Essential role of a ThPOK autoregulatory loop in the maintenance of mature CD4+ T cell identity and function

Abstract

The transcription factor ThPOK (encoded by the Zbtb7b gene) controls homeostasis and differentiation of mature helper T cells, while opposing their differentiation to CD4⁺ intraepithelial lymphocytes (IELs) in the intestinal mucosa. Thus CD4 IEL differentiation requires ThPOK transcriptional repression via reactivation of the ThPOK transcriptional silencer element (Sil^ThPOK). In the present study, we describe a new autoregulatory loop whereby ThPOK binds to the Sil^ThPOK to maintain its own long-term expression in CD4 T cells. Disruption of this loop in vivo prevents persistent ThPOK expression, leads to genome-wide changes in chromatin accessibility and derepresses the colonic regulatory T (T_reg) cell gene expression signature. This promotes selective differentiation of naive CD4 T cells into GITR^loPD-1^loCD25^lo (Triple^lo) T_reg cells and conversion to CD4⁺ IELs in the gut, thereby providing dominant protection from colitis. Hence, the ThPOK autoregulatory loop represents a key mechanism to physiologically control ThPOK expression and T cell differentiation in the gut, with potential therapeutic relevance.

Fig. 1.. Genetic mapping of an anti-silencer element in mature CD4 T cells.

a) Organization of mouse ThPOK gene (top), and diagram of silencer deletion mutants (bottom). Black boxes, blue boxes, arrows, and red boxes indicate exons, enhancers, silencers and promoters, respectively. Deletions within the silencer are indicated by thin black lines. “R” indicates positions of conserved Runx binding sites. b) FACS analysis of CD4, and CD8a expression in gated TCRβ+ PBLs of WT mice and homozygous mutant lines, as designated in panel a. c) Plots indicate % of SP CD4, CD8, CD4+ 8+ (DP) and CD4^lo/− cells within gated TCRβ+ PBLs of indicated strains (n = 5, for each strain). Error bars represent standard deviations. d) RNA was collected from freshly isolated cells before probing for ThPOK expression by qPCR.

Fig. 2.. Regulation of anti-silencer by ThPOK binding.

a) Numbered bars (top) represent 15 different mouse ThPOK silencer fragments that were tested as bait in Y1H analyses against ThPOK as prey (Zbtb7b). Fragments that bind ThPOK, and/or the related LRF (Zbtb7a) factor are indicated by thick red bars, whereas those that do not show binding are marked as thin black lines. Thick black line (middle) represents the full-length silencer, with positions of consensus Zbtb7b/a binding sites (as predicted by JASPAR algorithm) marked by red arrows, and conserved Runx binding sites by blue ovals. Sequence of regions deleted in OB11 and PY3 mutants, with positions of predicted Zbtb7b/a binding sites marked in red. b) EMSA analysis, using the 100bp OB11 region (lanes 1–6) or EBNA control DNA (lanes 7–9) as probes. Biotinylated probes were incubated with cell extracts from NIH 3T3 cells transfected with empty vector, Fl-ThPOK or EBNA expression constructs, as indicated. In some lanes unlabeled competitor DNA (lanes 4, 9), anti-Fl-ThPOK antibody (lane 5), or mutant OB11 probe in which the consensus ThPOK binding sites are destroyed (lane 6), were added. c-f) ChIP analysis with antibodies against indicated FL-ThPOK (c, d) or Runx3 (e, f) for indicated sorted thymocyte (c, e) of LN T cell (d, f) subsets. Red and blue bars indicate WT and ThPOK^OB11/OB11 cells, respectively. Error bars represent standard deviations of 3 biological replicates. G) 3C assay-coupled with sorted peripheral CD4T cells from WT or ThPOK^OB11/OB11 mice. qPCR was performed to reveal interactions between indicated elements (lower panel). Primer positions relative to ThPOK enhancers and promoters are marked (blue arrows, Top panel).

Fig. 3.. Loss of anti-silencer destabilizes CD4 T cell phenotype.

