Expression of the IL-7R α-chain (IL-7Rα) is strictly regulated during the development and maturation of lymphocytes. Glucocorticoids (GC) have pleiotypic effects on the growth and function of lymphocytes. Although GC have been reported to induce the transcription of IL-7Rα gene in human T cells, its molecular mechanism is largely unknown. In this study, we show that GC up-regulate the levels of IL-7Rα mRNA and protein in mouse T cells. This effect does not require protein synthesis de novo, because protein synthesis inhibitors do not block the process. Mouse IL-7Rα promoter has striking homology with human and rat, containing consensus motifs of Ikaros, PU.1, and Runx1 transcription factors. In addition, a conserved noncoding sequence (CNS) of ∼270 bp was found 3.6-kb upstream of the promoter, which was designated as CNS-1. A GC receptor (GR) motif is present in the CNS-1 region. Importantly, we show by reporter assay that the IL-7Rα promoter has specific transcription activity in T cells. This activity highly depends on the PU.1 motif. Furthermore, GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We also demonstrate that GR binds to the GR motif by EMSA. In addition, by chromatin immunoprecipitation assay, we show that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. These results demonstrate that GR binds to the GR motif in the CNS-1 region after GC stimulation and then activates the transcription of the IL-7Rα promoter. Thus, this study identifies the IL-7Rα CNS-1 region as a GC-responsive element.

Interleukin-7 is an essential cytokine for development of early lymphocytes and maintenance of mature lymphocytes. At early stages, it controls proliferation of T and B cell precursors, positive selection of CD8+ thymocyte (1), and the recombination accessibility of the TCRγ and IgH loci (2, 3, 4). At later stages, IL-7 supports survival and homeostatic proliferation of naive and memory T cells (5). IL-7 exerts its effect through interaction with the IL-7R, consisting of a unique α-chain (IL-7R α-chain (IL-7Rα)3) (6) and common cytokine receptor γ-chain (7, 8). The IL-7Rα also heterodimerizes with the unique thymic stromal-derived lymphopoietin receptor chain (9, 10). IL-7 binding to the IL-7R activates the JAK-1 and -3, which then activate STAT5 (11), phosphoinoside-3 kinase, Ras, and MAPK/ERK.

Expression of the IL-7Rα is strictly regulated during the development and maturation of lymphocytes. It is induced at the stage of common lymphoid progenitors and maintained during whole T cell life except at two stages: CD4+8+ thymocytes and activated T cells (12, 13, 14). In B cell lineage, in contrast, the IL-7Rα is down-regulated at the pre-B cell stage and kept off throughout the later stages (15). It is supposed that the IL-7Rα is actively down-regulated not to transmit unnecessary survival signals, when T cells have to decide their fate solely by the affinity between their TCR and the peptide-MHC complex. Therefore, the strict control of IL-7Rα expression probably plays an important role in the regulation of selection and immune response. However, the molecular mechanism of IL-7Rα expression is largely unknown except for reports that an Ets family transcription factor, PU.1, can bind to a motif in the IL-7Rα promoter in mouse pro-B cells (16) and that GA binding protein binds to the same motif and is essential in the transcription of IL-7Rα promoter in T cells (17).

Glucocorticoids (GC), immunosuppressive and anti-inflammatory agents, have pleiotypic effects on the growth, differentiation, and function of lymphocytes (18, 19). GC bind to the GC receptor (GR) in cytoplasm, and GR subsequently undergoes nuclear translocation. GR exercises its function either through association with GR binding motif in the promoters or through interaction with other signal molecules and transcription factors. A typical GR motif consists of two six-base palindrome sequences interrupted by a three-base spacer (TGTACANNNTGTTCT). However, GR can also bind to half-palindrome motifs (20, 21). GR regulates the function of T cells either positively or negatively. For example, GC inhibit transcriptional up-regulation of T cell-derived cytokines, such as IL-2, IL-4, and IFN-γ by inhibiting AP-1 (22) and NF-κB (23). Administration of GC results in apoptosis of immature thymocytes and T cell hybridomas (18). In contrast, GC block activation-induced apoptosis by TCR stimulation, through inducing GC-induced leucine zipper gene (24) and GC-induced TNFR family-related gene (25). In addition, it has also been reported that GC induce the expression of the IL-7Rα in human peripheral T cells (14), which may block activation-induced apoptosis. Based on these findings of mutual inhibition between TCR and GC, the role of GC has received much attention in T cell development.

