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Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity

Abstract

The CD4+ and CD8+ T cell dichotomy is essential for effective cellular immunity. How individual T cell identity is established remains poorly understood. Here we show that the high-mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4+ lineage–associated genes including Cd4, Foxp3 and Rorc in CD8+ T cells. Tcf1- and Lef1-deficient CD8+ T cells exhibit histone hyperacetylation, which can be ascribed to intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutation of five conserved amino acids in the Tcf1 HDAC domain diminishes HDAC activity and the ability to suppress CD4+ lineage genes in CD8+ T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes.

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Figure 1: Tcf1 and Lef1 deficiency perturbs CD8+ T cell integrity.
Figure 2: Tcf7−/−Lef1−/− CD8+ T cells exhibit histone hyperacetylation.
Figure 3: Tcf1 is connected with histone acetylation status in CD8+ T cells.
Figure 4: Tcf1 has intrinsic HDAC activity.
Figure 5: Mapping the HDAC activity domain in Tcf1.
Figure 6: Tcf1 catalyzes deacetylation of histone protein/peptide substrates.
Figure 7: Homology modeling and sequence conservation predict an HDAC-like structure of Tcf1.
Figure 8: Tcf1 HDAC activity is essential for establishing CD8+ T cell identity.

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Acknowledgements

We thank A. Kalen (the Radiation Core facility, University of Iowa) for mouse irradiation and I. Antoshechkin (California Institute of Technology) for RNA-seq. We thank H. Kawamoto (Kyoto University) for Tcf1 antiserum, H. Habelhah for advice on purification of recombinant proteins, and J.T. Harty, J.D. Colgan and A. Bhandoola for critical reading and insightful discussion of the manuscript. We thank the Flow Cytometry Core Facility (J. Fishbaugh, H. Vignes and G. Rasmussen, University of Iowa) for cell sorting; the Flow Cytometry Core Facility is supported by the Carver College of Medicine/Holden Comprehensive Cancer Center (the University of Iowa), the Iowa City Veteran's Administration Medical Center, and the National Center for Research Resources of the NIH (1 S10 OD016199). The Proteomics Facility (University of Iowa) is supported by an endowment from the Carver Family trust. This study is supported by grants from the American Cancer Society (RSG-11-161-01-MPC to H.-H.X.), the NIH (AI112579, AI105351, AI115149 and AI119160 to H.-H.X. and AI113806 to W.P.), the US National Science Foundation (CAREER Award 1452411 to C.A.M.) and the Carver Trust Young Investigator Award (01-224) to H.H.Q.; J. Z. and C.L. are supported by the Intramural Research Program of the NHLBI.

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Authors and Affiliations

Authors

Contributions

S.X. and F.L. performed most of the experiments with the help of S.Y., Q.S. and F.C.P.; Z.Z. analyzed the high-throughput data under the supervision of W.P.; Y.Z. performed homology modeling and coevolution analyses under the supervision of C.Z.; Y.L. and R.M.P. performed the proteomics analysis; P.K.M., H.H.Q. and C.A.M. helped with purification of recombinant proteins; C.L. directed gene targeting; J.Z. directed high-throughput sequencing; W.P. and H.-H.X. supervised the overall study, analyzed the data and wrote the paper. All authors edited the manuscript.

Corresponding authors

Correspondence to Weiqun Peng or Hai-Hui Xue.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Impact of Tcf1 and Lef1 deficiency on the development and gene expression of CD8+ T cells.

(a) Derepression of the CD4 coreceptor in Tcf7–/–Lef1–/– CD8+ T cells. Thymocytes from Tcf7–/–Lef1–/– and control mice were surface-stained. Shown is the gating strategy to identify TCRβhi CD69CD44 mature thymocytes and further fractionation to identify CD4+ and CD8+ single positive cells. The CD8+ mature thymocytes were further analyzed for the CD44hiIL-2Rβ+ memory-phenotype subset. Our previous study (Steinke et al. Nat. Immunol. 15, 646-656, 2014) demonstrated that the CD4+CD8+ population within the mature thymocyte subset was due to derepression of the CD4 coreceptor in CD8+ cells, but not aberrant expression of the CD8 coreceptor in CD4+ cells. This gating strategy was also used to sort control CD4+, CD8+, and Tcf7–/–Lef1–/– CD8+ T cells for transcript and histone mark analyses.

(b) Tcf7–/–Lef1–/– CD8+ T cells preserved the expression of CD8+ signature genes. CD4+ and CD8+ mature thymocytes were sorted from control mice, and Tcf7–/–Lef1–/– CD8+ mature thymocytes were also sort-purified. The sorted cells were analyzed for gene expression with quantitative RT-PCR. Nkg7 and Itgae are CD8+-specific genes, and the Runx3d transcript from its distal promoter is specifically utilized in CD8+ T cells.

