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. 2014 Jul;15(7):646-656.
doi: 10.1038/ni.2897. Epub 2014 May 18.

TCF-1 and LEF-1 Act Upstream of Th-POK to Promote the CD4(+) T Cell Fate and Interact With Runx3 to Silence Cd4 in CD8(+) T Cells

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Free PMC article

TCF-1 and LEF-1 Act Upstream of Th-POK to Promote the CD4(+) T Cell Fate and Interact With Runx3 to Silence Cd4 in CD8(+) T Cells

Farrah C Steinke et al. Nat Immunol. .
Free PMC article

Abstract

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

Figures

Figure 1
Figure 1. CD4-Cre-mediated deletion of TCF-1 or both TCF-1 and LEF-1 impairs CD4+ SP thymocyte development
(a) Complete ablation of TCF-1 and LEF-1 proteins in post-select DP thymocytes. Thymocytes were surface-stained, FACS-sorted for TCRβlo-medCD69CD4+CD8+ as pre-select DP (PreDP), and TCRβhiCD24+CD69+CD4+CD8+ as post-select DP (PostDP) thymocytes. Note that both TCF-1 and LEF-1 are expressed in multiple isoforms in thymocytes due to differential promoter usage and alternative splicing. Data are representative from 2 experiments. (b) Total thymic cellularity. Data are means ± s.d. from ≥ 5 independent experiments. *, p < 0.05; **, p < 0.01. (c) and (d) Analysis of post-select thymocytes. The TCRβhi thymocytes (c) are further fractionated into immature (CD24+CD69+) and mature (CD24 CD69) subsets (d). The frequency of each subset is shown. (e) Loss of TCF-1 or both TCF-1 and LEF-1 diminishes the frequency of CD4+ SP thymocytes. The immature CD69+CD24+TCRβhi thymocytes were fractionated into DP, CD4+CD8lo, CD4+ SP and CD8+ SP subsets with their frequencies shown. (f) Loss of TCF-1 or both TCF-1 and LEF-1 causes derepression of the CD4 coreceptor in CD8+ lineage thymocytes. The mature CD69CD24TCRβhi thymocytes were separated into CD4+ and CD8+ subsets. The values are frequencies of CD4+ and CD8+ cells, with the latter including both CD8+CD4 and CD8+CD4+ cells that appear in Tcf7−/− and Tcf7−/−Lef1−/− mice. Data in c-f are representative of ≥ 5 independent experiments.
Figure 2
Figure 2. The CD4+CD8+ (CD8*4) mature thymocytes in Tcf7−/−Lef1−/− mice belong to the CD8+ lineage
(a) The Tcf7−/−Lef1−/− CD8*4 mature thymocytes express CD8β. Mature TCRβhi thymocytes were analyzed for CD4, CD8α, and CD8β expression. (b) The Tcf7−/−Lef1−/− CD8*4 mature thymocytes express CD8+-characteristic genes. Shown is relative expression of each gene after normalization to Hprt1. Mature CD4+, CD8+, and CD8*4 thymocytes were sorted from Tcf7−/−Lef1−/− mice and littermate controls and analyzed for gene expression (n ≥ 3). (c) The Tcf7−/−Lef1−/− CD8*4 subset persists in the absence of MHC-II I-A and I-E molecules. Tcf7−/−Lef1−/− and control mice were crossed to an H2-Ab1−/− background, and the mature TCRβhi thymocytes were analyzed for CD4+ and CD8+ lineage distribution. Shown are representative data from 3 experiments.
Figure 3
Figure 3. Deficiency in TCF-1 or both TCF-1 and LEF-1 redirects CD4+ T cells to the CD8+ lineage
(a) Loss of TCF-1 or both TCF-1 and LEF-1 greatly diminishes CD4+ T cell output. Numbers of mature CD4+ and CD8+ SP thymocytes are shown as means ± s.d. (n ≥ 6). (b) Loss of TCF-1 or both factors reverses the CD4/CD8 ratio. Ratio of mature CD4+ to CD8+ cells was calculated from a. The horizontal line denotes mean value. *, p<0.05; **, p<0.01; ***, p<0.001. (c) MHC-II-selected thymocytes are redirected to CD8+ lineage in the absence of TCF-1 or both factors. BM cells from Tcf7−/−, Tcf7−/−Lef1−/−, or littermate controls were transplanted into lethally irradiated CD45.1+ congenic β2m−/− mice. Six weeks later, donor-derived (CD45.2+) mature CD69CD24 TCRβhi thymocytes were analyzed for CD4+ and CD8+ lineage distribution. Representative contour plots (c) are from 4 independent experiments. Numbers of mature CD4+ and CD8+ thymocytes in the BM chimeras are shown in (d) as means ± s.d. (n ≥ 14). ***, p<0.001. (e) and (f) OT-II TCR transgenic T cells adopt a CD8+ fate in the absence of TCF-1 or both factors. The OT-II TG was crossed onto Tcf7−/−, Tcf7−/−Lef1−/−, or littermate controls. After gating on the Vα2+TCRβhi subset, mature CD24 thymocytes were analyzed for CD4+ and CD8+ lineage distribution. Representative contour plots (e) are shown (n ≥ 5 from 5 experiments). Frequencies of mature CD4+ and CD8+ OT-II thymocytes are summarized in (f). ***, p<0.001.
