The transcription factor Runx3 promotes differentiation of naive CD4+ T cells into type-1 effector T (TH1) cells at the expense of TH2. TH1 cells as well as CD8+ T cells express a subset-specific Runx3 transcript from a distal promoter, which is necessary for high protein expression. However, all T cell subsets, including naive CD4+ T cells and TH2 cells, express a distinct transcript of Runx3 that is derived from a proximal promoter and that produces functional protein in neurons. Therefore, accumulation of RUNX3 protein generated from the proximal transcript needs to be repressed at the posttranscriptional level to preserve CD4+ T cell capability of differentiating into TH2 cells. In this article, we show that expression of RUNX3 protein from the proximal Runx3 transcript is blocked at the level of translational initiation in T cells. A coding sequence for the proximal Runx3 mRNA is preceded by a nonoptimal context sequence for translational initiation, known as the Kozak sequence, and thus generates protein at low efficiencies and with multiple alternative translational initiations. Editing the endogenous initiation context to an “optimal” Kozak sequence in a human T cell line resulted in enhanced translation of a single RUNX3 protein derived from the proximal transcript. Furthermore, RUNX3 protein represses transcription from the proximal promoter in T cells. These results suggest that nonpermissive expression of RUNX3 protein is restricted at the translational level, and that the repression is further enforced by a transcriptional regulation for maintenance of diverse developmental plasticity of T cells for different effector subsets.

The RUNX family transcription factors regulate differentiation of various cell types, including T lymphocytes (1). During T cell differentiation, RUNX3 plays important roles in the development and function of CD8+ CTLs and CD4+ type-1 effector T (TH1) cells (28). RUNX3 directs the differentiation of MHC class I–selected thymocytes into the CD8+ T cell lineage via repression of ThPOK (9). RUNX3 activates expression of effector genes, Ifng, Eomes, and Gzmb, and directly represses Il4 (2, 6, 10, 11). Consistent with these requirements, high RUNX3 protein expression is detected specifically in CD8+ T cells and TH1 cells, but RUNX3 is absent or expressed at low levels in their counterparts, naive CD4+ T cells and TH2 cells (2, 3, 6, 7, 12).

Although RUNX3 protein is expressed at high levels specifically in CD8+ T cells and TH1 cells, expression of its mRNA is broadly detected in developing thymocytes and T cells (3, 8). Runx3 mRNA is transcribed from two distinct promoters (proximal and distal) (13). The Runx3 isoform transcribed from the distal promoter (Runx3d) is required and sufficient for prominent expression of RUNX3 protein in CD8+ T and TH1 cells (3, 12). In postselection thymocytes and naive CD4+ T cells, Runx3d is negatively regulated by ThPOK, a crucial lineage commitment factor for the Th cell lineage (12, 14, 15), resulting in specific expression of Runx3d in CD8+ T cells. In TH1 cells, Runx3d is upregulated in a T-BET–dependent manner (2), although it remains unknown how T-BET counteracts ThPOK-mediated repression of Runx3d. In addition to Runx3d, a Runx3 isoform transcribed from the proximal promoter (Runx3p) is expressed in all subsets of developing thymocytes, naive CD4+ T cells and TH2 cells, although protein expression derived from this transcript is substantially lower than that from Runx3d (3, 8). RUNX3 protein is also expressed in neural tissues and is essential for the development of TrkC+ proprioceptive neurons in dorsal root ganglia (DRG) (16, 17). Although germline deletion of the Runx3 gene results in perinatal lethality or growth retardation caused by defective axonal projection of proprioceptive neurons, mice specifically lacking Runx3d show normal survival without neurologic symptoms (12, 17, 18). These findings indicate that functional RUNX3 protein is expressed from Runx3p in TrkC+ proprioceptor neurons in DRG. Therefore, it remains unclear how RUNX3 protein expression from the proximal transcript is restricted in lymphocytes, although it is expressed at a requisite level in neurons. Because ectopic expression of an open reading frame of Runx3p in transgenic mice or retrovirally transduced cells allows T cells to express RUNX3p protein (3, 4), the low protein expression is caused by translational regulation rather than regulation via degradation of translated protein.

Ectopic expression of RUNX3 results in downregulation of CD4 in preselection CD4+ CD8+ double-positive thymocytes and impairs their subsequent development into the CD4+ T cell lineage due to inhibition of positive selection of MHC class II–restricted thymocytes (3, 4, 19). Furthermore, expression of RUNX3 protein in unpolarized CD4+ T cells results in skewed differentiation toward the IFN-γ+ TH1 lineage at the expense of TH2 differentiation (2, 5, 6). Therefore, expression of RUNX3 protein from Runx3p needs to be restricted in thymocytes and activated CD4+ T cells to assure normal development of CD4+ mature thymocytes and TH2 cells.

By using cell-number normalized quantitation of Runx3 transcript levels and measurement of translational efficiency, we show that translation of Runx3p is restricted because of lack of an efficient Kozak sequence for translational initiation. Introduction of an optimal Kozak sequence into the endogenous locus by genome editing enhanced the translation efficiency of RUNX3 in a human T cell line. Compared with T cells, DRG neurons express a substantially higher level of Runx3p, and thereby achieve the requisite level of RUNX3 protein. These results provide a unique mechanism by which transcription factor expression is regulated between different cell types.

