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Laboratory of Molecular Immunology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Received 20 April 2005/ Returned for modification 6 June 2005/ Accepted 25 August 2005
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ABSTRACT |
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INTRODUCTION |
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chain (35, 38, 39), and correspondingly, IL-21 is most similar to IL-2, IL-4, and IL-15 (38). Like IL-2, IL-4, IL-7, IL-9, and IL-15, the receptor for IL-21 also contains the common cytokine receptor 
chain (c), and IL-21 signals in part through the activation of Jak1 and Jak3 (2, 11, 24, 35). IL-21 is produced by activated CD4+ T cells (38, 39), and corresponding to the expression of its receptor, IL-21 has actions on T, B, and NK cells. It enhances the proliferation of both anti-CD3 activated thymocytes and peripheral T cells (16, 38), and it also acts synergistically with IL-7 or IL-15 to enhance CD8+ T-cell proliferation (38, 57). IL-21 can promote NK cell maturation from bone marrow progenitors and activate the cytolytic activity of peripheral NK cells, and it can reduce IL-15-induced expansion of resting NK cells (16, 38), although IL-21R–/– mice have normal NK cell development (37). IL-21 can augment B-cell death in vitro (31, 36) and in vivo (36), but it also promotes the differentiation of B cells into postswitch and plasma cells and is critical for antigen-specific immunoglobulin (Ig) production in vivo (36, 37). IL-21R–/– mice exhibited normal lymphocyte development but abnormal Ig production, with reduced serum levels of IgG1 and IgG2b but elevated IgE in response to antigen (37). Correspondingly, IL-21 can inhibit antigen-induced IgE production (48). IL-21R–/– IL-4–/– double knockout mice exhibit a severely impaired IgG response as well as diminished IgE levels, indicating that these two cytokines cooperatively regulate Ig production (37). In addition to its physiological roles in lymphoid biology, IL-21 has antitumor actions as well that correlate with its ability to activate NK and cytotoxic CD8+ T cells and to enhance gamma interferon production by these cells (29, 47, 52, 57). Given the range of actions of IL-21 and its importance in regulating the immune system, we investigated the molecular mechanism involved in IL21R gene regulation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Real-time PCR analysis. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA). First-strand cDNA was made from 2 µg of total RNA using random hexamers and Omniscript reverse transcriptase (QIAGEN, Valencia, CA), following the manufacturer's suggested protocol. Quantitation of specific mRNAs and 18S rRNA (as a control) was performed by real-time PCR using the 7900H sequence detection system (Applied Biosystems, Foster City, CA). cDNAs were amplified using the TaqMan universal PCR master mix (Applied Biosystems). The primers and probes used to detect human IL-21R, Sp1, and 18S rRNA are as follows: IL-21R forward primer (5'-TGTGGAGGCTATGGA AGAAGATATG-3'), reverse primer (5'-GTGCACCCACCCATTTCTTG-3'), and probe (5'-6-carboxyfluorescein [FAM]-CGGTTCTTCATGCCCCTGTAA AGGG-6-carboxytetramethylrhodamine [TAMRA]-3'); Sp1 forward primer (5'-CAGCTTCAGGCTGTTCCAAACT-3'), reverse primer (5'-CTGCCAACT GACCTGTCCATT-3'), and probe (5'-FAM-TGGTCCCATCATCATCCG GACACC-TAMRA-3'); and 18S rRNA forward primer (5'-TTCGGAACT GAGGCATGAT-3'), reverse primer (5'-TTTCGCTCTGGTCCGTCTTG-3'), and probe (5'-FAM-CGCCGCTAGAGGTGAAATTCTTGGACC-TAMRA-3').
5' RACE. Total RNA was isolated from activated human PB T cells, and rapid amplification of 5' cDNA ends (5' RACE) was performed with a GeneRACER kit (Invitrogen). A "RACER 5' primer" complementary to the RNA oligonucleotide sequence and a gene-specific primer (GSP) complementary to IL-21R cDNA (either GSP1, 5'-GAGGAGGGAGACACTTCTTGAGT-3', or GSP2, 5'-ACTGTCCTGAGCAGGTCACAGTC-3') were used in the PCR. The "RACER 5' nested primer" and either IL-21R GSP2 or GSP3 (5'-CGGCTT GATGCTCTCAGCCAGGA-3') were then used in a second, nested PCR. The PCR product was subcloned into pCR4-TOPO vector with a TOPO TA cloning kit (Invitrogen), and the nucleotide sequence was determined.