To identify the molecular mechanism of the induction of IL-7Rα by GC, we dissected the mouse IL-7Rα promoter and characterized the molecular mechanism of transcription of the IL-7Rα gene. In this study, we demonstrated that GC positively induce the expression of IL-7Rα in mouse T cells and that ligand-activated GR binds to a GR motif in a conserved noncoding sequence (CNS) upstream of the IL-7Rα promoter, which was designated as CNS-1, and then activates the transcription of the IL-7Rα promoter. Thus, this study explores the molecular mechanism of IL-7Rα induction by GC and demonstrates that the CNS-1 region is a GC-responsive element of the IL-7Rα locus.

Total splenic cells from C57BL/6 mice aged 6–8 wk were cultured in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-ME. A mouse pro-B cell line, 38B9 (26), was maintained in IMDM medium supplemented with 10% FBS and 50 μM 2-ME. A mouse immature thymocyte line, KKF (27), was maintained in RPMI 1640 medium supplemented with 10% FBS and 50 μM 2-ME. Before dexamethasone (Dex) treatment, cells were cultured with the medium containing 5% charcoal-stripped FBS (MultiSer) for overnight. Dex and cycloheximide (Sigma-Aldrich) were dissolved in ethanol, and diluted in the medium. Puromycin (Sigma-Aldrich) was dissolved in water. The cells were treated with 10−7 M Dex, 40 μg/ml cycloheximide, or 200 μg/ml puromycin. FACS analysis was performed as previously described (2). Cells were stained with either biotin-anti-mouse IL-7Rα (eBioscience) or isotype-matched biotin-rat IgG2a (BD Biosciences), followed by streptavidin-PBXL-3 (Martek). In total splenic T cells, FITC-anti-CD3 (eBioscience) and PE-anti-B220 (eBioscience) were also added. Viable cells were analyzed by a FACSCalibur with CellQuest software, version 3.1 (BD Biosciences). Dead cells were excluded from the analysis by forward and side scatter and propidium iodide gatings.

The transcription initiation sites were determined by cloning and sequencing the full-length IL-7Rα cDNA from mouse spleen T cells with GeneRacer kit (Invitrogen Life Technologies). Nucleotide sequence of the IL-7Rα promoter was compared between mouse (GenBank accession no. NT_039618), human (GenBank accession no. NT_006576), and rat (GenBank accession no. NW_047622) by GENETYX-MAC, version 12.0, software (Software Development). DNA sequence motif search for putative transcription factor binding sites was performed with TESS and TFSEARCH web-based search programs.

Random-primed cDNA was amplified in triplicate using TaqMan Ribosomal RNA control reagents VIC probe or SYBR Green PCR Master Mix (Applied Biosystems) with 100 nM primers, for 40 cycles at 95°C for 30 s and 55°C for 30 s by ABI 7700 Sequence Detector (Applied Biosystems). The results were analyzed using the Sequence Detection System 1.7 software (Applied Biosystems). Serial dilution of 38B9 cDNA was used as a control for calibration of 18S rRNA and IL-7Rα mRNA. The level of IL-7Rα mRNA was normalized with that of rRNA. Sequences of the primers for the IL-7Rα are as follows: IL-7Rα, 5′-1, 5′-GGATGGAGACCTAGAAGATG-3′; IL-7Rα, 3′-1, 5′-GAGTTAGGCATTTCACTCGT-3′.

Mouse and human IL-7Rα genomic fragments were amplified by PCR. The IL-7Rα promoters (320 bp) were amplified and ligated into pGL3 vector (pGL3-IL-7Rαpr) (Promega) to generate IL-7Rα promoter-firefly luciferase reporter constructs. The IL-7Rα CNS-1 fragments (550 bp) were inserted into 3′ of luciferase gene of pGL3-IL-7Rαpr. A series of mutations was introduced using QuikChange site-directed mutagenesis kit (Stratagene). For mouse GR expression vector, GR cDNA was amplified by PCR and cloned into pcDNA3.1 vector (pcDNA3.1-mGR) (Invitrogen Life Technologies). Sequences of the primers for the IL-7Rα promoter and CNS-1 are as follows: mouse IL-7Rα promoter 5′-1, 5′-TTGTGATCCTGTTACATTGGACCC-3′; mouse IL-7Rα promoter 3′-1, 5′-AGAAAGAATAGAGAAGAAGCACGG-3′. Mouse IL-7Rα CNS-1 5′-1, 5′-GCAGTGCCATCCATGTCTGT-3′; mouse IL-7Rα CNS-1 3′-1, 5′-ACGTGTGATTCAACTTACAC-3′. Human IL-7Rα promoter 5′-1, 5′-TAGCCTCTAGCCTAAGATAG-3′; human IL-7Rα promoter 3′-1, 5′-CTCGGTCACACATACTTTAC-3′. Human IL-7Rα CNS-1 5′-1, 5′-AACTAGGTGGTTCTTCCTC-3′; human IL-7Rα CNS-1 3′-1, 5′-ACATGTGACTCGATTTATGG-3′. Site-directed mutagenesis was introduced as follows: Ikaros wild-type (GGGAA) into mutated (GAGAA) motif; PU.1 wild-type (CAGACTTCCTGTTT) into mutated (CAGACGTCGTGTTT) motif; Runx1 wild-type (TGTGGT) into mutated (TCTAAG) motif; GR wild-type in IL-7Rα CNS-1 (TGTTCTTTTACATCT) into mutated (CACTGCTTTGAGTGC).