Runx3dYFP and Runx3-flox (Runx3F) alleles were described previously (6, 12) and have been maintained in the C57BL/6 background. The Runx3F allele was crossed to Vav1-iCre (20) to delete Runx3 in all hematopoietic cells. C57BL/6 mice were purchased from the National Cancer Institute and Charles River and used as control. All mice were maintained in a specific pathogen-free facility at Washington University in St. Louis, and all experiments were performed according to a protocol approved by Washington University’s Animal Studies Committee.

Single-cell suspensions were prepared by mechanical disruption of spleens. The following mAbs were purchased from Biolegend: PerCP-Cy5.5– or allophycocyanin-conjugated anti-CD4 (RM4-5), PE-conjugated anti-CD25 (PC61), PE-Cy7–conjugated anti-CD8α (53-6.7), Pacific blue–conjugated anti-CD44 (IM7), allophycocyanin-conjugated anti-CD62L (MEL-14), FITC- or Alexa Fluor 647–conjugated anti-B220 (RA3-6B2), and PE-conjugated anti–IFN-γ (XMG1.2). For sorting of naive T cells, splenocyte samples were initially depleted of B220+ cells with Dynabeads Mouse Pan B (B220) magnetic beads (Life Technologies), stained with mAbs, and sorted as CD62L+CD44 CD4CD8+ naive CD8+ T cells or CD62L+CD44CD25CD8CD4+ naive CD4+ T cells on a FACSAria II (BD Biosciences). Purity of sorted populations was 95–99%. For intracellular cytokine staining, cells were restimulated for 5 h with 50 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of brefeldin A (5 μg/ml; Biolegend), fixed with 2% paraformaldehyde, made permeable with 0.03% saponin, and stained for IFN-γ in 0.3% saponin. Dead cells were excluded with staining with DAPI (Sigma) or Aqua LIVE/DEAD staining dye (Life Technology). Data were acquired with LSR Fortessa (BD Biosciences) and analyzed using FlowJo (Tree Star).

Naive T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Life Technologies) and 50 μM 2-ME in the presence of soluble anti-CD3 (0.1 μg/ml, 145-2C11; Biolegend) and anti-CD28 (1 μg/ml, 37.51; BioXCell) in multiwell tissue culture plates coated with rabbit Ab to hamster IgG (0855395; MP Biomedicals). CD4+ T cells were polarized to TH1 or TH2 as follows: for TH1 polarization, 20 ng/ml murine IL-12 (R&D) and 5 μg/ml anti–IL-4 (11B11; Biolegend); for TH2 polarization, 20 ng/ml murine IL-4 (eBioscience), 2 μg/ml anti–IL-12/23 p40 (C17.8; Biolegend), and anti–IFN-γ (XMG1.2; Biolegend). For nonpolarizing culture (TH0) of CD4+ T cells and CD8+ T cells, no cytokines or blocking Abs were added. Following culture for 3 d with TCR stimulation and polarization, the activated cells were rested for 3 d in the presence of IL-2 (25 U/ml; eBioscience). For retroviral transduction, viral supernatants were prepared by transfection of PlatE packaging cells (21) with TransIT 293 (Mirus Bio). After priming overnight under TH0 conditions, activated T cells were transduced by spin infection at 1,200 × g at 30°C for 90 min in the presence of 10 μg/ml polybrene (Sigma).

Cellular RNA was extracted with TRIzol (Life Technologies) and reverse-transcribed with qScript Supermix (Quanta Bio). DyNAmo ColorFlash SYBR green qPCR mix (Thermo Fisher) and a LightCycler 480 (Roche) were used for real-time quantitative RT-PCR (qRT-PCR). Quantities of transcripts were normalized to that of Gapdh mRNA unless specified otherwise. For quantification of cell-number normalized gene expression, 1 μl of 1:1000 dilution of ERCC (External RNA Controls Consortium) RNA Spike-In Control Mixes (Ambion) was added to cell lysates from 2 × 105 cells in TRIzol before RNA isolation and reverse transcription. The following primers were used: ERCC-00108, 5′-CTATCAGCTTGCGCCTATTAT-3′ and 5′-GTTGAGTCCACGGGATAGAGTC-3′; Gapdh, 5′-CTCACAATTT CCATCCCAGACC-3′ and 5′-CATCAATGGTGCAGCGAACTTTATTG ATG-3′; total Runx3, 5′-AGTGGGCGAGGGAAGAGTTTC-3′ and 5′-GCCTTGGTCTGGTCTTCTATCT-3′; distal Runx3, 5′-CAAAACAGCAGCCAACCAAGT-3′ and 5′-AGATGCTGTTGGAAGCCATGT-3′; proximal Runx3, 5′-CGTATTCCCGTAGACCCGAG-3′ and 5′-AGGGGAAGGCCGTGGAG-3′; firefly luciferase, 5′-CGCAGGTGTCGCAGGTCTTC-3′ and 5′-CCGTCATCGTCTTTCCGTGCTC-3′; human GAPDH, 5′-CCAGCAAGAGCACAAGAGGAAGAG-3′ and 5′-AGGAGGGGAGATTCAGTGTGGTG-3′; human RUNX3, 5′-CTCAGCACCACAAGCCACTT-3′ and 5′-GGGTCGGAGAATGGGTTCAG-3′; human RUNX3 3′ untranslated region (UTR), 5′-TCAAGACCAGTGATGGGCCG-3′ and 5′-GGAGCGCAGGTCCCATTC-3′; human RUNX3p, 5′-TATTCCCGTAGACCCAAGCACC-3′ and 5′-CCGGGGAGGGAGGTGTGA-3′.

