Repression of the Transcription Factor Th-POK by Runx Complexes in Cytotoxic T Cell Development

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Science  08 Feb 2008:
Vol. 319, Issue 5864, pp. 822-825
DOI: 10.1126/science.1151844


Mouse CD4+CD8+ double-positive (DP) thymocytes differentiate into CD4+ helper-lineage cells upon expression of the transcription factor Th-POK but commit to the CD8+ cytotoxic lineage in its absence. We report the redirected differentiation of class I–restricted thymocytes into CD4+CD8 helper-like T cells upon loss of Runx transcription factor complexes. A Runx-binding sequence within the Th-POK locus acts as a transcriptional silencer that is essential for Th-POK repression and for development of CD8+ T cells. Thus, Th-POK expression and genetic programming for T helper cell development are actively inhibited by Runx-dependent silencer activity, allowing for cytotoxic T cell differentiation. Identification of the transcription factors network in CD4 and CD8 lineage choice provides insight into how distinct T cell subsets are developed for regulating the adaptive immune system.

The peripheral T cell repertoire is formed after developing thymocytes have undergone a series of developmental selection processes. CD4+CD8+ double-positive (DP) thymocytes undergo positive selection through T cell receptor (TCR) interaction with major histocompatibility complex (MHC) proteins. This gives rise to two functionally distinct subsets: CD4+CD8 helper and CD4CD8+ cytotoxic T cells. Cells expressing MHC class II–restricted TCRs differentiate into the helper lineage and cease CD8 expression, whereas cells expressing class I–restricted TCRs differentiate into the cytotoxic linage and silence CD4 expression (13). Recently, gain or loss of function of the BTB/POZ domain–containing zinc finger transcription factor, Th-POK, revealed that its expression is essential and sufficient for development of helper-lineage cells (4, 5).

Runx transcription factor complexes are composed of heterodimers for one of three Runx proteins and their obligatory non–DNA-binding partner, Cbfβ protein (6). Because of the embryonic or neonatal lethality of mice deficient for any of Runx family genes, we used the Cre/loxP-mediated conditional gene inactivation (7) to clarify Runx complex function in silencing of the Cd4 gene (8) and recently reported that the combined inactivation of Runx1 and Runx3 in DP thymocytes resulted in a dramatic loss of CD8+ T cells (9). Runx proteins possess a conserved Val-Trp-Arg-Pro-Tyr (VWRPY) motif at the C-terminal end, allowing the recruitment of the Groucho/TLE co-repressor proteins to their target genes (10, 11). To test whether VWRPY-dependent repression might be involved in the loss of CD8+ T cells, we introduced the Runx1Δ446 allele (12) that generates a mutant Runx1 protein lacking the VWRPY motif on a Runx3-deficient background (Runx3f/f:Cd4 mice) (13). A marked reduction of splenic CD8+ T cells in Runx1Δ446/Δ446:Runx3f/f:Cd4 mice (Fig. 1A and fig. S1) indicated that VWRPY-dependent repression by Runx1 was involved in the generation of CD8+ T cells. Because the leaky CD4CD8+ subset that escaped Cre-mediated recombination (9) was less apparent in Runx1Δ446/Δ446:Runx3f/f:Cd4 mice (Fig. 1A), we used these mice for further analyses.

Fig. 1.

Differentiation of class I–restricted cells into CD4+CD8 helper-like cells by loss of Runx complex function. (A) CD4 and CD8 expression in lymph node αβT cells from mice with indicated genotypes. (B) CD4 and CD8 expression in mature thymocytes and LN TCRβ+ T cells either in the presence (II+) or absence (II°) of I-A MHC class II molecules. (C) Cell numbers of mature thymocytes and splenocytes showing CD4+CD8 αβT cells in class II+ control mice (lane 1), class II° control mice (lane 2), class II+ Runx1Δ446446:Runx3f/f:Cd4 mice (lane 3), and class II° Runx1Δ446446:Runx3f/f:Cd4 mice (lane 4). Error bars indicate standard deviation. (D) Expression of CD154 at 42 hours after in vitro TCR stimulation of control CD4+, CD8+, and class I–restricted CD4+CD8 cells. Intracellular staining of IL-4 and IFN-γ analyzed at 6 hours after re-stimulation of cells that were cultured for 5 days after initial TCR stimulation. Numbers in the plots in (A), (B), and (D) indicate the percentage of cells in each quadrant or region.