Transfection was done by electroporation as described previously (28). KKF cells (1 × 107) were transiently transfected by electroporation with 30 μg of luciferase reporter constructs and 100 ng of phRL-TK plasmid (Promega) driven by HSV thymidine kinase promoter by electroporation at 950 μF and 350 V. Reporter gene analysis was performed 24 h after transfection. In cotransfection studies, mixtures also contained 10 μg of mouse GR expression vector (pcDNA3.1-mGR). Dex (10−7 M) was added at 18 h after transfection, and the cells were harvested at 36 h after transfection. The total amount of DNA was kept constant with pGL3-basic or pcDNA3.1 vector. Cell lysates were then subjected to Dual-Luciferase Reporter Assay System (Promega), and luciferase activities were measured with a luminometer (Lumat LB9507; Berthold). Firefly luciferase activity was normalized by Renilla luciferase activity. In each experiment, samples were analyzed in triplicate, and each experiment was repeated at least twice.

Nuclear extract was prepared as previously described (29), and protein amount was quantitated using the DC protein assay (Bio-Rad). Nuclear extract (5 μg) was incubated for 30 min at room temperature with 2 ng of labeled oligonucleotide probe in 24 μl of binding buffer (20 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 4 mM DTT, 50 mM NaCl, 0.05% BSA, 10% glycerol, and 0.125% Nonidet P-40) containing 2 μg of poly(dI:dC). Binding reactions were electrophoresed through 5% gel (19:1 acrylamide/bis acrylamide) in 0.5× TBE buffer at 4°C. For competition assay, the reaction was preincubated with 50-fold molar excess of cold oligonucleotide for 5 min before the addition of labeled oligonucleotide probe. For Ab inhibition assay, the nuclear extract was preincubated with 5 μg of normal rabbit IgG (Upstate Biotechnology), rabbit anti-GR Ab (P-20; Santa Cruz Biotechnology), or mouse anti-Stat5 Ab (Santa Cruz Biotechnology) for 1 h on ice. Radioactivity was quantitated using a Bio-image Analyzer (BAS1500; Fuji Film). The sequences of double-strand oligonucleotides used as probes were as follows: GR consensus motif (GRE), 5′-AGAGGATCTGTACAGGATGTTCTAGAT-3′; GRE-mutated motif (GRE-mut), 5′-AGAGGATCTCAACAGGATCATCTAGAT-3′; mouse IL-7Rα CNS-1 GR, 5′-CTTAACTTTGTTCTTTTACATCTTCACAAAC-3′; mouse IL-7Rα CNS-1 GR-mut, 5′-CTTAACTTCACTCTTTTGAGTCTTCACAAAC-3′; human IL-7Rα CNS-1 GR, 5′-CTTGGCTTTGTTCTTTTACATCTTCACAAC-3′; human IL-7Rα CNS-1 GR-mut, 5′-CTTGGCTTCACTCTTTTGAGTCTTCACAAC-3′. Consensus nucleotides are underlined. Mutated nucleotides are in boldface.

ChIP was performed as previously described (30). Briefly, KKF cells (1 ∼ 2 × 107) were fixed with formaldehyde for 5 min at room temperature. Soluble chromatin-containing DNA of 200- to 1000-bp length was immunoprecipitated with 5 μg of anti-GR Ab (PA1-512; Affinity Bioreagents) or normal rabbit IgG (Upstate Biotechnology) overnight at 4°C. Purified ChIP DNA was measured by real-time quantitative PCR using iTaq SYBR Green supermix with ROX (Bio-Rad). PCR condition was 95°C for 10 min, followed by 40 cycles consisting of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. After amplification, a melting curve analysis was performed to verify the specificity of the reaction. Serial dilution of sonicated genomic DNA of Ba/F3 cells was used as a control for calibration. The level of ChIP DNA was normalized with that of input DNA. In each experiment, samples were analyzed in triplicate. The oligonucleotide primers used to amplify mouse IL-7Rα CNS-1 are as follows: IL-7Rα GR 5′-12, 5′-CCATTGCTCACCCACAATCT-3′; IL-7Rα GR 3′-1, 5′-GCTATCACTCCATGGTGAAC-3′. For control, the chromatin region from 1622 to 1808 bp upstream of ATG codon, which does not include GR motifs, was amplified with the primers as follows: IL-7Rα GR 5′-10, 5′-TGTCCTACCTACAAGATGTG-3′; IL-7Rα GR 3′-10, 5′-TGCTAGGTGAAACCTCTCAG-3′.