DNase I hypersensitivity assay. DNase I hypersensitivity assays were performed as described previously (17). Briefly, Molt-3 cells were activated with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) plus 1 µg/ml of ionomycin for 4 h at 37°C. The nuclear pellet of the cells was digested with DNase I. The purified genomic DNA was digested with NdeI (–7617 and +6809) and BglI (–8118 and +3147) and Southern blotted (Nytran Plus, Schleicher & Schuell, Keene, NH) using random primer-labeled probe 1 (+6331 to +6754) and probe 2 (+1051 to +1352) and Quickhyb solution (Stratagene, La Jolla, CA).
Restriction enzyme accessibility assay. A restriction enzyme accessibility assay was performed as described previously (8). Briefly, human PB T cells were activated with plate-bound anti-CD3 (2 µg/ml) and 1 µg/ml of anti-CD28 for 4 h, followed by digestion with 5 U of BstEII for 10 min at 37°C. Two micrograms of the DNA was digested with 10 U of StuI for 2 h at 37°C. Following blunting of the DNA ends with Klenow, the DNA was ligated to a universal linker (34). The cleavage sites were detected by PCR (34 cycles) with linker primer and a downstream antisense GSP (from +58 to +80, 5'-GAGCTTACGGTCACTCAG CAGAG-3'). 32P-labeled downstream antisense primer (from +39 to +62, 5'-CAGAGAGACGCCAGTGGGTCTGTC-3') was added to the PCR for the last two cycles, and the PCR products were separated on polyacrylamide gels, visualized by autoradiography, and quantitated using a PhosphorImager.
IL-21R luciferase reporter assays. The 5' regulatory region of the IL21R gene from –2300 to +350 was subcloned between the BglII and XhoI polylinker sites in the pREP4 luciferase reporter vector (27). Site-directed mutagenesis or deletion of the IL-21R –61 to –32 region were performed using a QuickChange kit (Stratagene).
Molt-3 cells were transfected using DEAE-dextran with 10 µg of the reporter construct plasmid and 40 ng of pREP7 renilla luciferase vector as a transfection efficiency control. After 24 h, cells were stimulated with medium alone or PMA and ionomycin (PI). Luciferase activity was measured (18) after 18 h.
Transient transfection of normal murine splenic T cells was performed by electroporation, as described previously (18). Preactivated murine splenic T cells were mixed with 30 µg of the IL-21R promoter reporter plasmid and 500 ng of pREP7 renilla luciferase vector. After electroporation, cells were cultured for 3 h, and then half of the cells were stimulated with anti-CD3 plus anti-CD28. After 18 h, the cells were harvested and analyzed for luciferase activity.
EMSAs and Western blotting. Nuclear extracts were prepared as described previously (25) from untreated naïve T cells or PI-activated T cells, and 10 µg of nuclear extracts and 20,000 cpm of 32P-labeled probe were used in electrophoretic mobility shift assays (EMSAs). For competition and supershift assays, before adding the labeled probe, either excess unlabeled oligonucleotides or antibodies to Sp1 or Sp3 (Upstate Biotechnology, Lake Placid, NY) or Sp2 or Sp4 (Santa Cruz Biotechnology, Santa Cruz, CA) were preincubated with the nuclear extracts. Recombinant Sp1 was from Sigma-Aldrich (St. Louis, MO), and Sp3 was from Upstate Biotechnology.
