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. 2012 Sep 28;151(1):153-66.
doi: 10.1016/j.cell.2012.06.053.

Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification

Robert M Samstein  1 , Aaron Arvey, Steven Z Josefowicz, Xiao Peng, Alex Reynolds, Richard Sandstrom, Shane Neph, Peter Sabo, Jeong M Kim, Will Liao, Ming O Li, Christina Leslie, John A Stamatoyannopoulos, Alexander Y Rudensky
Affiliations

Foxp3 exploits a pre-existent enhancer landscape for regulatory T cell lineage specification

Robert M Samstein et al. Cell. .

Abstract

Regulatory T (Treg) cells, whose identity and function are defined by the transcription factor Foxp3, are indispensable for immune homeostasis. It is unclear whether Foxp3 exerts its Treg lineage specification function through active modification of the chromatin landscape and establishment of new enhancers or by exploiting a pre-existing enhancer landscape. Analysis of the chromatin accessibility of Foxp3-bound enhancers in Treg and Foxp3-negative T cells showed that Foxp3 was bound overwhelmingly to preaccessible enhancers occupied by its cofactors in precursor cells or a structurally related predecessor. Furthermore, the bulk of Foxp3-bound Treg cell enhancers lacking in Foxp3(-) CD4(+) cells became accessible upon T cell receptor activation prior to Foxp3 expression, and only a small subset associated with several functionally important genes were exclusively Treg cell specific. Thus, in a late cellular differentiation process, Foxp3 defines Treg cell functionality in an "opportunistic" manner by largely exploiting the preformed enhancer network instead of establishing a new enhancer landscape.