(c) Aberrant upregulation of CD8+ effector molecules in naïve Tcf7–/–Lef1–/– CD8+ T cells. Cells were sorted and analyzed for gene expression as in (b). Prdm1 and Fasl are virtually not expressed, and Prf1 is minimally expressed in naïve CD8+ T cells. Data of (b) and (c) are means ± s.d. from 4 independent experiments (n ≥ 6).

(d) Tcf7–/–Lef1–/– splenic CD8+ T cells were neither aberrantly activated nor enriched in memory-phenotype cells. CD8+TCRβ+ splenocytes from Tcf7–/–Lef1–/– and control mice were identified (top panels) and further fractionated to CD62L+CD44lo naïve, CD62L+CD44hi memory-phenotype, CD62LCD44hi effector-phenotype cells (bottom panels). The CD8+ T cells were also analyzed for the CD44hiIL-2Rβ+ memory-phenotype subset. The frequency of each subset is shown in representative contour plots. Data are from ≥ 3 independent experiments with similar results.

Note that CD44hiIL-2Rβ+ memory-phenotype population was not enriched but actually reduced in both Tcf7–/–Lef1–/– mature CD8+ thymocytes and splenic CD8+ T cells. This observation is consistent with our previous finding that longevity of memory CD8+ T cells depends on Tcf1 (Zhou et al. Immunity 33, 229, 2010).

(e)-(f) Survey of Vβ TCR repertoire in Tcf7–/–Lef1–/– CD8+ T cells. CD8+ mature thymocytes from control and Tcf7–/–Lef1–/– mice were further stained with mouse Vβ TCR screening panel, and the percentage of each Vβ subtype was determined. Representative data for select Vβ TCRs are shown in histograms (e), and cumulative data from 2 experiments (n = 3) are summarized in (f). *, p<0.05; **, p<0.01 by student’s t-test.

Supplementary Figure 2 Tcf1 and Lef1 deficiency perturbs the lineage integrity of naive CD8+ T cells.

(a) Aberrant expression of CD4+ signature genes in Tcf7–/–Lef1–/– CD8+ T cells. CD4+ and CD8+ mature thymocytes were sorted from control mice, and Tcf7–/–Lef1–/– CD8+ mature thymocytes were also sort-purified. The sorted cells were analyzed for gene expression with quantitative RT-PCR. CD4+ signature genes including Cd40lg, St8sia6, Lgmn and Itgb3 were derepressed in Tcf7–/–Lef1–/– CD8+ T cells, and the derepression of Thpok was negligible. Gata3 was also up-regulated in Tcf7–/–Lef1–/– CD8+ T cells. Data are means ± s.d. from 4 independent experiments (n ≥ 6).

(b) Aberrant expression of Foxp3 and Rorγt in Tcf7-/-Lef1-/- CD8+ T cells. Foxp3 and Rorγt proteins were detected by intracellular staining in CD8+ mature thymocytes. Similar results were obtained for splenic TCRβ+CD8+ cells (not shown). Representative contour plots are shown from ≥ 4 experiments.

(c)-(e) Protein expression of FasL, Granzyme B and CD40L in naïve and activated Tcf7–/–Lef1–/– splenic CD8+ T cells. Naïve splenic CD8+ T cells were isolated from the spleens of control or Tcf7–/–Lef1–/– mice. A portion of the cells were activated with plate-bound anti-CD3 (10 μg/ml) in the presence of soluble anti-CD28 (1 μg/ml) and IL-2 (10 U/ml) for 2 days, followed by 1 day expansion in IL-2 only, and used as Day 3 effector CD8+ T cells. The naïve and effector CD8+ T cells were surface-stained for FasL and CD40L, or intracellularly stained for granzyme (shaded areas) or corresponding isotype controls (dotted lines). Values in the histograms are differences in mean fluorescence intensity (ΔMFI) between antibody and isotype control staining. Representative data from 2 independent experiments (n ≥ 3) are shown.

Supplementary Figure 3 Tcf1 and Lef1 deficiency perturbs the lineage integrity of naive CD8+ T cells.

WT CD4+ and CD8+, Tcf7–/–Lef1–/– CD8+ mature thymocytes were sort-purified and analyzed for H3K4me3, H3K27me3, and H3K27Ac by ChIP-Seq. (a) Analysis of H3K4me3 and H3K27me3 at the 108 CD4+ characteristic genes. Normalized read counts of H3K4me3 and H3K27me3 signals surrounding the TSSs of the 108 genes in the CD4+ T cell gene set were plotted.