Cell extracts were lysed cells for 10 min at 4°C in radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS [NaDodSO4]) with a protease inhibitor mixture (P8340; Sigma). Lysates were cleared by centrifugation at 21,000 × g for 10 min at 4°C. Lysates from equal numbers of cells were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride membranes (GE Healthcare). The membranes were incubated with primary Abs (identified later), followed by detection with an HRP-conjugated Ab against rabbit Ig L chain (211-032-171; Jackson Immunoresearch) and a Luminata HRP substrate (Millipore). Rabbit anti-RUNX3 mAb (9647, clone D6E2), which recognizes a peptide containing alanine at position 40 of human RUNX3, and rabbit anti-HDAC1 (ab7028) polyclonal Ab were purchased from Cell Signaling Technology and Abcam, respectively. Protein expression was quantitated with an ImageJ program (National Institutes of Health).

A coding sequence (CDS) of firefly luciferase ligated with a 5′ UTR and/or Kozak sequences of Runx3d or Runx3p was inserted into a murine stem cell virus–based retroviral backbone containing ires EGFP. The 1200M thymoma cells (22) were spin-infected as described earlier and were harvested for analyses 48 h postinfection. The translational efficiency of the luciferase was assessed by calculating ratios of luciferase activity to a quantity of the luciferase transcript determined by qRT-PCR.

A guide RNA targeting the sequence around the canonical initiation codon of RUNX3p (5′-TACGGGAATACGCATAACAG-3′) was designed using the CRISPR design tool (23) and inserted into pQCiG (24). A single-strand oligonucleotide, which contains gccaccATGGC flanked by 60-nt homology sequences on both sides was synthesized (Sigma) and used as a homology-directed repair template. A total of 5 × 105 Jurkat cells in 10 μl R buffer (supplied as part of the Neon Transfection System, Life Technologies) were mixed with 1 μg pQCiG containing the guide RNA and 20 pmol single-strand oligonucleotide, and electroporated using the Neon Transfection System (Life Technologies) under the following conditions: pulse voltage, 1400 V; pulse width, 10 ms; and pulse number, 3. GFP+ cells were single-cell sorted 2 d after electroporation. Correctly edited clones were screened for by genomic PCR using primers (TTCTGCTTTCCCGCTTCTCGCGGCAG and AGCACGTCCACCATCGAGCGCA CCTC) located outside of the homology regions, followed by NcoI digestion.

The p values were calculated with an unpaired two-tailed Student t test for two-group comparisons and by one-way ANOVA for multiple-group comparisons with the Tukey post hoc test in Prism 6 software (GraphPad). The p values <0.05 were considered significant.

To determine whether the cell-type–specific expression of RUNX3 is regulated at a translation level, we measured protein-to-transcript ratios by quantitating cell-number normalized levels of RUNX3 protein and transcripts. As a control, we used cells from Runx3dYFP/YFP mice, in which the first exon for Runx3d was replaced with an EYFP reporter followed by a polyadenylation sequence (12). As a result, all Runx3 protein in cells from these animals is derived exclusively from Runx3p.

As previously reported (3, 7), expression of full-length 48-kDa RUNX3 protein was detected at high levels specifically in CD8+ T and TH1 cells (Fig. 1A). Expression of the 48-kDa product was also detected at an intermediate level in TH0 cells, whereas its expression in TH2 cells was substantially lower compared with other subsets. Expression of 48-kDa RUNX3 was severely reduced in CD8+ T cells, TH0, and TH1 cells derived from Runx3dYFP/YFP mice, indicating that a large proportion of this full-length product is derived from Runx3d, as previously demonstrated (12). In addition, all WT CD4+ T cell subsets expressed a form of RUNX3 protein that was ∼42 kDa in its molecular mass, as well as two additional 46- to 48-kDa isoforms, both at substantially lower amounts compared with the 42-kDa isoform. Because CD8+ T cells lacking Runx3d also expressed RUNX3 proteins of similar molecular masses, the observed products must be derived from Runx3p.

FIGURE 1.

Translation of RUNX3 protein from the endogenous Runx3p is inefficient. (A) Immunoblot analysis of RUNX3 protein in CD8+ T cells, CD4+ T cells, and DRG from wild-type (+) and Runx3dYFP/YFP (−) mice. Lysates of 1 × 105 cells for indicated populations were loaded per lane, and expression of RUNX3 (top) and HDAC1 (bottom, loading control) was detected. Data shown are representative of three experiments. (B) qRT-PCR quantitation of expression of total and promoter-specific Runx3 transcripts. cDNA from indicated cell subsets was subjected to qRT-PCR using primers indicated in the schematic (top). Levels of expression were normalized to those of “spiked-in” control RNA added in proportion to cell numbers before RNA preparation. Data are shown as mean and SD from three independent samples. (C) Ratios between RUNX3 protein and total Runx3 mRNA presented as a surrogate for translation efficiency in cells shown in (A) and (B). Values were calculated by dividing density of total Runx3 protein bands in (A) by levels of the total Runx3 transcript in (B) determined by qRT-PCR. Data are shown as mean and SD from three independent samples. *p < 0.05. n.d., not detectable.

FIGURE 1.