Nuclear extracts (15 µg) were resolved on 8% Novex Tris-glycine gel or 8% NuPAGE Tris-acetate gel (Invitrogen) and blotted onto Immobilon-P membranes (Millipore Corporation, Bedford, MA). The membranes were then blotted with anti--tubulin (Santa Cruz Biotechnology) as a control or with anti-Sp1 and anti-Sp3 and developed by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
DNA affinity chromatography and mass spectrometry. DNA affinity chromatography was performed as described previously (55). Briefly, the protein binding site was concatemerized by a self-priming PCR technique using a wild-type sequence (5'-bio-CCCAGCTGCGGGTGGGCGGGGCTGGCGGGG-3') or a similar mutant oligonucleotide (5'-biotin-CCCAGCTGCGGGGTTGCGGGG CTGGCGGGG-3'). Six milligrams of nuclear extract from PI-activated human PB T cells was incubated with streptavidin-conjugated biotinylated PCR product. The bound proteins were eluted with binding buffer containing 600 mM KCl and then dialyzed at 4°C overnight. The final eluate was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver stained. The bands enriched with wild-type oligonucleotides were identified by liquid chromatography mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) (Lerner Research Institute, Mass Spectrometry Laboratory for Protein Sequencing, the Cleveland Clinic Foundation, Cleveland, Ohio).
Small interfering RNA (siRNA)-based inhibition. Purified human PB T cells were transfected using the human T cell Nucleofector kit (Amaxa, Gaithersburg, MD) with 100 nM siSp1, siSp3 (siGENOME SMARTpool siRNA, Dharmacon, Lafayette, CO), or siControl (Dharmacon). Since the transfection efficiency in human primary T cells was only 30 to 40%, we cotransfected the cells with pEYFP-N1 (BD Clontech, Franklin Lakes, NJ), which expresses enhanced yellow fluorescent protein (YFP). After culturing in antibiotic-free medium for 36 h, cells positive for YFP were isolated by cell sorting and cultured in medium alone or stimulated with anti-CD3 plus anti-CD28. After 2 h of incubation, total RNA was extracted and analyzed by quantitative real-time PCR.
ChIP. Chromatin immunoprecipitation (ChIP) assays were performed as described previously (27). Human PB T cells were activated with anti-CD3 plus anti-CD28 for 2 h, followed by cross-linking with formaldehyde. Immunoprecipitations were performed with 6 µg of anti-Sp1 (Upstate Biotechnology) or normal rabbit IgG (Santa Cruz Biotechnology) conjugated to Dynabeads protein A (Dynal Biotech, Brown Deer, WI). Immunoprecipitated DNA samples were analyzed by real-time PCR for IL-21R promoter or ß-actin as a control. The sequences of the primers and TaqMan probes were as follows: IL-21R promoter forward primer, 5'-GGTCCCTAAGAGGGAAGTGTCA-3'; reverse primer, 5'-GCACCCACTGTCACCAAAGG-3'; probe, 5'-FAM-CCCGATGGCCCC AAATGTCTTACTTG-TAMRA-3'; ß-actin forward primer, 5'-TCCACCTTCCAGCAGATGTG-3'; reverse primer, 5'-GCAACTAAGTCATAGTCCGCC TAGA-3'; and probe, 5'-FAM-AGCAGGAGTATGACGAGTCCGGCCC- TAMRA-3'.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TGGGCG motif in the promoter region is important for IL-21R expression. To identify the sequences critical for IL-21R promoter activity, the –2300 to +350 region was cloned in a luciferase reporter vector. Transient transfection of Molt-3 cells with this construct showed a potent increase in PI-induced activity (Fig. 4A). Analysis of a series of 5' deletion constructs revealed that potent reporter activity was present in a construct extending to –80 but not to –20 (Fig. 4A), indicating a critical role for the –80 to –20 region in PI-induced IL-21R promoter activity.
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To further define the factor binding sites, we made a series of mutant oligonucleotide probes by changing every three nucleotides within the –61 to –32 region (Fig. 5A) and incubated these probes with nuclear extracts from PI-activated human PB T cells. Mutation of either TGG to GTT (Mut5) or GCG to TAT (Mut6) greatly diminished DNA-protein complex formation (Fig. 5B, lanes 7 and 8). Similar findings were observed in Molt-3 cells (data not shown).