Figure 4
Figure 4. TCF-1 deficiency decreases Thpok but increases Runx3d expression in the bipotent precursors
Thymocytes from Tcf7−/− and littermate controls were sorted for 3 subsets, pre-select DP (PreDP), post-select DP (PostDP), and CD4+8lo intermediate (IM). The expression of indicated genes was measured by quantitative RT-PCR. To demonstrate kinetic changes of each gene during these developmental stages, the expression of each gene in control post-select DP (after normalization to Hprt1) was arbitrarily set to 1, and the relative expression of this gene in all other control or Tcf7−/− subsets was then normalized accordingly and presented as fold changes. Data are means ± s.d. from 4 independent experiments (n ≥ 6). ND, not reliably detected. *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 5
Figure 5. Ectopic expression of Th-POK rectifies defects in CD4+ T cell differentiation caused by loss of TCF-1
(a) Th-POK TG represses CD8+ differentiation in the absence of TCF-1 or both TCF-1 and LEF-1. Th-POK TG was crossed onto Tcf7−/− or Tcf7−/−Lef1−/− mice. The immature and mature TCRβhi thymocytes were analyzed for CD4+ and CD8+ lineage distribution. Frequency of each subset is shown in the representative data from ≥ 3 experiments. (b) Cumulative data on the impact of Th-POK TG on the numbers of total, mature CD4+ and CD8+ SP thymocytes. Data are means ± s.d. from 5 experiments (n ≥ 6). *, p<0.05; **, p<0.01; ***, p<0.001; NS, not statistically significant. (c) Th-POK TG prevents CD4+ to CD8+ lineage redirection caused by deficiency in TCF-1 or both TCF-1 and LEF-1. BM cells from Th-POK TG, Th-POK TG Tcf7−/−, or Th-POK TG Tcf7−/−Lef1−/− were transplanted into irradiated CD45.1+ β2m−/− recipients. Six weeks later, CD45.2+ mature TCRβhi thymocytes were analyzed for CD4+ and CD8+ distribution. Representative data from ≥ 3 experiments are shown. (d) Ectopic expression of Th-POK represses Runx3d in the presence or absence of TCF-1. Post-select DP (PostDP) and CD4+8lo intermediate (IM) thymocytes were sorted and analyzed for gene expression. Shown is relative expression of each gene after normalization to Hprt1. Data are representative from 3 independent experiments. ***, p<0.001.
Figure 6
Figure 6. TCF-1 acts through the GTE in the Thpok gene locus
(a) Identification of conserved TCF-LEF motifs in the Thpok gene locus, as marked by arrows (A through G). Partial structure of the Thpok gene is shown, with filled bars denoting exons. Open bars denote the following cis-elements, DRE (distal regulatory element, also known as Thpok silencer), GTE, and PRE (proximal regulatory element). The sizes of exons and regulatory elements are not drawn to scale. (b) TCF-1 binds to the GTE in the Thpok locus. Post-select DP and CD4+8lo thymocytes were sorted together from WT or Tcf7−/− mice and used in ChIP with anti-TCF-1 or control IgG followed by quantitative PCR. Enrichment by TCF-1 antibody at each motif or locus was first normalized to IgG, and then normalized to that at the Gapdh locus. Two known TCF-1 target genes, Lef1 and Axin2, were detected as positive controls. Data are pooled results from ≥3 experiments. **, p<0.01. (c) Mutation of TCF-1 sites in the GTE abrogates its enhancer activity. The luciferase reporter constructs (shown on the left) were transfected into the EL-4 cells by electroporation, and 48 hrs later the luciferase activity was measured. Luciferase activity driven by the SV40 alone (pGL3 promoter) is arbitrarily set to 1, and that containing WT or mutant (Mut) GTE was normalized accordingly. Data are means ± s.d. from 2 experiments (n = 3). *, p<0.05; **, p<0.01.
Figure 7