Translation of RUNX3 protein from the endogenous Runx3p is inefficient. (A) Immunoblot analysis of RUNX3 protein in CD8+ T cells, CD4+ T cells, and DRG from wild-type (+) and Runx3dYFP/YFP (−) mice. Lysates of 1 × 105 cells for indicated populations were loaded per lane, and expression of RUNX3 (top) and HDAC1 (bottom, loading control) was detected. Data shown are representative of three experiments. (B) qRT-PCR quantitation of expression of total and promoter-specific Runx3 transcripts. cDNA from indicated cell subsets was subjected to qRT-PCR using primers indicated in the schematic (top). Levels of expression were normalized to those of “spiked-in” control RNA added in proportion to cell numbers before RNA preparation. Data are shown as mean and SD from three independent samples. (C) Ratios between RUNX3 protein and total Runx3 mRNA presented as a surrogate for translation efficiency in cells shown in (A) and (B). Values were calculated by dividing density of total Runx3 protein bands in (A) by levels of the total Runx3 transcript in (B) determined by qRT-PCR. Data are shown as mean and SD from three independent samples. *p < 0.05. n.d., not detectable.

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Levels of total Runx3 transcript, as normalized to fixed cell numbers using exogenous control RNA, were similar among the subsets of activated T cells or between naive CD4+ and CD8+ T cells (Fig. 1B). High RUNX3 protein levels correlated with elevated expression of Runx3d and with T cell activation, whereas levels of the 42-kDa product was linked to Runx3p expression. Ratios between steady-state levels of RUNX3 protein and total Runx3 transcripts in WT or Runx3dYFP/YFP CD8+ T cells suggested a low translation efficiency of Runx3p in lymphocytes (Fig. 1C). RUNX3 protein was expressed in pooled DRG neurons predominantly from Runx3p and the protein-to-transcript ratio was similar to that of TH2 cells (Fig. 1A, 1C), even though only 10–15% of DRG neurons express Runx3 at an mRNA or protein level (25). Therefore, a subset of DRG neurons appeared to express Runx3p mRNA at substantially higher levels than T cells to achieve high RUNX3 protein expression. These results collectively suggest that translation of Runx3p is substantially less efficient compared with that of Runx3d.

Runx3p and Runx3d transcripts have distinct 5′ UTRs and sequence contexts for translation initiation (Kozak sequences) (26). To determine whether the 5′ UTR, Kozak sequences, or both contribute to translational restriction of Runx3, we first generated retroviral constructs, in which a Runx3 CDS was inserted downstream of the corresponding sequences from the Runx3d or Runx3p transcripts. These expression vectors were introduced into a thymoma cell, 1200M, which expresses only Runx3p and little endogenous Runx3 protein (Fig. 2A, 2B). The Runx3d construct was translated into a single 48-kDa protein. By contrast, Runx3p was translated into three proteins when expressed from a construct with its own 5′ UTR and Kozak sequence, recapitulating endogenous protein expression from Runx3p (Fig. 2B). Based on sequence predictions, translation of the Runx3p transcript could be initiated at two alternative nucleotide positions, −78 and +88, in addition to the canonical AUG (+1) that generates a 409-aa RUNX3 protein starting with an MPIPV sequence (Fig. 2A). The molecular masses of each product expressed from Runx3p corresponded to isoforms initiated at −78 (referred to as Runx3p [−26 to 409]), +1, and +88 (Runx3p [+30–409]) positions (Fig. 2B). Furthermore, consistent with endogenous protein patterns, the amounts of RUNX3 expressed from the Runx3p retroviral construct harboring its 5′UTR and Kozak sequence were lower than those of the Runx3d construct and Runx3p expression constructs with an optimal Kozak sequence (Fig. 2B). These data suggest that the restricted translation of Runx3p results from impaired translational initiation by 5′ UTR or Kozak sequence, which causes translational initiation at two additional alternative sites and reduced overall translation.

FIGURE 2.

Protein expression from Runx3p is restricted by translational initiation. (A) Schematic representation of alternative translational initiation sites of the Runx3p transcript. Numbers indicate positions (nucleotide) relative to the canonical AUG (+1) of Runx3p. (B) Immunoblot analysis of RUNX3 proteins expressed from retroviral constructs with or without their 5′UTRs and/or initiation context sequences (Kozak) in 1200M thymoma cell line (left). Lysates of CD8+ T cells from wild-type (B6), Runx3dYFP/YFP, and Runx3f/f: Vav1-iCre (Runx3Δ/Δ) mice were used as control (right). Asterisk indicates truncated RUNX3 protein from the Runx3Δ allele. Data are representative of two experiments. (C) Translational efficiency of a luciferase reporter retrovirally expressed with or without 5′UTR with an endogenous or optimal Kozak sequence in 1200M cells. (D) Translational efficiency of a luciferase reporter placed at the alternative initiation site at +87 in retrovirally transduced 1200M cells. Translational efficiencies in (C) and (D) were calculated by luciferase activities normalized by the luciferase mRNA levels determined by qRT-PCR. Data are normalized to a value of a control construct without a 5′UTR and with an optimal Kozak sequence and shown by mean and SD from three experiments. *p < 0.01, **p < 0.0001. D, distal; O, optimal Kozak sequence (gccacc); P, proximal.

FIGURE 2.

Protein expression from Runx3p is restricted by translational initiation. (A) Schematic representation of alternative translational initiation sites of the Runx3p transcript. Numbers indicate positions (nucleotide) relative to the canonical AUG (+1) of Runx3p. (B) Immunoblot analysis of RUNX3 proteins expressed from retroviral constructs with or without their 5′UTRs and/or initiation context sequences (Kozak) in 1200M thymoma cell line (left). Lysates of CD8+ T cells from wild-type (B6), Runx3dYFP/YFP, and Runx3f/f: Vav1-iCre (Runx3Δ/Δ) mice were used as control (right). Asterisk indicates truncated RUNX3 protein from the Runx3Δ allele. Data are representative of two experiments. (C) Translational efficiency of a luciferase reporter retrovirally expressed with or without 5′UTR with an endogenous or optimal Kozak sequence in 1200M cells. (D) Translational efficiency of a luciferase reporter placed at the alternative initiation site at +87 in retrovirally transduced 1200M cells. Translational efficiencies in (C) and (D) were calculated by luciferase activities normalized by the luciferase mRNA levels determined by qRT-PCR. Data are normalized to a value of a control construct without a 5′UTR and with an optimal Kozak sequence and shown by mean and SD from three experiments. *p < 0.01, **p < 0.0001. D, distal; O, optimal Kozak sequence (gccacc); P, proximal.