Potentially, the loss of CD8+ T cells could occur either by a developmental block of class I–restricted cells or by a redirection of class I–restricted cells toward the CD4+CD8 lineage. To determine whether CD4+CD8 cells that emerge in Runx mutant mice are class II–restricted or redirected class I–restricted cells, we crossed Runx1Δ446/Δ446:Runx3f/f:Cd4 mice onto a MHC class II–deficient background (14). Although there was a marked decrease in CD4+CD8 T cell numbers in control class II–deficient mice, the predominance of CD4+CD8 T cells persisted in class II–deficient Runx1Δ446/Δ446:Runx3f/f:Cd4 mice in both the thymus and the periphery (Fig. 1, B and C). These results indicated that the absence of Runx complexes forced the majority of class I–restricted cells to differentiate into CD4+CD8 T cells.

We next examined the functional properties of these CD4+CD8 cells. One of the characteristic features of CD4+ helper-lineage T cells is the early induction of CD154, the ligand for CD40, after TCR stimulation (15) and the production of interleukin-4 (IL-4). These were observed in control CD4+ T cells as well as in class I–restricted CD4+CD8 cells, but not in control CD8+ T cells (Fig. 1D). In contrast, although high interferon-γ (IFN-γ) production was detected in control CD8+ T cells, it was absent in both wild-type CD4+ T cells and in class I–restricted CD4+CD8 cells (Fig. 1D). We conclude from these results that class I–restricted CD4+CD8 cells that develop in Runx mutant animals are functionally helper-like T cells.

Because ectopic expression of Th-POK has been shown to redirect class I–restricted cells to become CD4+CD8 cells (4, 5), we measured the expression of Th-POK in several Runx mutant mice, including a strain in which the Cbf β gene is conditionally inactivated by either a Lck-Cre or a Cd4-Cre transgene (13). Consistent with a previous report (4), Th-POK expression was not detected in control CD69 DP thymocytes. In contrast, a 40-fold increase in Th-POK transcript abundances was detected in CD69 DP thymocytes in which Runx complexes were disrupted either by combined Runx1 mutations with a Runx3 deficiency or by loss of Cbfβ protein (Cbfβf/f:Lck mice) (Fig. 2A). A modest Th-POK de-repression by inactivation of Runx1 alone indicated a redundant function of Runx3 in the repression of Th-POK.

Fig. 2.

De-repression of Th-POK by loss of Runx complex function. (A and B) Relative Th-POK expression abundances (normalized to hprt) in sorted CD69 DP thymocytes (A) from wild-type (lane 1), Runx1f/f:Cd4 (lane 2), Runx1Δ446/Δ446 (lane 3), Runx3f/f:Cd4 (lane 4), Runx1f/f:Runx3f/f:Cd4 (lane 5), Runx1Δ446/Δ446:Runx3 f/f:Cd4 (lane 6), Cbfβf/f:lck (lane 7), and Cbfβf/f:Cd4 (lane 8) mice and in CD4+ and CD8+ peripheral T cells in mice of the indicted genotype (B). One representative result out of three experiments is shown. Lane 9 in (A) indicates Th-POK expression in control CD4+CD8 SP thymocytes. (C) Relative Th-POK expression abundances after reconstitution of Runx complex function. Purified CD4+CD8 and CD4+CD8int cells from Cbfβf/f:Cd4 mice were transduced with control retroviral vector (GFP) or vector encoding Cbfβ (Cbfβ).