(b) H3K27Ac ChIP-Seq tracks at the St8sia6, Cd40lg, and Lgmn genes. Gene structures and transcriptional orientations were marked on top of each panel. These CD4+ signature genes were associated with strong H3K27Ac signals in WT CD4+ T cells, which were absent in WT CD8+ T cells. Tcf7–/–Lef1–/– CD8+ T cells, however, showed strong H3K27Ac signals similar to WT CD4+ T cells.

(c) Analysis of H3K4me3 and H3K27me3 at the 472 upregulated genes in Tcf7–/–Lef1–/– CD8+ T cells. Normalized read counts of H3K4me3 and H3K27me3 signals surrounding the TSSs of the 472 upregulated genes in Tcf7–/–Lef1–/– CD8+ T cells were plotted.

(d) H3K27Ac ChIP-Seq tracks at the Foxp3 and Rorc genes. Gene structures and transcriptional orientations were marked on top of each panel. Compared with WT CD8+ T cells, Tcf7–/–Lef1–/– CD8+ T cells showed increased H3K27Ac signals at the 5’-regulatory region of Foxp3 and the gene body of Rorc. In b and d, also shown are the Tcf1 ChIP-Seq tracks in splenic CD8+ T cells, with the MACS-called Tcf1 binding peaks marked by green rectangles.

Supplementary Figure 4 Validation of H3K4me3 and H3K27me3 marks at select gene loci.

Splenic WT CD4+ and CD8+, Tcf7–/–Lef1–/– CD8+ T cells were sort-purified from the spleens and subjected to ChIP analysis using antibodies to H3K4me3 and H3K27me3 or control IgG. The signals of H3K4me3 and H3K27me3 at the indicated genomic locations were normalized to that obtained from IgG ChIP at the same location to calculate their enriched signals.

It is of note that unlike uniformly increased H3K27Ac signals at the loci of upregulated genes in Tcf7–/–Lef1–/– CD8+ T cells (compare with Fig. 2e), changes in H3K4me3 and H3K27me3 were more dependent on the gene context. St8sia6 and Fasl showed clear increase in H3K4me3 and decrease in H3K27me3 in Tcf7–/–Lef1–/– CD8+ T cells compared with WT CD8+ T cells. Similar changes were found at the Itgb3 and Prdm1 genes but were less pronounced. On the other hand, at the Cd4 TSS or silencer, both WT and Tcf7–/–Lef1–/– CD8+ T cells had low levels of H3K4me3 and maintained high levels of H3K27me3 compared with WT CD4+ T cells. Consistent with lack of strong derepression of Thpok in Tcf7–/–Lef1–/– CD8+ T cells, the Thpok gene harbored little H3K4me3 but strong H3K27me3 signals in both WT and Tcf7–/–Lef1–/– CD8+ T cells. The Cd40lg upstream regulatory region was distal to the TSS or gene body and was used a negative control for H3K4me3 and H3K27me3 signals. Data are pooled results from 2 independent experiments with each sample measured in duplicate.

Supplementary Figure 5 Loss of Tcf1 and Lef1 does not cause severe dysregulation of histone acetyltransferase and HDAC genes.

(a-b) The RNA-Seq data from control, Tcf7–/–, and Tcf7–/–Lef1–/– CD8+ mature thymocytes were analyzed for expression of known histone acetyltransferase (a) and HDAC (b) genes. Gene expression was quantified by FPKM values from the RNA-Seq analysis. Shown in the heatmap are the log2-transformed expression values.

Note that there was an ~1.5-2 fold increase in Kat2b and Ncoa3 transcripts (a) and an ~1.5 fold decrease in the transcripts of Hdac2, Hdac6, Hdac10, Sirt3 and Sirt4 (b) in Tcf7–/–Lef1–/– CD8+ T cells, based on FPKM values from the RNA-Seq analysis. These moderate gene expression changes less likely accounted for the substantial increase of acetylation at H3K27 and H3K9 in Tcf7–/–Lef1–/– CD8+ T cells.

(c) Validated expression of selected HDAC genes. CD8+ mature thymocytes were sorted from control and Tcf7–/–Lef1–/– mice, and mature CD4+ thymocytes were from control mice. The expression of indicated HDAC genes were determined by quantitative RT-PCR. All genes were normalized to Gapba in each sample. The expression of each gene in control CD8+ thymocytes was set as 1, and its expression in control CD4+ and Tcf7–/–Lef1–/– CD8+ thymocytes were calculated accordingly. Data are means ± s.d. from 2 independent experiments (n = 4). **, p<0.01; ***, p<0.001 by Student’s t-test.