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To quantitatively assess contribution of the Runx3 5′ UTR and Kozak sequences in translation in T cells, we generated retroviral luciferase reporter constructs, containing a 5′ UTR and a Kozak sequence from Runx3d or Runx3p (Fig. 2C). The translational efficiency for luciferase transcripts was assessed by calculating ratios of luciferase activity to its transcript as determined by qRT-PCR of transduced cells (27). Consistent with our experiments using the Runx3 CDS, the translational efficiency of luciferase was reduced by 5-fold when luciferase was expressed in the presence of the 5′UTR and Kozak sequence from Runx3p compared with those from Runx3d (Fig. 2C). Although we observed a trend for reduced translation efficiency of luciferase with the Runx3p 5′UTR alone compared with the Runx3d 5′UTR, this property was significantly reduced when the proximal rather than the distal Kozak sequence was used (Fig. 2C). Furthermore, luciferase translation efficiency was also reduced in the context of Runx3p (+30–409) compared with that of the Runx3d 5′UTR and Kozak sequence (Fig. 2D), suggesting that translation of Runx3p from all three initiation sites was less efficient compared with that of Runx3d. These results collectively indicate that protein expression from Runx3p is compromised because of inefficient translational initiation.

To determine whether the inefficient Kozak sequence of Runx3p is the primary determinant of low translation efficiency and alternative initiation, we used the CRISPR/Cas9 system to edit this proximal element in Jurkat cells (Fig. 3A). We isolated clones with biallelic editing of the initiation context sequence into an optimal Kozak sequence (“edited”) and a clone with deletion of a 28-bp sequence containing the putative translation initiation site and a branch point for splicing of RUNX3d (“deleted”), as confirmed by restriction enzyme digestion of PCR products and DNA sequencing (Fig. 3B and data not shown). In Jurkat cells, the endogenous RUNX3 was expressed as proteins with two distinct molecular masses, 48 and 42 kDa (Fig. 3C). Although translation of RUNX3p is potentially initiated at an alternative CUG codon located 167 nucleotides upstream of the canonical AUG, this alternative product would be terminated by a stop codon after being translated into a 12-aa peptide even if it is expressed (data not shown). The deleted clone, therefore, served as a control for the 42-kDa isoform because RUNX3 protein could be expressed only from the alternative initiation site downstream of the canonical AUG of the RUNX3p transcript. In all four clones containing the edited Kozak sequence, RUNX3 protein was expressed exclusively as a 48-kDa protein (Fig. 3C), indicating that alternative translational initiation resulted from an inefficient Kozak sequence preceding the RUNX3p CDS, which may be poorly recognized by the initiation complex. The edited Kozak sequence increased the protein-to-mRNA ratios, surrogates for translational efficiency, by 3-fold (Fig. 3D). These data suggest that the low translational efficiency of RUNX3p resulted, at least in part, from the inefficient Kozak sequence upstream of its putative initiation codon. Contrary to our prediction, however, the levels of RUNX3p protein did not change substantially between the parental cells and the edited clones even though RUNX3p was expressed as a single protein from the edited Kozak sequence (Fig. 3C). Instead, levels of both total RUNX3 mRNA and RUNX3p in the edited Jurkat clones decreased by ∼3-fold (Fig. 3E, 3F). Consistent with this observation, levels of endogenous RUNX3 mRNA in Jurkat cells transduced with a RUNX3-expressing retrovirus were also reduced by 2.5- to 3-fold (Fig. 3G). These findings also suggest that expression of RUNX3 protein derived from RUNX3p is tightly regulated by an additional feedback mechanism at the mRNA level.

FIGURE 3.

Editing the translational initiation context of RUNX3p increases translation efficiency and prevents alternative initiation. (A) Strategy to edit the Kozak sequence of RUNX3p using the CRISPR/Cas9 technology in Jurkat cells. Locations of the primers used in (B), the guide RNA target sequence, and the protospacer adjacent motif (PAM) and a repair template sequence are shown. (B) Detection of homology-mediated repair by an NcoI digestion of the PCR product encompassing the targeted sequence. (C) Immunoblot analysis and qRT-PCR analysis of RUNX3 in parental Jurkat cells and edited clones. A clone with a deletion of a sequence including the canonical ATG was used as a negative control for RUNX3p (1–409). (DF) RUNX3 protein to transcript ratios (D) and RT-PCR analysis of the total RUNX3 (E) and RUNX3p transcripts (F) in parental Jurkat cells and edited clones. (G) RT-PCR analysis of endogenous RUNX3 expression in Jurkat cells infected with an empty or RUNX3 retrovirus. Data are shown by mean and SD from two experiments. *p < 0.001.

FIGURE 3.

