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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

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.

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Figure 1: CD4-Cre–mediated deletion of TCF-1 alone or of both TCF-1 and LEF-1 impairs the development of CD4+ SP thymocytes.
Figure 2: The CD8*4 mature thymocytes in Tcf7−/−Lef1−/− mice belong to the CD8+ lineage.
Figure 3: Deficiency in TCF-1 alone or in both TCF-1 and LEF-1 redirects CD4+ T cells to the CD8+ lineage.
Figure 4: TCF-1 deficiency decreases Thpok expression but increases Runx3d expression in bipotent precursor cells.
Figure 5: Ectopic expression of Th-POK rectifies the defects in CD4+ T cell differentiation caused by loss of TCF-1.
Figure 6: TCF-1 acts through the GTE in the Thpok locus.
Figure 7: Deletion of Runx3 does not rectify the defects in differentiation into the CD4+ lineage caused by the loss of TCF-1 and LEF-1.
Figure 8: TCF-LEF and Runx factors act together in silencing Cd4 in CD8+ T cells.

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Acknowledgements

We thank R. Bosselut (National Cancer Institute of the US National Institutes of Health) for mice expressing the transgene encoding Th-POK; S.-C. Bae (Chungbuh National University) for the Myc-tagged Runx3 expression plasmid; B.J. Fowlkes for input and discussions; Y. Wakabayashi and Y. Luo (NHLBI) for high-throughput sequencing and data processing; T. Zhao for animal husbandry; the Flow Cytometry Core facility at the University of Iowa (J. Fishbaugh, H. Vignes and G. Rasmussen) for support for cell sorting; and Radiation Core facility at the University of Iowa (A. Kalen) for mouse irradiation. Supported by the American Cancer Society (RSG-11-161-01-MPC to H.-H.X.) and the US National Institutes of Health (HL095540 and AI105351 to H.-H.X.; HG006130 to K.T.; AI007485 (for support of F.C.S.); and P30CA086862 and 1S10 RR027219 to the Flow Core Facility at the University of Iowa). The content is solely the responsibility of the authors and does not necessarily represent the official views of the US National Institutes of Health.

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Authors

Contributions

F.C.S. and S.Y. did experiments and analyzed the data; X.Z. and B.Z. did the coimmunoprecipitation experiments; B.H. and W.Y. analyzed the ChIP-Seq data under the supervision of K.T. and J.Z.; H.K. provided anti-TCF-1; H.-H.X. designed and supervised the study and, with F.C.S. and S.Y., wrote the paper.

Corresponding author

Correspondence to Hai-Hui Xue.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conditional targeting of Tcf7.

(a) Targeting strategy. The Tcf7 gene was conditionally targeted by the International Knockout Mouse Consortium (IKMC, project 37596). Depicted on top is partial structure of the Tcf7 gene with filled rectangles in yellow denoting exons (all numbered). The exon 4 of Tcf7 was flanked by two LoxP sites, and deletion of this exon results in a nonsense frame-shift mutation. Also marked are key enzyme sites and relative locations of 5' and 3' probes used in Southern blotting. Shown in the middle is the structure of Tcf7-targeted allele, highlighting the targeting arms, locations of inserted LoxP sites (filled triangles in red), Frt sites (open triangles in blue), β-galactosidase-neomycin resistant gene (LacZ-Neo) cassettes. Note that two extra BamHI sites were embedded in the LacZ-Neo cassette, and these two sites were used to facilitate detection of the targeted allele by Southern blotting. By crossing with Rosa26-Flippase knock-in mice, the Frt site-flanked LacZ-Neo cassette was excised, giving rise to the Tcf7-floxed allele. (b) Identification of targeted mice. Genomic DNA was extracted from tails of targeted mice, digested with BamHI, and Southern-blotted with the 5' or 3' probes. Both probes detect the WT allele at approximately 13.4 kb. The 5'-probe detects the targeted allele at 5.2 kb (top panel), and the 3' probe detects the targeted allele at 8.7 kb (bottom). The probes were amplified with the following primers: 5' probe: 5'-agggtgggcacagagatatg and 5'-gccagagctcagctgctaat; 3' probe: 5'-agccaaggtcattgctgagt and 5'-ccttcctgtgttgaggtggt.

Supplementary Figure 2 Deficiency in both TCF-1 and LEF-1 does not affect TCR-dependent induction of the expression of GATA-3 and Tox.

Although CD69 expression was reduced in TCRβhi thymocytes from Tcf7-/-Lef1-/- mice (Fig. 1c), the combination of CD69 and CD24 was sufficient to distinguish immature and mature subsets within the TCRβhi population. The surface-stained thymocytes from Tcf7-/-Lef1-/- and littermate controls were sorted for 3 subsets, pre-select DP (PreDP), post-select DP (PostDP), and CD4+8lo intermediate (IM). Gata3 and Tox expression was measured as an end outcome of TCR signaling in positively selected DP subsets. The relative expression of each gene was normalized to Hprt1. Data are pooled results from 3 independent experiments and shown as means ± s.d. (n ≥ 3). Gata3 and Tox expression between control and Tcf7-/-Lef1-/- within each subset was not statistically different (p>0.4). Note that the post-select DP thymocytes from Tcf7-/-Lef1-/- mice may contain a fraction of CD8+ T cells that had derepressed expression of CD4 (the CD8*4 cells). Because Gata3 and Tox were less abundantly expressed in CD8+ T cells compared with DP thymocytes, CD8*4 cells unlikely contributed to elevating Gata3 and Tox expression in Tcf7-/-Lef1-/- post-select DP thymocytes.