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Identification of Sp1 and Sp3 binding to the GC box in the IL-21R promoter in vitro. To characterize factor(s) binding to the TGGGCG region, we used DNA affinity chromatography (55). PCR-amplified concatemers spanning the entire –61 to –32 region (containing the TGGGCG motif) were biotinylated and tethered to streptavidin paramagnetic particles. A parallel control purification was performed in which TGG GCG was mutated to GTTGCG (Mut5). Nuclear extracts from PI-activated human PB T cells were separately incubated with each affinity resin and washed to remove the nonspecific binding, and eluates were resolved by SDS-PAGE and silver stained. Several bands were more enriched in the eluate from wild-type resin than in that from mutant resin. The major band of approximately 95 kDa that was present only in the wide-type resin eluate (Fig. 6A) was analyzed by LC-MS and MALDI-TOF mass spectrometry and identified as Sp1. The p80 band also contained Sp1 peptides and perhaps represents a degradation product of Sp1. No peptides were detected from the p40 band, so the identity of p40 is completely unclear. The inability to recover peptides may have been due to protein degradation or a low abundance of the material.
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We next examined whether the expression levels of Sp1 and Sp3 in primary T cells were changed upon TCR stimulation. Interestingly, in human PB T cells, the basal expression of Sp1 was variable among different donors. In those donors with low basal expression, Sp1 was potently induced after stimulation with anti-CD3 plus anti-CD28 (Fig. 6D, donor 1), whereas in other donors where Sp1 was expressed at a high level in resting cells, no inducibility was observed (Fig. 6D, donor 2). It is possible that the altered basal levels of Sp1 expression might reflect recent exposure of some donors to antigenic stimuli. Consistent with this, the level of Sp1 was low in naïve resting mouse splenic T cells but consistently induced after TCR activation (Fig. 6D). Our data thus indicated that Sp1 bound to the IL-21R promoter in vitro and that TCR stimulation augmented levels of Sp1. In contrast, the expression level of Sp3 was relatively stable upon TCR activation, and even in the donors with induced Sp1 expression, no inducibility of Sp3 was observed (Fig. 6E).
Suppression of TCR-mediated IL-21R induction by siSp1. To investigate the role of Sp1 and Sp3 in vivo for TCR-induced IL21R gene regulation, we used synthetic small interfering RNAs (21-nucleotide duplex siRNAs). As a control, we used an siRNA that, based on bioinformatic algorithms, was designed to have more than four mismatches with other known human genes. We transfected 25, 50, 100, 150, or 200 nM siSp1 or siSp3 into human primary T cells and found that 100 nM provided an optimal silencing effect by measuring the Sp1 or Sp3 mRNA levels using quantitative real-time PCR (data not shown), and we used this dose in subsequent experiments. In human PB T cells, when the Sp1 expression level was effectively silenced by siRNA transfection (Fig. 7B), the level of TCR-induced IL-21R expression was diminished by over 50% (Fig. 7A). However, when the expression level of Sp3 was effectively silenced by siSp3 (Fig. 7C), the TCR-induced IL-21R expression was at most minimally reduced. Moreover, the combination of siSp3 and siSp1 did not decrease the expression of IL-21R more than transfection of only siSp1 (Fig. 7A). The control siRNA did not affect Sp1 (Fig. 7B), Sp3 (Fig. 7C), or IL-21R (Fig. 7A) expression. These results demonstrate that although both Sp1 and Sp3 bind to the IL-21R promoter, Sp1 plays a more critical role in regulating IL-21R expression in human T cells.