Although Th-POK mRNA was undetectable in control CD8+ T cells and in CD8+ T cells deficient for Runx1 or Runx3, it was present in Cbfβ-deficient CD4+CD8int T cells (Fig. 2B) that still developed in Cbf βf/f:Cd4 mice because of the slow turnover of Cbfβ protein after inactivation of the Cbf β gene (13). We therefore next examined whether Th-POK repression could be restored in these CD4+CD8int cells upon re-expression of Cbfβ protein. Purified CD4+CD8 and CD4+CD8int cells were transduced with a retroviral vector encoding Cbfβ or with an empty vector control. In these experiments, expression of Th-POK was markedly reduced upon re-expression of Cbfβ in CD4+CD8int cells, with no detectable effect in CD4+CD8 cells (Fig. 2C). These results suggest that Runx-mediated Th-POK repression operates in peripheral CD8+ T cells.

To understand mechanisms underlying Runx-mediated repression of Th-POK, we examined whether Runx complexes directly associate with the Th-POK locus. Using a ChIP-on-chip (ChIP indicates chromatin immunoprecipitation) approach with an antibody against Cbfβ2, we detected two regions occupied by Runx complexes within the Th-POK locus. Distal and proximal Runx-binding sequences (RBS-1 and RBS-2, respectively) are located ∼3.1 kb upstream and ∼7.4 kb downstream of exon Ia (Fig. 3A) and contain two or one conserved Runx motifs, respectively (Fig. 3A and figs. S2 and S3). By using ChIP analysis in T cell subsets, we confirmed an association between Runx complexes and these two regions (Fig. 3B). However, binding of Runx complexes to RBS-1 and RBS-2 was detected in both Th-POK–expressing and nonexpressing cells, revealing that the binding of Runx complexes to these regions did not correspond with Th-POK repression.

Fig. 3.

Identification and characterization of RBSs at the Th-POK locus. (A) The structure of the murine Th-POK locus is shown at the top. Circles represent putative Runx motifs, with those in red indicating evolutionarily conserved Runx motifs. Black boxes represent exons, and each green bar represents the signal intensity of an individual oligonucleotide probe in a ChIP-on-chip experiment. Blue boxes represent RBSs. The maps for each reporter transgene construct (Tg-a to Tg-g) are indicated. The restriction sites shown are Eco47III (E47), EcoRV (RV), HindIII (H), KpnI (Kp), and XhoI (X). (B) ChIP experiment showing binding of Runx complexes to RBS-1 and RBS-2 in the indicated cell subsets. The regions at 1 kb upstream of exon Ia (UP1) and the TCRβ enhancer (TCRβ) were used as negative and positive controls, respectively. (C) Histograms showing the GFP expression in the indicated T cell subsets from representative transgenic founder for each construct. The dashed line indicates nontransgenic littermate control. Numbers in the histogram indicate the percentage of GFP+ cells, and numbers in parenthesis indicate mean fluorescent intensity of GFP in GFP+ cells. The numbers of transgenic founders expressing GFP among the total transgenic founders are indicated at right.

To better understand the functional activities of RBS-1 and RBS-2 in light of these results, we performed transgenic reporter assays. A 15.5-kb genomic fragment encompassing the RBSs and exons Ia and Ib was linked to a green fluorescent protein (GFP) reporter transgene cassette (Tg-a in Fig. 3A). In all transgenic mouse founders obtained with Tg-a, GFP expression was first detected in postselection CD4+CD8int thymocytes and was up-regulated in CD4+ SP thymocytes, remaining high in splenic CD4+ T cells, whereas it was almost undetectable in splenic CD8+ T cells (Fig. 3C). The 15.5-kb fragment thus contains the major cis-regulatory regions that direct expression of Th-POK in the helper lineage.

To further narrow down the critical Th-POK regulatory regions, we deleted either 5′ or 3′ sequences as well as RBS-1 from the 15.5-kb fragment. Whereas RBS-2 (fig. S3) was found to be required for positive transcriptional regulatory activity (as in the Tg-c and Tg-d constructs), deletion of a 674-bp fragment of RBS-1 (Tg-b) resulted in GFP expression both in CD4+ helper-lineage and in CD8+ cytotoxic-lineage cells, indicating that RBS-1 is a transcriptional silencer required to repress the reporter gene in CD8 lineage cells. Efficient repression of GFP in CD8+ T cells by a 562-bp fragment of RBS-1 (fig. S2) in the context of Tg-e construct required Runx motifs (Tg-f and Tg-g) (Fig. 3C), consistent with Runx-dependent activity of RBS-1 silencer.