Supplementary Figure 3 Lack of TCF-1 and LEF-1 diminishes CD4+ T cell output independently of their role in thymocyte survival.

(a) and (b) Loss of TCF-1 or both TCF-1 and LEF-1 diminishes CD4+ T cell output in the periphery. Splenocytes were surface-stained, and TCRβ+ cells were analyzed for CD4+ and CD8+ lineage distribution. Representative and cumulative data are shown in a and b, respectively. (c) Loss of TCF-1 and LEF-1 reverses the CD4/CD8 ratio in the periphery. The CD4+ to CD8+ ratio is calculated from b. Data are from ≥ 4 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001. (d) Germline deletion of TCF-1 results massive cell death in TCRβhi thymocytes. Thymocytes from germline-targeted TCF-1 knockout mice and littermate controls were harvested, and Caspase-3&7 activation was measured in the TCRβhi subset. (e) Late deletion of TCF-1 and LEF-1 alleviates death of thymocytes. TCRβhi thymocytes were analyzed as in d. The frequency of Caspase3&7-positive subset is shown. Data are representative from ≥ 3 experiments.(f) Deficiency in TCF-1 and LEF-1 does not cause preferential death of CD4+ SP T cells. CD4+ or CD8+ TCRβhi thymocytes were analyzed for Caspase activation. Cumulative data from 3 experiments are shown.

Supplementary Figure 4 The redirected CD8+ T cells in B2m–/– chimeras reconstituted with Tcf7–/–Lef1–/– BM exhibit true CD8+ T cell characteristics.

BM cells from Tcf7-/-, Tcf7-/-Lef1-/-, or littermate controls were transplanted into lethally irradiated CD45.1+ congenic β2m-/- mice. Six weeks later, splenocytes were isolated from the BM chimeras and used for downstream analysis. (a) and (b) The redirected CD8+ T cells in the absence of TCF-1 or both TCF-1 and LEF-1 persist in the periphery. Donor-derived CD45.2+TCRβ+ splenocytes were analyzed for CD4+ and CD8+ lineage distribution. Representative contour plots (a) are from 4 independent experiments with ≥ 4 recipients analyzed in each experiment. Numbers of mature CD4+ and CD8+ splenocytes in the BM chimeras are shown in b as means ± s.d. (n ≥ 14). *, p<0.05; **, p<0.01***; p<0.001. (c) The redirected Tcf7-/-Lef1-/- CD8+ T cells express CD8+ T cell-characteristic genes. CD4+ and CD8+ splenic T cells were sorted from WT C57BL/6 mice, and CD8+CD4 and CD8*4 CD45.2+TCRβ+ splenocytes were sorted from the Tcf7-/-Lef1-/--reconstituted β2m-/- BM chimeras (Tcf7-/-Lef1-/- BM chimeras), followed by gene expression analysis. (d) The redirected Tcf7-/-Lef1-/- CD8+ T cells proficiently produce granzyme B and interferon-γ upon stimulation. Splenic T cells were isolated from WT B6 mice or Tcf7-/-Lef1-/- BM chimeras, and then activated by plate-bound anti-CD3 antibody and soluble anti-CD28 antibody in the presence of IL-2. Three days later, the cells were stimulated with PMA/Ionomycin in the presence of Golgi plug, and then surface-stained for CD40L, intracellularly stained for granzyme B, interferon-γ, and IL-2. For c and d, similar results were obtained for redirected Tcf7-/- CD8+ T cells (not shown).

Supplementary Figure 5 Expression of a transgene encoding a TCR alters the timing of CD4-Cre–mediated deletion of Tcf7 and Lef1.