The induction of IL-7Rα expression by GC was previously reported in human T cells (14). Therefore, we first checked whether GC up-regulate the level of IL-7Rα mRNA and protein in mouse spleen T cells. Freshly isolated total mouse spleen cells were cultured with 10−7 M Dex, and cell surface level of IL-7Rα on T cells was analyzed by flow cytometry. IL-7Rα expression was not changed at 2 and 6 h but slightly increased at 12 h by Dex treatment (∼1.5-fold at the peak fluorescence intensity) (Fig. 1,A and data not shown). To confirm whether GC induce IL-7Rα mRNA in mouse T cells, we analyzed the levels of IL-7Rα mRNA after Dex treatment by real-time quantitative PCR (Fig. 1 B). The IL-7Rα mRNA level started to increase within 1 h after Dex treatment, and soared up to 6-fold at 2 h. The level gradually decreased afterward. These results indicate that GC rapidly and transiently increase IL-7Rα mRNA in mouse peripheral T cells.

FIGURE 1.

GC induce IL-7Rα expression on mouse T cells. A, Induction of IL-7Rα expression in mouse spleen T cells by GC treatment. Mouse spleen T cells were cultured with or without 10−7 M Dex for 12 h, and stained with either biotin-anti-IL-7Rα (thick line) or isotype-matched biotin-IgG (dotted line) as control, followed by streptavidin-PBXL-3 and FITC-anti-CD3, then analyzed by flow cytometry. The CD3+ cells were gated, and their IL-7Rα expression is shown. B, Induction of IL-7Rα mRNA by GC treatment. Mouse spleen T cells were cultured with 10−7 M Dex for the indicated time course. Total RNA was extracted, and IL-7Rα mRNA was measured by real-time quantitative PCR. IL-7Rα mRNA level was normalized with 18S rRNA level. Data are the mean ± SE of triplicate data points from a representative experiment. C, Induction of IL-7Rα expression in a mouse T cell line by GC treatment. KKF cells were cultured with or without 10−7 M Dex for 16 h. The cells were stained with biotin-anti-IL-7Rα (thick line) or isotype-matched biotin-IgG (dotted line) as control, followed by streptavidin-PBXL-3, and analyzed by flow cytometry. D, Effect of protein synthesis inhibitor on induction of IL-7Rα mRNA by GC. KKF cells were preincubated with 40 μg/ml cycloheximide (CHX) or 200 μg/ml Puromycin (PURO) for 10 min before stimulation with 10−7 M Dex for the indicated time course. Total RNA was extracted, and IL-7Rα mRNA was measured by real-time quantitative PCR. IL-7Rα mRNA level was normalized with 18S rRNA level.

FIGURE 1.

GC induce IL-7Rα expression on mouse T cells. A, Induction of IL-7Rα expression in mouse spleen T cells by GC treatment. Mouse spleen T cells were cultured with or without 10−7 M Dex for 12 h, and stained with either biotin-anti-IL-7Rα (thick line) or isotype-matched biotin-IgG (dotted line) as control, followed by streptavidin-PBXL-3 and FITC-anti-CD3, then analyzed by flow cytometry. The CD3+ cells were gated, and their IL-7Rα expression is shown. B, Induction of IL-7Rα mRNA by GC treatment. Mouse spleen T cells were cultured with 10−7 M Dex for the indicated time course. Total RNA was extracted, and IL-7Rα mRNA was measured by real-time quantitative PCR. IL-7Rα mRNA level was normalized with 18S rRNA level. Data are the mean ± SE of triplicate data points from a representative experiment. C, Induction of IL-7Rα expression in a mouse T cell line by GC treatment. KKF cells were cultured with or without 10−7 M Dex for 16 h. The cells were stained with biotin-anti-IL-7Rα (thick line) or isotype-matched biotin-IgG (dotted line) as control, followed by streptavidin-PBXL-3, and analyzed by flow cytometry. D, Effect of protein synthesis inhibitor on induction of IL-7Rα mRNA by GC. KKF cells were preincubated with 40 μg/ml cycloheximide (CHX) or 200 μg/ml Puromycin (PURO) for 10 min before stimulation with 10−7 M Dex for the indicated time course. Total RNA was extracted, and IL-7Rα mRNA was measured by real-time quantitative PCR. IL-7Rα mRNA level was normalized with 18S rRNA level.