Editing the translational initiation context of RUNX3p increases translation efficiency and prevents alternative initiation. (A) Strategy to edit the Kozak sequence of RUNX3p using the CRISPR/Cas9 technology in Jurkat cells. Locations of the primers used in (B), the guide RNA target sequence, and the protospacer adjacent motif (PAM) and a repair template sequence are shown. (B) Detection of homology-mediated repair by an NcoI digestion of the PCR product encompassing the targeted sequence. (C) Immunoblot analysis and qRT-PCR analysis of RUNX3 in parental Jurkat cells and edited clones. A clone with a deletion of a sequence including the canonical ATG was used as a negative control for RUNX3p (1–409). (DF) RUNX3 protein to transcript ratios (D) and RT-PCR analysis of the total RUNX3 (E) and RUNX3p transcripts (F) in parental Jurkat cells and edited clones. (G) RT-PCR analysis of endogenous RUNX3 expression in Jurkat cells infected with an empty or RUNX3 retrovirus. Data are shown by mean and SD from two experiments. *p < 0.001.

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To test the functionality of the RUNX3 isoforms translated from Runx3p, we placed a CDS corresponding to each downstream of the optimized Kozak element and they were retrovirally expressed in primary CD4+ T cells (Fig. 4A). Retroviral expression of Runx3d in CD4+ T cells cultured under neutral TH0 conditions increased the percentage of IFN-γ+ cells compared with empty virus-infected control. CD4+ T cells expressing each of the three Runx3p-derived isoforms also expressed IFN-γ at frequencies comparable with those of Runx3d-expressing CD4+ T cells. Next, to test the repressor activity associated with each Runx3 isoform, we retrovirally expressed these proteins in the AKR1 CD4+ CD8+ thymoma cell line and assessed downregulation of CD4 expression. As shown in Fig. 4B, Runx3d and all three alternatively initiated Runx3p isoforms induced CD4 downregulation with comparable efficiency. These results suggest that differences in protein sequences at the N terminus of RUNX3 had little effect on their functionality and that distinct contribution to gene regulation between Runx3d and Runx3p transcripts is primarily dependent on their expression levels.

FIGURE 4.

Alternatively initiated RUNX3 protein isoforms are functionally intact. (A) Intracellular staining for IFN-γ in CD4+ T cells transduced with retrovirus expressing each RUNX3 isoform after 6 d of culture under neutral conditions. Percentages of GFP+ IFN-γ+ and GFP+ IFN-γ cells in viable cells, as determined by LIVE/DEAD Aqua staining, are shown in representative plots from two independent experiments with more than two biological replicates each. Statistical analysis of percentages of IFN-γ+ cells in GFP+ cells is shown in the right panel with mean and SD. (B) Surface CD4 staining of the AKR1 thymoma cell line transduced with retrovirus expressing each RUNX3 isoform at 72 h postinfection. Percentages of GFP+ CD4+ and GFP+ CD4lo/− cells in DAPI viable cells are shown in representative plots from two independent experiments. Statistical analysis of percentages of CD4lo/− cells in GFP+ cells is shown in the right panel with mean and SD. *p < 0.0001.

FIGURE 4.

Alternatively initiated RUNX3 protein isoforms are functionally intact. (A) Intracellular staining for IFN-γ in CD4+ T cells transduced with retrovirus expressing each RUNX3 isoform after 6 d of culture under neutral conditions. Percentages of GFP+ IFN-γ+ and GFP+ IFN-γ cells in viable cells, as determined by LIVE/DEAD Aqua staining, are shown in representative plots from two independent experiments with more than two biological replicates each. Statistical analysis of percentages of IFN-γ+ cells in GFP+ cells is shown in the right panel with mean and SD. (B) Surface CD4 staining of the AKR1 thymoma cell line transduced with retrovirus expressing each RUNX3 isoform at 72 h postinfection. Percentages of GFP+ CD4+ and GFP+ CD4lo/− cells in DAPI viable cells are shown in representative plots from two independent experiments. Statistical analysis of percentages of CD4lo/− cells in GFP+ cells is shown in the right panel with mean and SD. *p < 0.0001.

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Regulation of RUNX3 protein levels is critical to CD4 versus CD8 and TH1 versus TH2 lineage decisions. Whereas high RUNX3 protein expression is essential for the development and function of CD8+ T cells and TH1 differentiation (2, 3, 58, 1012), expression of RUNX3 protein needs to be restricted during CD4+ T cell development and TH2 differentiation despite constitutive expression of Runx3 mRNA. Runx3 is expressed as two major transcripts originating from distinct promoter regions (3, 17). As we previously demonstrated, the high RUNX3 expression is dependent on transcription of Runx3d, which is efficiently translated into protein (12). By contrast, naive CD4+ T cells and TH2 cells express only Runx3p (3, 6), which is translated inefficiently. Our data suggested that the initiation context sequence of Runx3p contributes to low protein translation. Editing the endogenous Kozak sequence in a T cell line resulted in increased translation efficiency from the Runx3p transcript. Furthermore, levels of the Runx3p transcript are negatively regulated directly or indirectly by RUNX3 protein, which further provides tighter regulation of RUNX3 levels in these cell types. Even though the protein-to-transcript ratio increased by editing the Kozak sequence, levels of both RUNX3p and total RUNX3 mRNA were reduced, resulting in a modest change in the RUNX3 protein level. These results suggest that transcription of the proximal message is also regulated by a negative feedback loop, which keeps RUNX3 protein levels sufficiently low to maintain T cell plasticity for non-TH1 lineages. This interpretation is also consistent with increased Runx3p in the presence of reduced RUNX3 protein in Runx3dYFP/YFP CD8+ T cells and the decreased RUNX3 transcript in Jurkat cells expressing retroviral RUNX3. RUNX3 binds to a few regions in the Runx3 locus based on ChIP-seq analysis using CD8+ T cells (28). However, all binding sites were located in the distal promoter and its upstream region, whereas there is no binding site near the proximal promoter or downstream region. Therefore, it seems likely that the feedback repression of the proximal transcript is mediated through indirect mechanisms. However, we cannot rule out the possibility that reduced RUNX3p levels in the presence of RUNX3 protein result from cleavage or degradation of the transcript rather than diminished transcription. In contrast with T cells, DRG neurons express requisite levels of RUNX3 protein from the proximal transcript. This seems to be achieved by substantially higher levels of Runx3p in TrkC+ DRG neurons. Amounts of the Runx3p transcript may thus be regulated differently in neurons from lymphocytes possibly independent of the negative feedback mechanism.