(a) Expression of the OT-II TG greatly diminished total thymocyte numbers in Tcf7-/- and Tcf7-/-Lef1-/- mice compared with littermate controls. Also compare with Fig. 1b.(b) CD4-Cre initiates deletion of Tcf7 and Lef1 at the DN stage in the presence of OT-II TG. Thymocytes from Tcf7-/-Lef1-/- and littermate controls with or without the OT-II TG were isolated and surface-stained. Lineage-negative CD4CD8 thymocytes were sorted as DN, and TCRβ+CD69CD4+CD8+ cells sorted as pre-select DP (PreDP) subsets. The expression of Tcf7 and Lef1 was measured by quantitative RT-PCR. Data are duplicate measurements of two samples. *, p<0.05; **, p<0.01; ***, p<0.001. Note that without the OT-II TG, CD4-Cre did not excise Tcf7 and Lef1 at the DN stage but initiated the deletion from the pre-select DP stage, consistent with our Western blot data in Fig. 1a. In contrast, in the presence of the OT-II TG, CD4-Cre initiated deletion of both Tcf7 and Lef1 from the DN stage. Because TCF-1 is critical for survival of early thymocytes (as seen in Supplementary Fig. 3d), early deletion of TCF-1 and LEF-1 in the presence of OT-II TG at least partly account for more severe reduction of total thymocytes as shown in (a). (c) OT-II TG T cells adopt CD8+ T cell fate in the absence of TCF-1 or both TCF-1 and LEF-1. The numbers of CD4+ or CD8+ SP thymocytes were calculated from the mature Vα2+TCRβhiCD24 thymic subset. Data are means ± s.d. (n ≥ 5-10). In spite of reduced total thymic cellularity upon deletion of TCF-1 or both TCF-1 and LEF-1, the mature OT-II+ thymocytes were predominantly CD8+.

Supplementary Figure 6 ChIP-Seq analysis of the binding of TCF-1 to the Thpok and Cd4 loci

ChIP-Seq of TCF-1 in whole thymocytes was reported by Li L et al. (Blood 122, 902, 2013), and ChIP-Seq of Runx3 in CD8+ T cells was reported by Lotem J et al (PLoS One, 8, e80467, 2013). The data were downloaded and processed for peak calling using MACS. Using the same stringent criteria (detailed in Supplementary Fig. 8), wherein 2,827 TCF-1 binding peaks were identified in CD8+ T cells, we found 32,663 peaks in whole thymocytes. Possible reasons for the higher numbers of TCF-1 binding peaks in whole thymocytes include: 1) TCF-1 may regulate different target genes during thymocyte maturation stages. The binding events detected in whole thymocytes are a collection of all TCF-1 binding events at different stages; and 2) the ChIP-Seq control sample was from input DNA for peak calling, whereas ChIP-Seq of TCF-1 and Runx3 by us and Lotem J et al used IgG or non-immune serum-immunoprecipitated samples as control. The ChIP-Seq track wiggle files were uploaded to the UCSC genome browser for visualization of enriched binding by the transcription factors. For the select gene locus, the transcription start site (TSS) and orientation are marked by arrows. The horizontal bars over TCF-1 or Runx3 tracks indicate the enriched binding peaks identified by MACS. (a) shows enriched binding of TCF-1 at the Thpok GTE in whole thymocytes but not in CD8+ T cells. (b) shows co-occupancy of TCF-1 and Runx3 at the Cd4 silencer in all cell types. TCF-1 is also associated with Cd4 enhancer and weakly with Cd4 promoter in whole thymocytes, consistent with reported TCF-1 binding to these regions by Huang Z et al (J. Immunol. 176, 4880, 2006). Note that no TCF-1 binding to Cd4 enhancer and promoter in CD8+ T cells.

Supplementary Figure 7 The TCF-1-binding sites in the Thpok GTE are important for the GTE enhancer activity.

(a) The TCF-1 sites in the 473-bp Thpok GTE. The two TCF-1 sites are marked and mutant sequence aligned. (b) Conservation of TCF-1 sites across different species, with core sequence highlighted. (c) Mutation of TCF-1 sites in the Thpok GTE abrogates its enhancer activity. 293T cells were transfected with the indicated luciferase reporter constructs along with an internal control pRL-TK. Forty-eight hours later, luciferase activity was measured as in Fig. 6c.

Supplementary Figure 8 ChIP-Seq analysis of TCF-1 in CD8+ T cells.

(a) Numbers of TCF-1 peaks identified using stringent and permissive settings of the MACS algorithm. Under the stringent setting, 2,827 peaks were defined as stringent TCF-1 peaks, and under the permissive settings, 6,577 additional peaks were defined as permissive TCF-1 peaks. (b) Genomic distribution of all TCF-1 binding peaks. Promoter region is defined as “-5 kb to +1 kb” flanking the transcription start sites of known RefSeq genes. (c) Overlap of TCF-1 binding peaks with H3K4me3 and H3K27me3 peaks in human naïve CD8+ T cells. The homologous regions of the H3K4me3 and H3K27me3 peaks from human CD8+ T cells were identified in mouse genome using the LiftOver tool. TCF-1 binding peaks in different genomic regions were then assessed for peak overlapping. The criterion for overlapping is that the closest boundary-to-boundary distance of two peaks is within 400 bp. (d) TCF-LEF and (e) Runx motifs found in the permissive TCF-1 binding peaks. The motif logos are shown. Note that the motifs found in the permissive TCF-1 peaks are consistent with those found in the stringent TCF-1 peaks (Fig. 8c and 8d). (f) Venn diagram showing the motif distribution in the permissive TCF-1 peaks.

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Steinke, F., Yu, S., Zhou, X. et al. 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. Nat Immunol 15, 646–656 (2014). https://doi.org/10.1038/ni.2897

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