To examine the physiological function of the RBS-1 silencer, we deleted the 674-bp KpnI-Eco47III sequences from the Th-POK locus by homologous recombination in embryonic stem (ES) cells (Fig. 4A and fig. S4). Deletion of RBS-1 from one Th-POK allele led to the loss of peripheral CD8+ T cells (Fig. 4B) and to the Th-POK de-repression in CD69 DP thymocytes (Fig. 4C). We further investigated Th-POK de-repression by using mice in which the coding sequence for Th-POK was replaced with the gfp gene (Th-POKGFP locus). GFP expression in mice heterozygous for Th-POKGFP allows us to examine expression of Th-POK at the single-cell level. Although GFP expression from the Th-POKGFP locus was not detected in CD69 DP thymocytes, deletion of RBS-1 (Th-POKGFP:SΔ locus in Fig. 4A) resulted in uniform de-repression of GFP in CD69 DP thymocytes, followed by high GFP expression in both helper- and cytotoxic-lineage mature thymocytes (Fig. 4D).

Fig. 4.

Essential requirement of the Th-POK silencer for development of CD8+ T cells. (A) Schematic structure of the Th-POK locus and targeted alleles Th-POKSΔ, Th-POKGFP, and Th-POKGFP:SΔ. Exons and loxP sequences are indicated as black boxes and black triangles, respectively. (B) CD4 and CD8 expression in lymph node αβT cells from wild-type (+/+), Th-POKSΔ heterozygous (SΔ/+), and Th-POK hemizygous transgenic (Th-POK Tg) mice. (C) Relative Th-POK expression abundances in sorted CD69 DP thymocytes showing derepression of Th-POK upon deletion of the Th-POK silencer. (D) GFP expression from the Th-POKGFP and Th-POKGFP:SΔ alleles in indicated thymocyte subsets. Dashed and bold lines indicate GFP expression in control mice and Th-POK+/GFP (+/GFP) or Th-POK+/GFP:SΔ (+/GFP:SΔ) mice, respectively. The numbers in parenthesis indicate mean fluorescent intensity of GFP in total CD69 DP thymocytes.

Our results reveal that helper lineage–specific expression of Th-POK is regulated by the RBS-1 silencer, whose activity depends on binding of Runx complexes. We therefore refer to RBS-1 as the Th-POK silencer (fig. S5). The association of Runx complexes with the Th-POK silencer in cells expressing Th-POK indicates that specificity of silencer activity is not regulated at the level of Runx complex binding. Additional molecules that interact with Runx factors bound to the Th-POK silencer may therefore have a central role in regulating Th-POK silencer activity.

The antagonistic interplay between primary lineage-determining factors is often observed when two opposing fates are induced in progenitor cells (16, 17). Th-POK was recently described as an inhibitor of Runx-dependent Cd4 silencer activity (18), consistent with an antagonistic interplay between these two factors. Identification of Th-POK and Runx complex target genes will help to further unravel the transcription factors network regulating lineage specification of DP thymocytes.

Uniform de-repression of Th-POK in CD69 DP thymocytes upon deletion of the Th-POK silencer indicates that silencer-mediated Th-POK repression operates in all pre-selection DP thymocytes. It is therefore possible that TCR signals after engagement of MHC class II result in antagonism of Th-POK silencer activity and thus induce Th-POK expression. Given that sustained class II–specific TCR signals are thought to be necessary for specification of the helper lineage (1921), reversal of silencer-mediated Th-POK repression may require class II–specific TCR signals during a specified time window. Our results suggest that a mechanism regulating Th-POK silencer activity acts as a sensor to distinguish qualitative differences in TCR signaling. Further studies on the regulatory pathways of Th-POK repression will shed light on how signals initiated by external stimuli are converted into genetic programs in the cell nucleus.

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Material and Methods

Figs. S1 to S5


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