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Next, we checked whether IL-7Rα expression is augmented by GC treatment in mouse T cell lines. An IL-7Rα+ immature thymocyte line, KKF, was treated with 10−7 M Dex for 16 h, and the levels of IL-7Rα mRNA and protein were analyzed. Dex treatment induced a 4-fold increase of cell surface IL-7Rα expression (Fig. 1,C). IL-7Rα mRNA was also induced within 1 h after the addition of Dex, peaked at 2 h by ∼5-fold, and gradually decreased thereafter (Fig. 1 D). These results demonstrate that IL-7Rα mRNA is rapidly induced by GC not only in human but also in mouse T cells.

To test whether there is need for new protein synthesis during mouse IL-7Rα mRNA induction by GC, we used cycloheximide and puromycin, which inhibit protein synthesis by blocking the peptidyl synthesis activity of eukaryotic ribosome (Fig. 1 D). When cycloheximide and Dex were simultaneously added, Dex-mediated induction of IL-7Rα mRNA was not blocked. The treatment with cycloheximide alone did not change the IL-7Rα mRNA level. Similar results were obtained with puromycin. These observations are comparable with the previous report (14). These results indicate that GC directly induce the IL-7R mRNA, not through de novo protein synthesis.

Because IL-7Rα expression is regulated at the level of transcript, we next characterized cis-control elements of the IL-7Rα locus (Fig. 2). Another group previously reported a transcription initiation site 945 bp upstream of the translation initiation site (31). However, we could not detect any IL-7Rα transcripts in this region by RT-PCR analysis (data not shown). Therefore, we determined transcription initiation sites by isolating mouse full-length IL-7Rα cDNA from 5′-capped mRNA. IL-7Rα mRNA started from several sites within the region between 46 and 130 bp upstream of the translation initiation site (Fig. 2, A and B, arrows). One of these transcription start sites is very close to the major site reported previously (17). Search between mouse, human, and rat sequences revealed striking homology in the region spanning ∼320 bp from the translation initiation site. Percent homology was 75% for 197 bp between mouse and human. Consensus motifs of PU.1 and Runx1, indispensable transcription factors for development of hemopoietic stem cells and lymphocyte progenitors (32, 33), were conserved in this region. In addition, Ikaros motif was conserved between mouse and human.

FIGURE 2.

Comparison of cis-control elements in the mouse, rat, and human IL-7Rα loci. A, Schematic representation of cis-control elements of the mouse IL-7Rα locus. Open boxes indicate CNS-1 and the promoter. Closed box indicates the first exon. Horizontal arrows indicate the transcription initiation sites. B and C, The mouse IL-7Rα promoter (B) and CNS-1 (C) sequences, aligned with corresponding regions of rat and human. Asterisk (∗) indicates aligned identical nucleotides relative to the mouse sequence. Dashed line indicates a gap. The open boxes indicate the conserved transcription factor motifs and the ATG codon. Horizontal arrows indicate the transcription initiation sites.

FIGURE 2.

Comparison of cis-control elements in the mouse, rat, and human IL-7Rα loci. A, Schematic representation of cis-control elements of the mouse IL-7Rα locus. Open boxes indicate CNS-1 and the promoter. Closed box indicates the first exon. Horizontal arrows indicate the transcription initiation sites. B and C, The mouse IL-7Rα promoter (B) and CNS-1 (C) sequences, aligned with corresponding regions of rat and human. Asterisk (∗) indicates aligned identical nucleotides relative to the mouse sequence. Dashed line indicates a gap. The open boxes indicate the conserved transcription factor motifs and the ATG codon. Horizontal arrows indicate the transcription initiation sites.

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Because a CNS identified by cross-species sequence comparisons has much possibility to be a control element (34), we compared the DNA sequence upstream of the IL-7Rα promoter between mouse, rat, and human. In addition to the promoter, a CNS of ∼270 bp was identified at 3.6 kb upstream of the translation initiation site (Fig. 2, A and C). We designate this region as CNS-1. Percent homology was 86% for 300 bp between mouse and human. Consensus motifs of GATA, NF-κB, GR, and Evi-1 transcription factors were conserved in CNS-1.

To elucidate the molecular mechanism of transcriptional activation of the mouse IL-7Rα promoter, we first conducted reporter assay with the KKF cell line. A 320-bp fragment of IL-7Rα promoter region was cloned into a luciferase reporter vector. This plasmid DNA was transfected into KKF cells by electroporation. As shown in Fig. 3 A, the promoter showed specific transcription activity. In contrast, previously reported promoter (31) did not reveal any activity (data not shown).