a) Structure of ThPOK^GFP, ThPOK^ΔSil.GFP, and ThPOK^DOB11.GFP reporter alleles, showing insertion site of GFP-encoding exon (labeling of elements as in Fig. 1a). b) FACS analysis of CD4, and CD8a expression in total PBLs of WT mice and heterozygous ThPOK^ΔOB11.GFP/+ mice (top), and GFP expression of gated SP CD4 and CD8 population from PBLs of same mice (bottom rows). c) FACS analysis of CD4, and CD8a expression in gated TCRβ+ PBLs (top), or GFP expression of gated CD4^hi (green), CD4^lo/− (orange), CD4+ CD8+ (red), or CD8 PBLs (middle), of WT mice and indicated compound heterozygous reporter lines. Bottom row illustrates ThPOK alleles of indicated mouse. Red box represents silencer (thin black line shows extent of deletion); green and black boxes indicate whether allele expresses GFP or ThPOK mRNA. d) RNA was collected from freshly sorted CD4 T cells from ThPOK^GFP/+ (white), ThPOK^{ΔOB11.GFP/ΔOB11}(red), or ThPOK^GFP/ΔOB11 (blue) mice and probed for GFP mRNA expression by qPCR. e) Reconstitution of RAG−/− hosts with CD4^hi, CD4^lo and SP CD8 cells from ThPOK^{ΔOB11.GFP/ΔOB11} donor mice. FACS analysis showing sort gates for isolation of donor cells (upper left panel). FACS analysis of CD4 and CD8 expression by PBLs from reconstituted hosts at indicated time after transfer (next 3 columns). Plots at right indicate % of CD4^hi, CD4^lo/−, CD4+ 8+ (DP) and CD8 cells within gated TCRβ+ PBLs of mice reconstituted with indicated ThPOK^{ΔOB11/ΔOB11} donor cells, at each time point. Error bars represent standard deviations of 4 biological replicates. f) Sorted T cell population from indicated mice were stimulated with anti-TCRβ. RNA was prepared at different hour of probed for ThPOK mRNA expression by qPCR.

Fig. 4.. Ablation of anti-silencer leads to deregulation of CD4 T cell gene expression program.

a) Venn diagram illustrating intersection between gene subsets that are differentially expressed (according to RNA-seq analysis) between indicated T cell subsets from ThPOK^{ΔOB11/ΔOB11} mice and WT CD4 T cells. Number of genes that are differentially expressed for each subset is shown in brackets below subset name. Bar graph at bottom indicates number of 789 commonly misregulated (upward and downward arrow indicate up regulated and down regulated genes respectively) that are direct target of ThPOK and Runx3 or both ThPOK and Runx3, as indicated. b) Volcano plot illustrating gene expression differences between OB11 CD4^lo and WT CD4 T cells. The grey dots represent genes differentially expressed (adjusted P<0.05) between samples. Genes with the largest negative or positive standardized mean difference are marked. c) Heat map displaying hierarchical clustering of DEGs for indicated OB11 and WT T cell subsets. Analysis is restricted to the union of all 2769 genes differing in expression between any OB11 T cell subset and WT CD4 T cells (FDR 5%). Red indicates increased gene expression levels; blue indicates decreased levels. d-e) Heat maps displaying hierarchical clustering of DEGs for indicated OB11 and WT T cell subsets, that are commonly misregulated between any OB11 T cell subset and WT CD4 T cells (789 genes), and are targets of ThPOK alone (318 genes) (d) or both ThPOK and Runx3 (212 genes) (e) (FDR 5%). Red indicates increased gene expression levels; blue indicates decreased levels. f) Pie charts illustrating distribution of ThPOK binding sites within target DEGs that are direct target of ThPOK (right) or ThPOK+ Runx3 (left) g) Heat map displaying hierarchical clustering of DEGs for indicated ThPOK^{ΔOB11/ΔOB11} and WT T cell subsets. Analysis is restricted to the subset of 1042 DEGs whose expression differs between any ThPOK^{ΔOB11/ΔOB11} subset versus WT CD4 T cells, but NOT between WT CD4 and WT CD8 T cells, i.e. genes that are not CD8 like (FDR 5%). Red indicates increased gene expression levels; blue indicates decreased levels. h) Plots illustrating codistribution of ThPOK ChIP-seq peaks with other TF binding sites (defined by ChIP-seq). Same analysis was carried out for ThPOK peaks associated with Runx consensus motifs (bottom), not associated with Runx motifs (middle), or random genomic fragments, i.e. NOT bound by ThPOK (top).

Fig. 5.. Loss of anti-silencer function causes genome-wide change in chromatin accessibility.

a) Circular plot of mouse chromosomes. Outer 2 rings represent ATAC-seq peaks. Inner 2 rings represent RNA-seq peaks. Blue and red rings represent WT CD4 and OB11 CD4^lo T cells, respectively. b) Venn diagram indicating intersection between ATAC-seq peaks that are selectively open in WT CD4 and OB11 CD4^lo T cells and α-ThPOK ChIP-seq peaks, as indicated. c) Relative enrichment of TF binding sites associated with open chromatin region in WT CD4 T cells (top) or ThPOK^{ΔOB11/ΔOB11} CD4^lo T cells (bottom). d) Relative distribution of DACRs in ThPOK^ΔOB11/ΔOB1 CD4^lo (red) versus WT CD4 T cells (blue) with respect to gene organization (TSS = transcriptional start site). e) Venn diagram indicating intersection between DACRs that are selectively open in WT CD4 and ThPOK^{ΔOB11/ΔOB11} CD4^lo T cells, and Th cell associated super enhancers, as indicated. f) Heat maps showing relative expression of genes associated with 67 and 98 SEs that are selectively open in ThPOK^{ΔOB11/ΔOB11} CD4^lo versus WT CD4 T cell subsets, respectively (from panel e). g) Heat map showing top 250 regions with highest differential accessibility between WT CD4 and ThPOK^{ΔOB11/ΔOB11} CD4^lo T cells, filtered for genes that are also differentially expressed between these cell types (but NOT differentially expressed between WT CD4 and WT CD8 T cells) (left panel). Comparison of chromatin accessibility of these 250 regions with a T cell developmental accessibility panorama (Immgen) (right panel).

