Supplementary Figure 8: Impact of mutating key residues in the Tcf1 HDAC domain.

(a) Experimental design for genetic complementation. A bicistronic retroviral vector MIG-R1 was used to achieve forced expression of WT or mutant Tcf1 proteins in BM progenitor cells from Tcf7^–/–Lef1^–/– mice. Six weeks later, the donor-derived CD45.2⁺GFP⁺TCRβ⁺ T cells in the spleens of BM chimeras were analyzed.

(b) Mutation of 5 amino acids in Tcf1 HDAC domain does not affect its DNA binding capacity. Recombinant WT Tcf1 p45 or Tcf1 Mut5aa proteins were purified, and 0.6 µg protein was incubated with biotin-labeled TOP probe in the absence or presence of 200 fold molar excess of unlabeled TOP nucleotides as a competitor. The protein-DNA complex was identified on a native PAGE. Data are representative of two experiments.

(c) WT p45 Tcf1 and Tcf1 Mut5aa bind to target gene loci with similar capacity in vivo. BM cells from Tcf7^–/–Lef1^–/– mice (CD45.2⁺) were infected with retrovirus expressing WT p45 Tcf1 or Tcf1 Mut5aa followed by transplantation into CD45.1⁺ congenic recipient mice. Six weeks later, splenic CD45.2⁺GFP⁺TCRβ⁺CD8⁺ T cells were sorted from gene-complemented recipient mice and used in ChIP with anti-Tcf1 or control IgG. Enriched Tcf1 binding at Hprt, Lef1 or Axin2 gene loci in each cell type was first calculated by normalizing anti-Tcf1 to IgG, and the relative enrichment was then determined by normalizing signals at Lef1 (left panel) or Axin2 (right panel) to that at the Hprt locus. WT CD8⁺ T cells were used as a positive control. Data are from 2 independent experiments with each sample measured in duplicates or triplicates. ***, p<0.001 compared with empty vector (Vec)-complemented Tcf7^–/–Lef1^–/– CD8⁺ T cells. ns, not statistically significant between WT p45 Tcf1- and Tcf1 Mut5aa-complemented Tcf7^–/–Lef1^–/– CD8⁺ T cells.

(d) WT p45 Tcf1 and Tcf1 Mut5aa proteins are expressed at similar levels in gene-complemented Tcf7^–/–Lef1^–/– CD8⁺ T cells. Tcf1 expression in CD45.2⁺GFP⁺TCRβ⁺CD8⁺ splenic T cells from the gene-complemented BM chimeras was determined by intranuclear staining. Numbers in the histograms denote the ΔMFI between Tcf1 antibody and control IgG staining. The Tcf1 antibody (C63D9) specifically recognizes the N-terminus of full-length p45 Tcf1 protein.

Note that the expression levels of Tcf1, WT or mutant, driven by the retroviral LTRs remained lower than that driven by Tcf1 native promoter as shown in WT CD8⁺ T cells (the far-right panel).

(e-f) Analysis of CD4⁺ and CD8⁺ lineage distribution. Splenic CD45.2⁺GFP⁺TCRβ⁺ T cells from the BM chimeras were analyzed for CD4 and CD8 expression. Values in (e) denote frequency of CD4⁺ and CD8⁺ T cells. Cumulative data on CD4⁺ T cell frequency and numbers are shown in Fig. 8a, and those on CD8⁺ T cell frequency and numbers are in (f). Data are from 3 experiments (n = 4).

(g) Analysis of CD4 derepression in CD8⁺ T cells. Splenic CD45.2⁺GFP⁺TCRβ⁺CD8⁺ T cells from the BM chimeras were analyzed for frequencies of CD8⁺CD4^– and CD8⁺CD4⁺ subsets. The cumulative data on frequency of CD8⁺CD4⁺ subset are in the right panel. Data are from 3 experiments (n = 4).

Note that Cd4 gene silencing in CD8⁺ T cells is known to be established and maintained by epigenetic mechanisms. In addition to histone modification, DNA methylation was recently reported to play a critical role in this process (Sellars M et al. Nat. Immunol. 16, 746, 2015). This may account for the relative moderate effect of WT p45 Tcf1 overexpression in repressing CD4 expression in CD8⁺ T cells. Nonetheless, the effect of WT Tcf1 was abrogated in the Tcf1mut5aa mutant.

(h) Analysis of Foxp3 derepression in CD8⁺ T cells. Splenic CD45.2⁺GFP⁺TCRβ⁺CD8⁺ T cells were sorted from the BM chimeras followed by intranuclear staining for Foxp3. Frequency of Foxp3⁺ cells is shown in histograms, and cumulative data are in the right panel. Data are from 2 experiments (n = 3-4).

(i) Analysis of FasL expression in CD8⁺ T cells. Splenic CD45.2⁺GFP⁺TCRβ⁺CD8⁺ T cells from the BM chimeras were analyzed for FasL expression. Values in the histograms are ΔMFI between FasL antibody and isotype control staining. Representative data from 2 independent experiments (n = 4-5) are shown. In each experiment, the ΔMFI in empty vector-complemented Tcf7^–/–Lef1^–/– CD8⁺ T cells was set at 1, and that in WT Tcf1- or Tcf1 Mut5aa-complemented cells was calculated accordingly. The cumulative data are in the right panel. For panels (d), (f)-(i), *, p<0.05; **, p<0.01; ***, p<0.001 unless specified otherwise.