FIGURE 3.

Transcriptional activation of the IL-7Rα promoter by GC. A, Transcriptional activation of the IL-7Rα promoter. Schematic illustration of reporter constructs with IL-7Rα promoter are shown on the left. The KKF cells were transfected with the mixture of the IL-7Rα promoter-luciferase reporter plasmid containing various mutations in binding motifs of transcription factors, and Renilla luciferase control vector. The cells were cultured for 24 h. Firefly luciferase activity in the whole-cell lysate was normalized with Renilla luciferase activity. Data are the mean ± SE of triplicate data points from a representative experiment. B, Transcriptional activation of the IL-7Rα promoter through the GR motif in the CNS-1 region by GC. Schematic illustration of reporter constructs with mouse and human IL-7Rα promoter and CNS-1 region are shown on the left. The KKF cells were transfected with the mixture of the IL-7Rα promoter and CNS-1 region luciferase reporter plasmid, mouse GR expression vector, and Renilla luciferase control vector. The cells were cultured for 18 h with 10−7 M Dex. Firefly luciferase activity was normalized with Renilla luciferase activity. Data are the mean ± SE of triplicate data points from a representative experiment.

FIGURE 3.

Transcriptional activation of the IL-7Rα promoter by GC. A, Transcriptional activation of the IL-7Rα promoter. Schematic illustration of reporter constructs with IL-7Rα promoter are shown on the left. The KKF cells were transfected with the mixture of the IL-7Rα promoter-luciferase reporter plasmid containing various mutations in binding motifs of transcription factors, and Renilla luciferase control vector. The cells were cultured for 24 h. Firefly luciferase activity in the whole-cell lysate was normalized with Renilla luciferase activity. Data are the mean ± SE of triplicate data points from a representative experiment. B, Transcriptional activation of the IL-7Rα promoter through the GR motif in the CNS-1 region by GC. Schematic illustration of reporter constructs with mouse and human IL-7Rα promoter and CNS-1 region are shown on the left. The KKF cells were transfected with the mixture of the IL-7Rα promoter and CNS-1 region luciferase reporter plasmid, mouse GR expression vector, and Renilla luciferase control vector. The cells were cultured for 18 h with 10−7 M Dex. Firefly luciferase activity was normalized with Renilla luciferase activity. Data are the mean ± SE of triplicate data points from a representative experiment.

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We next tested whether the conserved motifs are important for this transactivation. A series of mutations was introduced in the motifs of Ikaros, PU.1, and Runx1, and these reporter constructs were transfected into KKF cells. As shown in Fig. 3 A, mutations in the Runx1 motif slightly decreased the transcriptional activity of the IL-7Rα promoter. In contrast, the activity was significantly decreased with mutation of the PU.1 motif, suggesting that the PU.1 motif plays the major role in activation of the IL-7Rα promoter. We also obtained similar results in a pro-B cell line, 38B9, and a pre-B cell line, NSF 5.3 (data not shown). These results suggest that the PU.1 motif is important for the activity of the IL-7Rα promoter, and that the Runx1 motif may have positive effects.

Next, we checked whether the GR motif in the CNS-1 region plays an important role in transcriptional activation of the IL-7Rα promoter by GC stimulation. The reporter constructs of the mouse IL-7Rα promoter with or without the CNS-1 region were transfected with GR expression vector into KKF cells. After 18 h, the cells were cultured with Dex for 18 h. As shown in Fig. 3 B, Dex treatment only slightly increased the transcriptional activity without CNS-1, but greatly increased it by 3.6-fold with CNS-1. This induction by Dex was diminished in the construct with mutation in the GR motif in CNS-1. In addition, similar results were obtained with human counterpart promoter and CNS-1 region. These results indicate that GR, activated by GC, transactivates the IL-7Rα promoter through the GR motif in CNS-1. It is also suggested that the CNS-1 serves as a GC-responsive element in the IL-7Rα locus.

To test whether GR binds directly to the GR motif in the IL-7Rα CNS-1 region, we performed EMSA with IL-7Rα CNS-1 GR motif oligonucleotide (Fig. 4,A). Nuclear extract of Dex-treated KKF cells showed two kinds of DNA-protein complex with CNS-1 GR motif oligonucleotide probe, which probably represent monomer and dimer of GR as previously reported (35) (Fig. 4,B, lane 1, arrows). GC treatment did not change these binding activities (Fig. 4,B, lane 2). These activities were reduced by addition of the same GR motif oligonucleotide competitor, but not mutated oligonucleotide competitor (Fig. 4, B, lanes 3 and 4; C and D). Similar results were obtained with cold competitor oligonucleotide of human CNS-1 GR motif and GR consensus motif (Fig. 4, B, lanes 5–8; C and D). The binding activity was further confirmed by inhibition assay using an anti-GR Ab (Fig. 4, EG). Incubation with the anti-GR Ab resulted in reduced levels of binding activities, whereas a control anti-Stat5 Ab did not affect the activities. The anti-GR Ab probably inhibited the DNA binding of GR. These results, taken together, indicate that GR protein binds to the GR motif in the IL-7Rα CNS-1 region in vitro.