In summary, the results from this study reveal a unique mechanism by which expression of a transcription factor is regulated at the posttranscriptional level and provide insights into lineage- or cell-type–specific transcription factor expression.

We thank Chun Chou and Kenneth M. Murphy for discussion, Sunnie Hsiung for technical assistance, Dan R. Littman (New York University) and Jerry Pelletier (McGill University) for providing reagents, and Chyi-song Hsieh and Eugene M. Oltz for critical reading of the manuscript.

This work was supported by National Institutes of Health Grant AI097244 (to T.E.), a grant from the Edward Mallinckrodt Jr. Foundation (to T.E.), and a fellowship from the National Research Foundation of Korea (2013R1A6A3A03058430 to B.K.).

Abbreviations used in this article:

CDS

coding sequence

DRG

dorsal root ganglia

qRT-PCR

quantitative RT-PCR

Runx3d

Runx3 isoform transcribed from the distal promoter

Runx3p

Runx3 isoform transcribed from the proximal promoter

TH1

type-1 effector T

UTR

untranslated region.

1
Collins
A.
,
Littman
D. R.
,
Taniuchi
I.
.
2009
.
RUNX proteins in transcription factor networks that regulate T-cell lineage choice.
Nat. Rev. Immunol.
9
:
106
115
.
2
Djuretic
I. M.
,
Levanon
D.
,
Negreanu
V.
,
Groner
Y.
,
Rao
A.
,
Ansel
K. M.
.
2007
.
Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells.
Nat. Immunol.
8
:
145
153
.
3
Egawa
T.
,
Tillman
R. E.
,
Naoe
Y.
,
Taniuchi
I.
,
Littman
D. R.
.
2007
.
The role of the Runx transcription factors in thymocyte differentiation and in homeostasis of naive T cells.
J. Exp. Med.
204
:
1945
1957
.
4
Grueter
B.
,
Petter
M.
,
Egawa
T.
,
Laule-Kilian
K.
,
Aldrian
C. J.
,
Wuerch
A.
,
Ludwig
Y.
,
Fukuyama
H.
,
Wardemann
H.
,
Waldschuetz
R.
, et al
.
2005
.
Runx3 regulates integrin alpha E/CD103 and CD4 expression during development of CD4-/CD8+ T cells.
J. Immunol.
175
:
1694
1705
.
5
Kohu
K.
,
Ohmori
H.
,
Wong
W. F.
,
Onda
D.
,
Wakoh
T.
,
Kon
S.
,
Yamashita
M.
,
Nakayama
T.
,
Kubo
M.
,
Satake
M.
.
2009
.
The Runx3 transcription factor augments Th1 and down-modulates Th2 phenotypes by interacting with and attenuating GATA3.
J. Immunol.
183
:
7817
7824
.
6
Naoe
Y.
,
Setoguchi
R.
,
Akiyama
K.
,
Muroi
S.
,
Kuroda
M.
,
Hatam
F.
,
Littman
D. R.
,
Taniuchi
I.
.
2007
.
Repression of interleukin-4 in T helper type 1 cells by Runx/Cbf beta binding to the Il4 silencer.
J. Exp. Med.
204
:
1749
1755
.
7
Sato
T.
,
Ohno
S.
,
Hayashi
T.
,
Sato
C.
,
Kohu
K.
,
Satake
M.
,
Habu
S.
.
2005
.
Dual functions of Runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes.
Immunity
22
:
317
328
.
8
Taniuchi
I.
,
Osato
M.
,
Egawa
T.
,
Sunshine
M. J.
,
Bae
S. C.
,
Komori
T.
,
Ito
Y.
,
Littman
D. R.
.
2002
.
Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.
Cell
111
:
621
633
.
9
Setoguchi
R.
,
Tachibana
M.
,
Naoe
Y.
,
Muroi
S.
,
Akiyama
K.
,
Tezuka
C.
,
Okuda
T.
,
Taniuchi
I.
.
2008
.
Repression of the transcription factor Th-POK by Runx complexes in cytotoxic T cell development.
Science
319
:
822
825
.
10
Cruz-Guilloty
F.
,
Pipkin
M. E.
,
Djuretic
I. M.
,
Levanon
D.
,
Lotem
J.
,
Lichtenheld
M. G.
,
Groner
Y.
,
Rao
A.
.
2009
.
Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs.
J. Exp. Med.
206
:
51
59
.
11
Yagi
R.
,
Junttila
I. S.
,
Wei
G.
,
Urban
J. F.
 Jr.
,
Zhao
K.
,
Paul
W. E.
,
Zhu
J.
.
2010
.
The transcription factor GATA3 actively represses RUNX3 protein-regulated production of interferon-gamma.
Immunity
32
:
507
517
.
12
Egawa
T.
,
Littman
D. R.
.
2008
.