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TCR-induced IL-21R expression depends on the dephosphorylation of Sp1. Posttranslational modification of Sp1 is known to regulate its transcriptional activity. For example, lipopolysaccharide treatment reduced Sp1 DNA-binding activity and transcriptional activation by dephosphorylation of Sp1 at serine and threonine residues (56). In addition, it was reported that in human T lymphocytes treated with anti-CD2 plus anti-CD28, Sp1 was dephosphorylated, correlating with cell cycle progression. The dephosphorylation of Sp1, which was mediated by protein phosphatase 2A (PP2A), increased its transcriptional activity in a reporter assay in Kit225 cells (22). We thus examined the effect of calyculin A, a potent inhibitor of both PP2A and protein phosphatase 1 (PP1) that specifically inhibits dephosphorylation at serine/threonine residues (43), on TCR-induced IL-21R expression. When 10 nM calyculin A was incubated with human PB T cells stimulated with anti-CD3 plus anti-CD28 for 2 h, it blocked the IL-21R mRNA induction (Fig. 8A) without affecting viability or expression of 18S rRNA (data not shown). A second phosphatase inhibitor, okadaic acid, which binds to the catalytic subunits of PP1 and PP2A (with a 200-fold-higher affinity for PP2A) (22), caused a similar reduction of TCR-induced IL-21R expression (Fig. 8A). By EMSA, the DNA-binding activity of Sp1 was decreased by calyculin A or okadaic acid treatment in T cells activated with anti-CD3 plus anti-CD28 (Fig. 8B, lane 4 versus lane 3 and lane 8 versus lane 7). The decrease in DNA-binding activity did not result from protein degradation after cytotoxic calyculin A treatment, as Sp1 protein expression was if anything somewhat increased after calyculin A treatment, as detected by Western blotting (Fig. 8C, lane 2 versus lane 1 and lane 4 versus lane 3). By Western blotting, two Sp1 bands were detected in resting T cells (Fig. 8C, lane 1), with the lower-molecular-weight form most likely arising from dephosphorylation, given the increase in the upper band after calyculin A treatment (Fig. 8C, lane 2). TCR stimulation augmented Sp1 with a relative increase in the lower dephosphorylated band (Fig. 8C, lane 3), and the addition of calyculin A again increased the higher-molecular-weight phosphorylated form (Fig. 8C, lane 4). Similar effects on Sp1 expression level were observed in okadaic acid-treated T cells (Fig. 8C). Together, these results indicate that TCR stimulation induces Sp1 with a relative increase in the dephosphorylated form, and the dephosphorylation of Sp1 may be required for IL-21R promoter activity.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In examining the molecular mechanism underlying the transcriptional regulation of the IL21R gene, we found that Sp1 binds to a GC-rich motif in the proximal promoter and mediates IL-21R induction in activated human T cells. Sp1 is known to activate many vital genes, including genes involved in cell growth and development (5, 15, 21), and homozygous deletion of Sp1 in mice causes severe lethal embryonic malformation (30). Classically, Sp1 was viewed as a constitutive transcription factor that regulates the basal expression of many cellular genes (7, 14). However, it has now been found to be involved in tissue-specific gene expression and in the control of transcription following different stimuli (3, 40, 51).
In the immune system, Sp1 has been reported to control the expression of genes that mediate important cellular functions. For example, Sp1 regulates the expression of genes encoding IL-2Rß (26), TCR V (19), and FasL (54) and plays a critical role in regulating the division of T lymphocytes after costimulation with anti-CD2 and anti-CD28 (22). We now show that Sp1 plays an essential role in regulating TCR-induced IL-21R expression. Mutation of the Sp1 binding sites in the IL-21R proximal promoter region diminished PI-induced reporter activity. Strikingly, Sp1 expression was significantly increased in primary T cells after TCR stimulation. The TCR-induced augmentation of Sp1 transcriptional activity correlated with induction of IL-21R mRNA, and transfection of Sp1 siRNAs into primary human T cells significantly decreased TCR-induced IL-21R mRNA expression. Unexpectedly, however, Sp1 expression is only slightly increased 2 h after TCR activation, while IL-21R mRNA levels were highly induced at this time point. We therefore hypothesized that posttranslational modification of Sp1 might play a critical role for IL-21R gene expression. Depending on cell type and stimuli, phosphorylation of Sp1 has been reported to either increase (41, 45) or decrease (1, 23, 32, 58) Sp1 transcriptional activity. We thus examined the effect of the phosphatase inhibitors calyculin A and okadaic acid, which inhibit PP1 and PP2A activity, and found that they decreased IL-21R expression. Moreover, the phosphatase inhibitors increased the phosphorylation of Sp1 and correspondingly decreased Sp1 DNA-binding activity for the IL-21R promoter. These results suggest that posttranslational modification of Sp1 is critical for TCR-induced IL-21R expression.
