice following Dox treatment. (e) ATAC-seq signal in cTEC near the TSS of high confidence Foxn1 target genes (red) and all other genes (gray; 1.54-median fold change). (f) Scatter plot analysis of expression levels of high confidence Foxn1 targets (red: >2-fold; blue: <2-fold differential expression). (g) Gene ontology analysis of high confidence Foxn1 gene targets. The number of high confidence targets in each term (> 2 fold enrichment) are: threonine peptidase 8 (representing 38.1% of genes in that gene ontology category); proteasome 9 (16.7%); transit peptides 23 (5.0%); protein trafficking 23 (5.0%) and Golgi apparatus 26 (4.7%). *p<0.05. (Student’s t-test (a,b); Benjamini-Hochberg (g)).

Figure 5

Psmb11 and Cd83 are direct targets of Foxn1. (a) Left: Foxn1 ChIP-seq binding profiles for Psmb11 and Cd83. Scale bars 2kb (top) and 5kb (bottom). Right: RT-qPCR analysis of gene expression in cTEC isolated from one week old iFoxn1^∆7,8 mice at the indicated times after treatment with a single dose of Dox relative to untreated iFoxn1^∆7,8 mice. Measurements of transcripts of the rtTA transgene replacing the endogenous Psmb11 locus in homozygous iFoxn1^∆7,8 mice. (b,c) Luciferase assays for the Foxn1-dependent activation of wild type (wt) Psmb11 and Cd83 minimal promoters and of Psmb11 and Cd83 promoter mutants (mut1-4) containing alterations in the GACGC (black bars) and GAAGC motifs (black ovals). (d-h) Comparative analysis of the impact of a loss of Psmb11 expression and expression of a hypomorphic Foxn1 allele, respectively, on intrathymic T cell development of 5 week old mice with indicated genotype for (d) CD4 and CD8 expression on thymocytes, (e) CD69 and TCR β-chain expression on thymocytes, (f) Helios and PD-1 on Foxp3^-CCR7^- thymocytes, (g) CD5 and TCR β-chain expression CD4⁺CD8⁺ thymocytes, and (h) CD24 and CCR7 expression on CD8SP thymocytes. *p<0.05. (Student’s t-test). Data is representative of two independent experiments (mean ±SD) with sample sizes of four (d-h) and three measurements each (a-c). Contour plots (d-h) are representative of data in bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a representative experiment.

Figure 6

Comparative analysis of the impact of a loss of Cd83 expression and expression of a hypomorphic Foxn1 allele, respectively, on intrathymic T cell development. Analysis of 5 (Foxn1^wt*/-) and 7 (Cd83^+/+, Cd83^-/-) week old mice with indicated genotype for (a) EpCAM and CD83 expression on cTEC (CD45^-EpCAM⁺MHCII⁺UEA1^-Ly51⁺), (b) CD4 and CD8 expression on total thymocytes, (c) CD69 and TCR β-chain expression on total thymocytes, (d) CD24 and CCR7 expression on CD4SP thymocytes. *p<0.05. (Student’s t-test (a-d). Data in bar graphs is representative of two independent experiments (mean ±SD ) with sample sizes of three. Contour plots are representative of data in bar graphs. Numbers shown in individual gates and quadrants of flow cytometry plots represent the frequencies observed in a representative experiment.