Foxp3 occupancy and regulation of key target genes during T-cell stimulation

Abstract

Foxp3+CD4+CD25+ regulatory T (T(reg)) cells are essential for the prevention of autoimmunity. T(reg) cells have an attenuated cytokine response to T-cell receptor stimulation, and can suppress the proliferation and effector function of neighbouring T cells. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in T(reg) cells, is required for T(reg) development and function, and is sufficient to induce a T(reg) phenotype in conventional CD4+CD25- T cells. Mutations in Foxp3 cause severe, multi-organ autoimmunity in both human and mouse. FOXP3 can cooperate in a DNA-binding complex with NFAT (nuclear factor of activated T cells) to regulate the transcription of several known target genes. However, the global set of genes regulated directly by Foxp3 is not known and consequently, how this transcription factor controls the gene expression programme for T(reg) function is not understood. Here we identify Foxp3 target genes and report that many of these are key modulators of T-cell activation and function. Remarkably, the predominant, although not exclusive, effect of Foxp3 occupancy is to suppress the activation of target genes on T-cell stimulation. Foxp3 suppression of its targets appears to be crucial for the normal function of T(reg) cells, because overactive variants of some target genes are known to be associated with autoimmune disease.

Figure 1. Strategy to identify direct Foxp3 transcriptional effects

Genetically matched Foxp3⁺ and Foxp3⁻ cell populations were generated by transduction of FLAG-tagged Foxp3 into a Foxp3⁻ murine T-cell hybridoma. Foxp3 binding sites at promoters across the genome were identified by ChIP experiments with an anti-FLAG antibody. Foxp3 dependent transcriptional regulation was identified by gene expression profiling performed on each of these cell types.

Figure 2. Direct Foxp3 targets include key modulators of T-cell function

a, Foxp3 ChIP enrichment ratios (ChIP-enriched versus total genomic DNA) across indicated promoters are shown for stimulated (pink) and unstimulated (blue) cells. Exons (blocks) and introns (lines) of genes and the mir-155 precursor (grey) are drawn to scale below the plots, with direction of transcription noted by an arrow. b, Foxp3 bound genomic regions are enriched for the presence of a forkhead DNA motif, represented here in WebLogo (http://weblogo.berkeley.edu). c, The KEGG TCR signalling pathway, enriched (P = 1.4 × 10⁻⁵) for proteins encoded by direct targets of Foxp3 (blue outline), is displayed.

Figure 3. Foxp3 directly suppresses the activation of target genes

a, Replicate expression data for the 125 genes with Foxp3 dependent differential expression in stimulated hybridomas (false disovery rate, FDR < 0.05) were hierarchically clustered and displayed. The Z-score normalized induction (red) or repression (green) is shown for each gene. Direct targets of Foxp3 in stimulated hybridomas are indicated (dark blue for FDR < 0.05, light blue for FDR < 0.10). b, For repressed (green) and induced (red) Foxp3-bound targets in a, log₂(fold difference) in expression between stimulated ex vivo effector (T_eff) T cells and T_reg cells is displayed. Slc17a6 and Adam10 are not expressed in the ex vivo samples. c, Site-specific PCR on 10 ng of ChIP DNA confirmed selected targets. Immunoprecipitated (IP) DNA was compared to serial dilutions (90, 30 and 10 ng of DNA) of unenriched whole cell extract (WCE) DNA. Enrichment ratios, shown at top right of each sub-panel, are normalized relative to the unenriched β actin control. DNA fragment size (bp) is indicated on the left of each row. d, The transcript levels of the panel of Foxp3 targets presented in c and of Foxp3 were analysed by real-time RT–PCR in stimulated and unstimulated cells, with (grey) and without (black) cyclosporin A. Mean values ± s.d. of relative expression, determined in triplicate, are shown for indicated genes.

Figure 4. Core direct regulatory effects of Foxp3

a, Shown here are a subset of direct Foxp3 targets that exhibit consistent transcriptional behaviour in hybridomas and in ex vivo T cells (Supplementary Fig. S5). b, Foxp3 binds to a large set of promoters both in unstimulated and stimulated T cells, but Foxp3 transcriptional regulation is more extensive in stimulated T cells. The genomic regions where Foxp3 binds are enriched for an Nfat binding site DNA motif. In the hybridomas, Foxp3 predominantly acts to directly suppress the activation of its target genes.