Sp1 has been reported to exert its transcriptional activation with the help of other comodulators or cofactors, such as NF-
B (20), STAT proteins (6, 28), Smad family factors (33), Egr family factors (26, 50), NFAT family proteins (54), or other members of the Sp family (9, 46). Complex C2, as detected in EMSA, appears to represent Sp3, since anti-Sp3 diminished the formation of C2. However, the DNA-binding activity of complex C2 was only slightly induced by PI activation, in contrast to Sp1, whose binding activity was markedly induced. In addition, siSp3 had little effect on TCR-induced IL-21R expression, and the cotransfection of siSp1 and siSp3 was not more potent than siSp1 transfection alone. Thus, unlike Sp1, Sp3 plays only a minor role in regulating IL-21R repression. So far, we do not yet have definitive data that support factors other than Sp1 as critically contributing to the regulation of IL-21R. Since PI activation is associated with protein kinase C activation and calcium/calcineurin pathway, it is conceivable that other factors, such as AP-1, NF-B, or NFAT (10, 13, 42, 49), might be involved in IL-21R regulation, an area for future investigation.
Taken together, our results reveal that Sp1 is indeed a critical regulator of IL21R gene expression, a finding with implications as to how the expression of this critical receptor can be controlled. Moreover, the observation that Sp1 is potently induced and dephosphorylated in response to anti-CD3 plus anti-CD28 has much broader implications for the regulation of Sp1-dependent genes in antigen-activated T cells. Further studies are needed to clarify how TCR activation induces the dephosphorylation of Sp1 and to identify the relevant target residues. Such studies will not only further clarify the basis for IL-21R regulation but also help us to understand how Sp1-regulated genes in general are broadly controlled in the immune system.
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ACKNOWLEDGMENTS |
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This research was supported by the Intramural Research Program of the NHLBI, NIH.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2. Asao, H., C. Okuyama, S. Kumaki, N. Ishii, S. Tsuchiya, D. Foster, and K. Sugamura. 2001. Cutting edge: the common gamma-chain is an indispensable subunit of the IL-21 receptor complex. J. Immunol. 167:1-5.
3. Black, A. R., J. D. Black, and J. Azizkhan-Clifford. 2001. Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J. Cell. Physiol. 188:143-160.[CrossRef][Medline]
4. Bouwman, P., and S. Philipsen. 2002. Regulation of the activity of Sp1-related transcription factors. Mol. Cell. Endocrinol. 195:27-38.[CrossRef][Medline]
5. Briggs, M. R., J. T. Kadonaga, S. P. Bell, and R. Tjian. 1986. Purification and biochemical characterization of the promoter-specific transcription factor, Sp1. Science 234:47-52.
6. Cantwell, C. A., E. Sterneck, and P. F. Johnson. 1998. Interleukin-6-specific activation of the C/EBPdelta gene in hepatocytes is mediated by Stat3 and Sp1. Mol. Cell. Biol. 18:2108-2117.
7. Cook, T., B. Gebelein, and R. Urrutia. 1999. Sp1 and its likes: biochemical and functional predictions for a growing family of zinc finger transcription factors. Ann. N. Y. Acad. Sci. 880:94-102.
8. Cui, K., P. Tailor, H. Liu, X. Chen, K. Ozato, and K. Zhao. 2004. The chromatin-remodeling BAF complex mediates cellular antiviral activities by promoter priming. Mol. Cell. Biol. 24:4476-4486.
9. Dong, S., T. Kojima, M. Shiraiwa, M. C. Mechin, S. Chavanas, G. Serre, M. Simon, A. Kawada, and H. Takahara. 2005. Regulation of the expression of peptidylarginine deiminase type II gene (PADI2) in human keratinocytes involves Sp1 and Sp3 transcription factors. J. Investig. Dermatol. 124:1026-1033.[CrossRef][Medline]
10. Favero, J., and V. Lafont. 1998. Effector pathways regulating T cell activation. Biochem. Pharmacol. 56:1539-1547.[CrossRef][Medline]
11. Habib, T., S. Senadheera, K. Weinberg, and K. Kaushansky. 2002. The common gamma chain (gamma c) is a required signaling component of the IL-21 receptor and supports IL-21-induced cell proliferation via JAK3. Biochemistry 41:8725-8731.[CrossRef][Medline]
12. Hempel, W. M., and P. Ferrier. 2004. Restriction endonuclease accessibility as a determinant of altered chromatin structure. Methods Mol. Biol. 287:53-63.[Medline]
13. Hogan, P. G., L. Chen, J. Nardone, and A. Rao. 2003. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17:2205-2232.