Figure 7. Deletion of Runx3 does not rescue CD4+ differentiation defects caused by loss of TCF-1 and LEF-1
(a) CD4-Cre-Runx3fl/fl (Runx3−/−) mice were crossed with Tcf7−/− and Tcf7−/−Lef1−/− strains to acquire Tcf7−/−Runx3−/− and Tcf7−/−Lef1−/−Runx3−/− animals. The immature and mature TCRβhi thymocytes were analyzed for CD4+ and CD8+ lineage distribution. Frequency of each subset is shown in the representative data from 4 experiments. (b) Cumulative data on the impact of Runx3 deletion on numbers of mature CD4+ and CD8+ SP thymocytes (n ≥ 5 from 4 experiments). (c) Deletion of Runx3 does not reverse the decrease in Thpok expression caused by TCF-1 deficiency. Post-select DP (PostDP) and CD4+8lo intermediate (IM) thymocytes were sorted and analyzed for Thpok gene expression. Shown is relative Thpok expression after normalization to Hprt1. *, p<0.05; **, p<0.01; ***, p<0.001. NS, not statistically significant. (d) Deletion of Runx3 does not prevent CD4+ to CD8+ lineage redirection caused by deficiency in TCF-1 or both TCF-1 and LEF-1. BM cells from Runx3−/−, Tcf7−/−Runx3−/− and Tcf7−/−Lef1−/−Runx3−/− mice were transplanted into irradiated CD45.1+ β2m−/− recipients. Six weeks later, CD45.2+ mature TCRβhi thymocytes were analyzed for CD4+ and CD8+ distribution. Data are representative from 3 experiments (n ≥ 6).
Figure 8
Figure 8. TCF-LEF and Runx factors cooperate in Cd4 silencing in CD8+ lineage T cells
(a) Genome-wide mapping of TCF-1 occupancy reveals its direct association with the Cd4 silencer. Splenic CD8+ T cells were used in ChIP followed by high throughput sequencing, and ChIP-Seq track wiggle files were uploaded to the UCSC genome browser for visualization of enriched TCF-1 binding peaks. Top two tracks are sequencing reads from ChIP with anti-TCF-1 and IgG, respectively. The bottom track shows fold enrichment of TCF-1 binding peaks. All tracks are shown for the Axin2 and Cd4 gene loci, with their transcription orientations marked with arrows. (b) Binding of TCF-1 to the Cd4 silencer is specific to CD8+ T cells. CD8+ or CD4+ SP thymocytes were sorted from WT or Tcf7−/− mice and used in ChIP with anti-TCF-1 or control IgG. Enrichment at the Gapdh and Lef1 loci was measured as negative and positive controls, respectively. Data are pooled results from at least 3 independent experiments. *, p<0.05; **, p<0.01. (c) TCF-LEF motif and (d) Runx motif enriched in the TCF-1 ChIP-seq peaks. (e) Distribution of TCF-LEF and Runx motifs in the TCF-1 binding peaks is summarized in a Venn diagram. (f) Runx3 is coimmunoprecipitated with TCF-1. Myc-tagged Runx3 and Flag-tagged full-length TCF-1 were overexpressed in 293T cells, and the lysates were immunoprecipitated with anti-Flag or control IgG followed by immunoblotting with anti-Myc. Lysate input without immunoprecipitation was blotted to detect the expressed proteins. (g) Both p45 and p33 TCF-1 isoforms are coimmunoprecipitated with Runx3. Myc-tagged Runx3 was overexpressed together with Flag-tagged full length (p45) or p33 TCF-1 isoform. The lysates were immunoprecipitated with anti-Myc and then immunoblotted with anti-Flag. As negative controls, pCMV-Myc was transfected in place of Myc-Runx3. (h) TCF-1 is coimmunoprecipitated with full length Runx3d or Runx3d lacking the VWRPY motif (Runx3ΔC). Runx3d or Runx3ΔC were overexpressed together with Flag-tagged full length TCF-1 in 293T cells. The cell lysates were immunoprecipitated with anti-Runx and then blotted with anti-Flag. Data in (f)-(h) are representative of at least 3 independent experiments. (i) and (j) TCF-1 and LEF-1 cooperate with Runx3 in Cd4 gene silencing. Mature TCRβhi thymocytes were analyzed in the compound knockout mice. Representative contour plots are shown in (i), and the values are percentages of CD8+CD4 and CD8*4 subsets within the CD8+ population (to avoid the influence of varied CD4+ frequency). Cumulative data on the frequency of the CD8*4 subset are in (j). n ≥ 5. ***, p < 0.001.

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