ThPOK acts late in specification of the helper T cell lineage and suppresses Runx-mediated commitment to the cytotoxic T cell lineage.
Nat. Immunol.
9
:
1131
1139
.
13
Levanon
D.
,
Groner
Y.
.
2004
.
Structure and regulated expression of mammalian RUNX genes.
Oncogene
23
:
4211
4219
.
14
Vacchio
M. S.
,
Wang
L.
,
Bouladoux
N.
,
Carpenter
A. C.
,
Xiong
Y.
,
Williams
L. C.
,
Wohlfert
E.
,
Song
K. D.
,
Belkaid
Y.
,
Love
P. E.
,
Bosselut
R.
.
2014
.
A ThPOK-LRF transcriptional node maintains the integrity and effector potential of post-thymic CD4+ T cells.
Nat. Immunol.
15
:
947
956
.
15
Wang
L.
,
Wildt
K. F.
,
Castro
E.
,
Xiong
Y.
,
Feigenbaum
L.
,
Tessarollo
L.
,
Bosselut
R.
.
2008
.
The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4+ T cells.
Immunity
29
:
876
887
.
16
Inoue
K.
,
Ozaki
S.
,
Shiga
T.
,
Ito
K.
,
Masuda
T.
,
Okado
N.
,
Iseda
T.
,
Kawaguchi
S.
,
Ogawa
M.
,
Bae
S. C.
, et al
.
2002
.
Runx3 controls the axonal projection of proprioceptive dorsal root ganglion neurons.
Nat. Neurosci.
5
:
946
954
.
17
Levanon
D.
,
Bettoun
D.
,
Harris-Cerruti
C.
,
Woolf
E.
,
Negreanu
V.
,
Eilam
R.
,
Bernstein
Y.
,
Goldenberg
D.
,
Xiao
C.
,
Fliegauf
M.
, et al
.
2002
.
The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons.
EMBO J.
21
:
3454
3463
.
18
Li
Q. L.
,
Ito
K.
,
Sakakura
C.
,
Fukamachi
H.
,
Inoue
Ki.
,
Chi
X. Z.
,
Lee
K. Y.
,
Nomura
S.
,
Lee
C. W.
,
Han
S. B.
, et al
.
2002
.
Causal relationship between the loss of RUNX3 expression and gastric cancer.
Cell
109
:
113
124
.
19
Kohu
K.
,
Sato
T.
,
Ohno
S.
,
Hayashi
K.
,
Uchino
R.
,
Abe
N.
,
Nakazato
M.
,
Yoshida
N.
,
Kikuchi
T.
,
Iwakura
Y.
, et al
.
2005
.
Overexpression of the Runx3 transcription factor increases the proportion of mature thymocytes of the CD8 single-positive lineage.
J. Immunol.
174
:
2627
2636
.
20
de Boer
J.
,
Williams
A.
,
Skavdis
G.
,
Harker
N.
,
Coles
M.
,
Tolaini
M.
,
Norton
T.
,
Williams
K.
,
Roderick
K.
,
Potocnik
A. J.
,
Kioussis
D.
.
2003
.
Transgenic mice with hematopoietic and lymphoid specific expression of Cre.
Eur. J. Immunol.
33
:
314
325
.
21
Morita
S.
,
Kojima
T.
,
Kitamura
T.
.
2000
.
Plat-E: an efficient and stable system for transient packaging of retroviruses.
Gene Ther.
7
:
1063
1066
.
22
Sawada
S.
,
Scarborough
J. D.
,
Killeen
N.
,
Littman
D. R.
.
1994
.
A lineage-specific transcriptional silencer regulates CD4 gene expression during T lymphocyte development.
Cell
77
:
917
929
.
23
Hsu
P. D.
,
Scott
D. A.
,
Weinstein
J. A.
,
Ran
F. A.
,
Konermann
S.
,
Agarwala
V.
,
Li
Y.
,
Fine
E. J.
,
Wu
X.
,
Shalem
O.
, et al
.
2013
.
DNA targeting specificity of RNA-guided Cas9 nucleases.
Nat. Biotechnol.
31
:
827
832
.
24
Malina
A.
,
Mills
J. R.
,
Cencic
R.
,
Yan
Y.
,
Fraser
J.
,
Schippers
L. M.
,
Paquet
M.
,
Dostie
J.
,
Pelletier
J.
.
2013
.
Repurposing CRISPR/Cas9 for in situ functional assays.
Genes Dev.
27
:
2602
2614
.
25
Yoshikawa
M.
,
Murakami
Y.
,
Senzaki
K.
,
Masuda
T.
,
Ozaki
S.
,
Ito
Y.
,
Shiga
T.
.
2013
.
Coexpression of Runx1 and Runx3 in mechanoreceptive dorsal root ganglion neurons.
Dev. Neurobiol.
73
:
469
479
.
26
Kozak
M.
1991
.
Structural features in eukaryotic mRNAs that modulate the initiation of translation.
J. Biol. Chem.
266
:
19867
19870
.
27
Lee
M. S.
,
Kim
B.
,
Oh
G. T.
,
Kim
Y. J.
.
2013
.
OASL1 inhibits translation of the type I interferon-regulating transcription factor IRF7.
Nat. Immunol.
14
:
346
355
.
28
Lotem
J.
,
Levanon
D.
,
Negreanu
V.
,
Leshkowitz
D.
,
Friedlander
G.
,
Groner
Y.
.
2013
.
Runx3-mediated transcriptional program in cytotoxic lymphocytes.
PLoS ONE
8
:
e80467
.

The authors have no financial conflicts of interest.