14. Kaczynski, J., T. Cook, and R. Urrutia. 2003. Sp1- and Kruppel-like transcription factors. Genome Biol. 4:206.[CrossRef][Medline]
15. Kadonaga, J. T., K. R. Carner, F. R. Masiarz, and R. Tjian. 1987. Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51:1079-1090.[CrossRef][Medline]
16. Kasaian, M. T., M. J. Whitters, L. L. Carter, L. D. Lowe, J. M. Jussif, B. Deng, K. A. Johnson, J. S. Witek, M. Senices, R. F. Konz, A. L. Wurster, D. D. Donaldson, M. Collins, D. A. Young, and M. J. Grusby. 2002. IL-21 limits NK cell responses and promotes antigen-specific T cell activation: a mediator of the transition from innate to adaptive immunity. Immunity 16:559-569.[CrossRef][Medline]
17. Kim, H. P., J. Kelly, and W. J. Leonard. 2001. The basis for IL-2-induced IL-2 receptor alpha chain gene regulation: importance of two widely separated IL-2 response elements. Immunity 15:159-172.[CrossRef][Medline]
18. Kim, H. P., and W. J. Leonard. 2002. The basis for TCR-mediated regulation of the IL-2 receptor alpha chain gene: role of widely separated regulatory elements. EMBO J. 21:3051-3059.[CrossRef][Medline]
19. Kingsley, C., and A. Winoto. 1992. Cloning of GT box-binding proteins: a novel Sp1 multigene family regulating T-cell receptor gene expression. Mol. Cell. Biol. 12:4251-4261.
20. Krehan, A., H. Ansuini, O. Bocher, S. Grein, U. Wirkner, and W. Pyerin. 2000. Transcription factors ets1, NF-kappa B, and Sp1 are major determinants of the promoter activity of the human protein kinase CK2alpha gene. J. Biol. Chem. 275:18327-18336.
21. Kriwacki, R. W., S. C. Schultz, T. A. Steitz, and J. P. Caradonna. 1992. Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1. Proc. Natl. Acad. Sci. USA 89:9759-9763.
22. Lacroix, I., C. Lipcey, J. Imbert, and B. Kahn-Perles. 2002. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes. J. Biol. Chem. 277:9598-9605.
23. Leggett, R. W., S. A. Armstrong, D. Barry, and C. R. Mueller. 1995. Sp1 is phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of the liver. J. Biol. Chem. 270:25879-25884.
24. Leonard, W. J. 2001. Cytokines and immunodeficiency diseases. Nat. Rev. Immunol. 1:200-208.[CrossRef][Medline]
25. Lin, J.-X., N. K. Bhat, S. John, W. S. Queale, and W. J. Leonard. 1993. Characterization of the human interleukin-2 receptor ß-chain gene promoter: regulation of promoter activity by ets gene products. Mol. Cell. Biol. 13:6201-6210.
26. Lin, J. X., and W. J. Leonard. 1997. The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites. Mol. Cell. Biol. 17:3714-3722.[Abstract]
27. Liu, R., H. Liu, X. Chen, M. Kirby, P. O. Brown, and K. Zhao. 2001. Regulation of CSF1 promoter by the SWI/SNF-like BAF complex. Cell 106:309-318.[CrossRef][Medline]
28. Look, D. C., M. R. Pelletier, R. M. Tidwell, W. T. Roswit, and M. J. Holtzman. 1995. Stat1 depends on transcriptional synergy with Sp1. J. Biol. Chem. 270: 30264-30267.