Fig. 6.. Ablation of anti-silencer promotes differentiation of Triple^lo Treg subset.

a) FACS analysis of Foxp3 versus CD4 (top row), PD-1 versus GITR (second row), CD4 versus CD25 (third row), or CD25 expression by gated GITR^loPD-1^lo or GITR^hiPD-1^hi LN (mesenteric) T cells. Bar graphs at right represent % of Tregs amongst total CD4 lymphocytes (top), or % of Triple^lo and Triple^hi cells amongst Foxp3+ CD4+ T cells, of ThPOK^ΔOB11/+ or ThPOK^{ΔOB11/ΔOB11} mice. b) RNA was collected from WT CD4⁺ Foxp3+T cells subsetted into Triple^hi, Triple^INT and Triple^lo subsets, as indicated, and assayed for ThPOK mRNA expression by qPCR. c) FACS analysis of Foxp3 versus CD25 (top row), PD-1 versus GITR (second row), CD4 versus CD25 (third, fourth rows), and CD73 versus FR4 expression for WT or ThPOK^{ΔOB11/ΔOB11} CD4 LN T cells following in vitro Treg polarization. Plots are for total population or gated subsets, as indicated. d) FACS analysis of Foxp3 versus forward scatter (FSC) (top row), CD4 versus CD8α (second row), and PD-1 versus GITR (bottom row) by total or gated Foxp3+ cLPLs or cIELs T cells, as indicated. Bar graphs at bottom represent % of Tregs amongst total population (left), or % of Triple^lo and Triple^hi cells amongst gated T cells, of ThPOK ^+/+ or ThPOK^{ΔOB11/ΔOB11} mice, as indicated. e) FACS analysis of CD4 versus CD8α (top row), or CD8α versus CD8β (second, third rows) expression, for either total TCRβ+ cells or indicated gated subset of freshly isolated iIEL, cIEL, iLPL or cLPL populations, as indicated. i-IEL =intestinal IEL; c-IEL = Colonic IEL; i-LPL = intestinal LPL, c-LPL = colonic -LPL.

Fig. 7.. Anti-silencer deficient CD4 T cells display anti-colitogenic activity.

a) Heat map displaying relative expression of colonic Treg signature genes for indicated ThPOK^{ΔOB11/ΔOB11} and WT T cell subsets. Red indicates increased gene expression levels; blue indicates decreased levels. b) Survival plot, c) Weight plot, and d) colitis severity score (based on colonic histology) after colitis induction with TNBS of indicated mouse strains. e) Weight plot of host RAG−/− mice after transfer of sorted naïve ThPOK^{ΔOB11/ΔOB11} or WT T cell subsets, as indicated. f) Similar analysis as in panel e, except all animals received a cotransfer of WT CD4 T cells. g) Bar graph indicating colitis severity score for same mice as in panel f, at 30 day timepoint. h) Histopathological analysis of colon at day 30, from same experiment as panel f. i) Bar graphs depicting proportions of CD45.1+ and CD45.2+ cells for indicated cell subset for each cotransfer at day 30 (same experiment as panel f). j) FACS analysis of PD-1 versus GITR expression, for gated CD45.1+ or CD45.2+ T cell subset, from cotransfer experiment (panel f). Bar graph at bottom indicates proportions of Triple^hi and Triple^lo cells within gated Foxp3+ Tregs. k) FACS analysis of CD4, CD8α and CD8β expression, for gated TCRβ+ cells, from co-transfer experiment (panel f) from different gut locations. l) Weight plot of host RAG−/− mice after transfer of sorted OX40ΔThPOK or WT CD4 T cells, as indicated. m) FACS analysis of CD8a versus Foxp3 expression, for indicated mice (right panels from cotransfer experiment in panel l). n) Bar graph indicating proportions of CD8aa and Foxp3+ cells among CD4+ IELs (same experiment as panel l). o) SMAD4 and ThPOK binding to the silencer were determined by ChIP assay performed using anti SMAD4 antibody and anti Flag antibody for ThPOK in anti CD3/CD28 stimulated CD4 T cells, cultured (48h) in presence and absence of 2ng/ml of TGFβ.