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Figures

Figure 1
Figure 1
The enhancer repertoire is predominantly identical in Treg and Foxp3− CD4+ T cells (A) Representative plots of DNase-seq reads aligned to the Ctla4 and Irf4 gene loci demonstrating DHS peaks that are shared by, or unique to, the indicated cell types. (B) Scatter plot of DHS peak reads per million (RPM) found in either Foxp3− or Foxp3+ T cells demonstrating new Treg-specific sites (red) and sites lost in Treg cells (blue). (C) Scatter plot of DHS peak RPM identified upon analysis of Treg and B cells demonstrating T-cell-specific sites (red) and B-cell-specific sites (blue). (D) Expression differences for genes with proximal DHSs with increased or decreased DNase accessibility (p-values are from one-sided KS-test).
Figure 2
Figure 2
Foxp3 predominantly binds pre-accessible sites. (A) Representative Foxp3 ChIP-seq reads aligned to the Ikzf2 gene locus. (B) Localization of Foxp3 and DNase accessible sites demonstrating enrichment at promoters and within the first intron. Downstream distal sites are enriched comparably to upstream distal sites. (C) Genes bound by Foxp3 in Treg cells are enriched for differential expression compared to Foxp3-negative CD4+ T cells. Venn diagrams show significant overlap between up and down regulated genes and Foxp3 bound genes (hypergeometric tests). Cumulative distributions show quantitative shifts in expression of genes bound by Foxp3 relative to all expressed genes (one-tailed KS-tests). (D) Representative plot of DNase-seq and ChIP-seq reads aligned to the Foxp3 locus illustrating Treg-specific DHS corresponding to a Foxp3 binding site. (E, F) Foxp3 binding site accessibility is largely similar in Foxp3+ Treg and Foxp3− CD4+ cells. Scatter plot of all DHS peaks (black) and those containing Foxp3 bound sites (pink) in the indicated cell populations.
Figure 3
Figure 3
Foxp3 cofactors are bound at pre-accessible enhancers prior to Foxp3 expression. (A) Motifs within Foxp3 binding sites identified by sequence analysis of ChIP-seq peaks and corresponding motif enrichment plots. Flanking regions were sampled 200bp away from centers of peaks. (B) Proportion of Foxp3 binding sites that contain the indicated motifs or their combinations. (C) Motif occurrence is enriched in Foxp3 peaks with more ChIP-seq reads. (D) Representative plots of Ets-1 and Elf-1 ChIP-seq reads aligned to the Foxp3 binding sites at the Junb promoter. (E) ChIP-seq analysis of Foxp3 cofactor occupancy at Foxp3 bound sites in Treg cells containing the corresponding motif demonstrates significant overlap between cofactor and Foxp3 binding. ChIP signal is shown in units of RPM within a 200bp window around Foxp3 peaks. Elf1 and Ets1 signal is transformed by Log2(x+0.5) and Cbfβ signal was transformed by Log2(x+1).
Figure 4
Figure 4
Foxp3 cofactors are present at sites of Foxp3 binding prior to Foxp3 expression. (A) Heatmaps showing DNase-seq read counts mapped to genome-wide DHSs containing FKHD and ETS motifs demonstrate increased DNase hypersensitivity at Foxp3 binding sites identified by ChIP-seq (grey tick marks). Conservation (phyloP) is increased at these sites and is shown in red. Heatmaps are shown with black indicating no signal and white/red as high signal. (B) Foxp3 binding sites containing indicated motifs are similarly protected in Foxp3−CD4+ and Foxp3+ Treg cells. Heatmaps show DNase-seq read counts mapped to individual Foxp3 binding sites in Treg cells and Foxp3-negative CD4+ T cells aligned to the corresponding motif. (C) ChIP-seq analysis of Ets1, Elf1, and Runx/Cbfb occupancy of Foxp3-bound sites containing ETS and RUNX motifs, respectively, in Foxp3-negative CD4+ T cells. Units and transforms are same as described in Figure 3 legend. (D) Elf1 binding at Foxp3-bound sites in both Foxp3-negative CD4+ T cells and Treg cells revealed by ChIP-seq analysis. Spearman’s rank correlation is greater for Foxp3 binding sites with ETS motif than without. Data is incremented by 0.5RPM to increase dynamic range.
Figure 5
Figure 5
The forkhead transcription factor family member Foxo1 serves as a “predecessor” of Foxp3 in precursor cells. (A) Representative plots of Foxp3 and Foxo1 ChIP-seq reads from Treg and Foxp3-negative CD4+ T cells aligned to the Lef1 and Jak1 gene loci. Foxo1 ChIP was normalized by RPM and percentage of tags in peaks. (B) Differential Foxo1 binding at Foxp3 binding sites in Foxp3+ Treg vs. Foxp3−CD4+ T cells (quantile normalized). (C) Foxp3-bound genes that show a decrease in Foxo1 binding in Treg cells vs. Foxp3-negative CD4+ T cells exhibit decreased gene expression in Treg cells compared to precursor cells.
Figure 6
Figure 6
The majority of “Treg-specific” DHS sites are established in a Foxp3- independent manner upon TCR dependent activation of Foxp3− CD4+ T cells. (A) Scatter plot of DHSs in Treg vs. Foxp3-negative CD4+ T cells. AP-1 motif was enriched at sites with an increased DNase sensitivity in Treg cells, while RUNX and HMG motifs were enriched at sites with diminished DNase sensitivity. (B, C) Scatter and CDF plots of DHS peaks found in naive and activated Foxp3− CD4+ T cells demonstrate that the majority of Treg-specific DNase sites are present in activated Foxp3− CD4+ T cells. (D) CDF plots of expression changes of genes containing “Treg-specific” DHS sites in GFP+Foxp3− T cells expressing Foxp3GFPKO reporter null allele vs. naïve Foxp3− CD4+ or Foxp3+ Treg cells. (E) CDF plots of expression changes of genes containing “Treg-specific” DHS sites in calcineurin B1-deficient and -sufficient Treg cells with impaired vs. unperturbed NFAT activation, respectively.
Figure 7
Figure 7
Potential modes of utilization of preformed enhancer network by Foxp3 during Treg cell differentiation. (A) The majority of Foxp3 binding sites are found within enhancers that are occupied by Foxp3 cofactors before Foxp3 is expressed and are accessible in precursor cells. (B) Foxp3 binds to sites occupied by a related forkhead transcription family member Foxo1, which serves as a “placeholder” and can be displaced by Foxp3. (C) Foxp3 is recruited to enhancers primed in a TCR signaling dependent manner during Treg cell differentiation likely via NFAT-AP1 assisted chromatin remodeling.
See this image and copyright information in PMC

Comment in

  • Designing an enhancer landscape.
    Xu J, Smale ST. Xu J, et al. Cell. 2012 Nov 21;151(5):929-31. doi: 10.1016/j.cell.2012.11.007. Cell. 2012. PMID: 23178114 Free PMC article.

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