FIGURE 4.

GR binds to the GR motif of the IL-7Rα CNS-1 region. A, Oligonucleotide probes for EMSA. B–G, Nuclear extracts of KKF cells were incubated with labeled oligonucleotide probes and subjected to EMSA. Arrows indicate specific binding activity. For competition assay (B), the reaction was preincubated with 50-fold molar excess of cold oligonucleotide for 5 min before the addition of labeled oligonucleotide probe. For Ab inhibition assay (E), nuclear extract was preincubated with 5 μg of rabbit IgG, rabbit anti-GR Ab, or mouse anti-Stat5 Ab for 1 h before the addition of labeled oligonucleotide probe. C and D, and F and G, represent the relative GR binding activity in EMSA quantitated from the data in B and E, respectively. The levels of GR binding before Dex treatment (B and E, lanes 1) were arbitrarily defined as 100.

FIGURE 4.

GR binds to the GR motif of the IL-7Rα CNS-1 region. A, Oligonucleotide probes for EMSA. B–G, Nuclear extracts of KKF cells were incubated with labeled oligonucleotide probes and subjected to EMSA. Arrows indicate specific binding activity. For competition assay (B), the reaction was preincubated with 50-fold molar excess of cold oligonucleotide for 5 min before the addition of labeled oligonucleotide probe. For Ab inhibition assay (E), nuclear extract was preincubated with 5 μg of rabbit IgG, rabbit anti-GR Ab, or mouse anti-Stat5 Ab for 1 h before the addition of labeled oligonucleotide probe. C and D, and F and G, represent the relative GR binding activity in EMSA quantitated from the data in B and E, respectively. The levels of GR binding before Dex treatment (B and E, lanes 1) were arbitrarily defined as 100.

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Next, we examined whether GR is recruited to endogenous IL-7Rα CNS-1 chromatin after GC treatment by ChIP assay. KKF cells were cultured with Dex, harvested at various time points, fixed with formalin, lysed, and sonicated to prepare soluble chromatin. The chromatin fraction was immunoprecipitated with anti-GR or control Ab, and purified genomic DNA was measured with the primers for the IL-7Rα CNS-1 or control region by real-time quantitative PCR. As shown in Fig. 5,A, Dex treatment induced accumulation of GR at the IL-7Rα CNS-1 region. The recruitment peaked at 2 h, and diminished by 4 h. This time course of GR binding to the IL-7Rα promoter correlated well with that of IL-7Rα mRNA levels (Fig. 1, B and D). This recruitment of GR was not observed with control primers, which amplify a region ∼1.7 kb upstream of the translation initiation site (Fig. 5 B). These results demonstrate that GR is rapidly recruited to endogenous IL-7Rα promoter after GC stimulation.

FIGURE 5.

Recruitment of GR to endogenous IL-7Rα CNS-1 region by GC treatment. KKF cells were cultured with 10−7 M Dex for the indicated time. Soluble chromatin preparation was immunoprecipitated with either anti-GR Ab or control normal rabbit IgG. Purified ChIP and input DNA were analyzed by real-time quantitative PCR with the primers for the CNS-1 region (A) and a negative control region (B) of mouse IL-7Rα locus. The amount of ChIP DNA was normalized with that of input DNA. The mean value of control Ab before Dex treatment was arbitrarily defined as 1. Data are the mean ± SE of triplicate data points from a representative experiment.

FIGURE 5.

Recruitment of GR to endogenous IL-7Rα CNS-1 region by GC treatment. KKF cells were cultured with 10−7 M Dex for the indicated time. Soluble chromatin preparation was immunoprecipitated with either anti-GR Ab or control normal rabbit IgG. Purified ChIP and input DNA were analyzed by real-time quantitative PCR with the primers for the CNS-1 region (A) and a negative control region (B) of mouse IL-7Rα locus. The amount of ChIP DNA was normalized with that of input DNA. The mean value of control Ab before Dex treatment was arbitrarily defined as 1. Data are the mean ± SE of triplicate data points from a representative experiment.