It is of interest to note that Hdac1 and Hdac2, which are known for critical regulation of CD4+ lineage integrity, are expressed at similar levels between mature CD4+ and CD8+ T cells. On the other hand, CD4+ T cells express moderately higher levels of Hdac6 and Hdac10, but lower levels of Hdac7 than CD8+ T cells. Consistent with the RNA-Seq results, among the validated HDAC genes, only Hdac7 showed a modestly increased expression in Tcf7–/–Lef1–/– CD8+ T cells.

Supplementary Figure 6 Highly purified Tcf1 catalyzes deacetylation of histone H3K9Ac peptide.

(a) Purification of Tcf1 by FPLC. Shown is the chromatogram elution profile for Tcf1 run over a superdex 75 10/300 GL column. The UV absorbance trace at 280 nm is shown in black and that at 260 nm is shown in gray. Fractions were collected every 0.5 ml starting at 1 ml. The major peak was eluted at 8–10 ml, corresponding to fractions 14-18, which were analyzed by SDS-PAGE and Coomassie Blue staining (see inset). Fraction 15 containing Tcf1 with the highest purity was used in all the HDAC assays with histone proteins or peptide as substrates (Fig. 6 and panels b-d in this figure).

Fraction 15 was immunoblotted with a house-made Tcf1 antiserum, which confirmed the major component in the preparation was Tcf1. The minor amounts of proteins of smaller molecular weights were also reactive to the Tcf1 antiserum, suggesting that these smaller proteins are likely degradation products from purified Tcf1.

(b) Purity of FPLC-purified Tcf1 as determined by silver staining. Fractions 14-17 from an independent FPLC purification was resolved on SDS-PAGE, and silver-stained using the “Pierce Silver Stain for Mass Spectrometry”.

(c) Detection of histone H3 (1-21) in K9 acetylated and deacetylated forms with MALDI. Commercially available histone H3(1-21)K9Ac and H3(1-21) peptides were suspended in an HDAC assay buffer at 100 µM and detected on MALDI at m/z of ~2296 and ~2254, respectively. Each peptide appears as a series of multiple consecutive peaks with an one Dalton spacing and a relative peak height determined by the natural abundance of principally carbon, hydrogen and sulfur isotopes.

(d) Validating the identity of the product in Tcf1-catalyzed HDAC assay using H3(1-21)K9Ac peptide. FPLC-purified Tcf1 was incubated with 100 µM H3(1-21)K9Ac peptide for 2 hrs, and the 2,254 Da product and 2,296 Da remaining substrate peptide were detected with MALDI as in Fig. 6b. The product and substrate were further analyzed with Laser-Induced Fragmentation Time of Flight (LIFT). The product (lower panel) had the identical amino acid sequence as the substrate (upper panel), except that the product sequence displays Lysine 9 in its deacetylated form, with 42 Da (equal to an acetyl group) reduction in mass at this particular residue.

(e) Kinetics calculation of Tcf1-catalyzed deacetylation of H3(1-21)K9Ac peptide. The reciprocal of reaction velocity (1/V) and that of H3(1-21)K9Ac peptide substrate concentration (1/[S]) were plotted using data from Fig. 6d. Linear trendline was added using Excel, and the equation and correlation coefficient (R2) are shown in the figure. Determined from the y-intercept, Vmax = 1/1,401,754 mM/sec = 0.71×10–6 mM/sec. Determined from the slope, Km = 30,758/1,401,754 mM =0.022 mM.

Supplementary Figure 7 Top 50 predicted coevolving residue pairs in Tcf1 and Lef1 orthologs.

Ninety-eight Tcf1 or Lef1 protein sequences were retrieved from 56 species and used in direct coupling analysis (DCA). In the output heatmap, the axes show the residues in the 30 aa Tcf1 HDAC domain and their positions. Each colored square represents the coordinates of the top 50 co-evolved pairs predicted by DCA. The color shade represents the strength of direct co-evolution coupling as measured by the direct information (DI).

Note that co-evolution may also play a role in the divergence of Tcf1 and Lef1 from conventional HDACs (Fig. 7b). Many residues that have a high degree of conservation within HDACs are involved in potential co-evolution in Tcf1 and Lef1. Examples include:

1. L196: L196-Q192, L196-T193, L196-D195, L196-S206;

2. G198: G198-T193, G198-P194, G198-S201, G198-S206, G198-V214;

3. T203: T203-T213, T203-V214;

4. L221: L221-P217,L221-S218, L221-P219, L221-P220;

5. P219: P219-P217, P219-S218, P219-P220, P219-L221.

All these predicted co-evolving pairs are in the columns marked by green arrows.

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.

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Xing, S., Li, F., Zeng, Z. et al. Tcf1 and Lef1 transcription factors establish CD8+ T cell identity through intrinsic HDAC activity. Nat Immunol 17, 695–703 (2016). https://doi.org/10.1038/ni.3456

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