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In this study, we first showed that GC up-regulate the levels of IL-7Rα mRNA and protein in mouse T cells. The induction of IL-7Rα mRNA does not require de novo protein synthesis, because protein synthesis inhibitors do not block the process. The mouse IL-7Rα promoter contains Ikaros, PU.1, and Runx1 consensus motifs, some of which are also conserved in rat and human. We also identified a highly conserved upstream region designated as CNS-1. The CNS-1 region contains a GR consensus motif. Importantly, we showed by reporter assay that the activity of the IL-7Rα promoter highly depends on the PU.1 motif and that GC treatment augments the transcriptional activity through the GR motif in the CNS-1 region. We further showed that GR binds to its motifs in the CNS-1 region and that GR is rapidly recruited to endogenous CNS-1 chromatin after GC stimulation. Thus, this study defines the IL-7Rα CNS-1 as a GC-responsive element, and provides further insights into the molecular mechanism of IL-7Rα induction by GC.

GC antagonize activation-induced cell death of T cells by TCR stimulation (18). Activation-induced apoptosis is caused by the up-regulation of Fas ligand expression (36). GC induce GC-induced leucine zipper gene, which then blocks the up-regulation of Fas ligand (24). GC-induced TNFR family-related gene may also participate in the antagonism (25). In contrast, TCR signaling transiently down-regulates expression of the IL-7Rα (13, 14), which probably ensures precise Ag-driven clonal expansion and subsequent activation-induced cell death. It is possible that GC may interfere with this regulation by directly inducing the transcription of IL-7Rα, as demonstrated by this study. Therefore, this can be one of the mechanisms how GC antagonize the activation-induced cell death of T cells.

GR knockout mice show normal intrathymic T cell development (37, 38). Therefore, it is probable that IL-7Rα expression in steady state does not require GR signaling. Because the PU.1 motif plays a significant role in the activation of the IL-7Rα promoter, Ets family transcription factors such as GA binding protein are probably involved in the GR-independent mechanism (17). Therefore, GR-mediated mechanism may be overestimated in our reporter assay. Or, in another way, it can be that the significance of the GR-mediated mechanism of IL-7Rα induction may be different between early and late stages of T cell development.

The PU.1 motif plays a significant role on the activation of the IL-7Rα promoter. PU.1 is critical for hemopoietic stem cell maintenance, myeloid and B cell lineage development, and may also support pro-T cell generation (39, 40, 41). PU.1−/− fetal hemopoietic progenitors fail to express IL-7Rα transcripts. In addition, it has been reported that PU.1 is bound to the same motif in the IL-7Rα promoter and that a multimerized binding site representing this sequence can stimulate transcription of a reporter gene in pro-B cells (16). Our observation that the IL-7Rα promoter activity highly depends on the PU.1 motif is comparable with these results. In the T cell lineage, however, PU.1 expression is severely reduced after the pro-T cell stage, and constitutive expression of PU.1 prevents fetal precursors from T cell development (42). Very recently, it has been reported that GA binding protein, a member of Ets transcription factor family, binds to this PU.1 motif and is essential in the transcription of IL-7Rα promoter in T cells (17).

Ikaros and Runx1 are involved in early hemopoietic and lymphoid development. Our result suggests that Runx1 may positively regulate the IL-7Rα promoter. Runx1 is expressed not only in hemopoietic progenitors and myeloid cells, but also in T cells in the thymus and spleen (33). Indeed, Runx1 is known to play a role in single-positive thymocytes and naive T cells (43), where IL-7Rα is highly expressed. Thus, it is possible that Runx family transcription factors may positively control the IL-7Rα promoter.

In this study, we characterized the IL-7Rα promoter and identified the molecular mechanism of induction of IL-7Rα gene by GC. Based on our data, GR is recruited to the GR motif of the CNS-1 region by GC treatment, and induces the transcription of the IL-7Rα promoter. This study indicates that the CNS-1 region is a GC-responsive element of the IL-7Rα locus.

We thank Dr. K. Igarashi for the 38B9 cell line, and Drs. T. Saito and A. Takeuchi for the KKF cell line; S. Hayashi and S. Kamioka for excellent technical assistance; members of K. Ikuta’s lab for discussion; and Drs. J. Bodor and N. Begum for critically reading the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This study was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by a grant provided by the Mochida Memorial Foundation for Medical and Pharmaceutical Research. H.-C.L. was supported by a Japan Society for the Promotion of Science postdoctoral fellowship for foreign researchers.

3

Abbreviations used in this paper: IL-7Rα, IL-7R α-chain; GC, glucocorticoid; GR, GC receptor; CNS, conserved noncoding sequence; Dex, dexamethasone; ChIP, chromatin immunoprecipitation.

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