29. Ma, H. L., M. J. Whitters, R. F. Konz, M. Senices, D. A. Young, M. J. Grusby, M. Collins, and K. Dunussi-Joannopoulos. 2003. IL-21 activates both innate and adaptive immunity to generate potent antitumor responses that require perforin but are independent of IFN-gamma. J. Immunol. 171:608-615.
30. Marin, M., A. Karis, P. Visser, F. Grosveld, and S. Philipsen. 1997. Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell 89:619-628.[CrossRef][Medline]
31. Mehta, D. S., A. L. Wurster, M. J. Whitters, D. A. Young, M. Collins, and M. J. Grusby. 2003. IL-21 induces the apoptosis of resting and activated primary B cells. J. Immunol. 170:4111-4118.
32. Mortensen, E. R., P. A. Marks, A. Shiotani, and J. L. Merchant. 1997. Epidermal growth factor and okadaic acid stimulate Sp1 proteolysis. J. Biol. Chem. 272:16540-16547.
33. Moustakas, A., and D. Kardassis. 1998. Regulation of the human p21/WAF1/Cip1 promoter in hepatic cells by functional interactions between Sp1 and Smad family members. Proc. Natl. Acad. Sci. USA 95:6733-6738.
34. Mueller, P. R., and B. Wold. 1989. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780-786.
35. Ozaki, K., K. Kikly, D. Michalovich, P. R. Young, and W. J. Leonard. 2000. Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain. Proc. Natl. Acad. Sci. USA 97:11439-11444.
36. Ozaki, K., R. Spolski, R. Ettinger, H. P. Kim, G. Wang, C. F. Qi, P. Hwu, D. J. Shaffer, S. Akilesh, D. C. Roopenian, H. C. Morse III, P. E. Lipsky, and W. J. Leonard. 2004. Regulation of B cell differentiation and plasma cell generation by IL-21, a novel inducer of Blimp-1 and Bcl-6. J. Immunol. 173:5361-5371.
37. Ozaki, K., R. Spolski, C. G. Feng, C. F. Qi, J. Cheng, A. Sher, H. C. Morse III, C. Liu, P. L. Schwartzberg, and W. J. Leonard. 2002. A critical role for IL-21 in regulating immunoglobulin production. Science 298:1630-1634.
38. Parrish-Novak, J., S. R. Dillon, A. Nelson, A. Hammond, C. Sprecher, J. A. Gross, J. Johnston, K. Madden, W. Xu, J. West, S. Schrader, S. Burkhead, M. Heipel, C. Brandt, J. L. Kuijper, J. Kramer, D. Conklin, S. R. Presnell, J. Berry, F. Shiota, S. Bort, K. Hambly, S. Mudri, C. Clegg, M. Moore, F. J. Grant, C. Lofton-Day, T. Gilbert, F. Rayond, A. Ching, L. Yao, D. Smith, P. Webster, T. Whitmore, M. Maurer, K. Kaushansky, R. D. Holly, and D. Foster. 2000. Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function. Nature 408:57-63.[CrossRef][Medline]
39. Parrish-Novak, J., D. C. Foster, R. D. Holly, and C. H. Clegg. 2002. Interleukin-21 and the IL-21 receptor: novel effectors of NK and T cell responses. J. Leukoc. Biol. 72:856-863.
40. Philipsen, S., and G. Suske. 1999. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 27:2991-3000.
41. Rafty, L. A., and L. M. Khachigian. 2001. Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-zeta. Nucleic Acids Res. 29:1027-1033.
42. Rao, A., C. Luo, and P. G. Hogan. 1997. Transcription factors of the NFAT family: regulation and function. Annu. Rev. Immunol. 15:707-747.[CrossRef][Medline]
43. Resjo, S., A. Oknianska, S. Zolnierowicz, V. Manganiello, and E. Degerman. 1999. Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A. Biochem. J. 341:839-845.
44. Roh, T. Y., S. Cuddapah, and K. Zhao. 2005. Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping. Genes Dev. 19:542-552.
45. Rohlff, C., S. Ahmad, F. Borellini, J. Lei, and R. I. Glazer. 1997. Modulation of transcription factor Sp1 by cAMP-dependent protein kinase. J. Biol. Chem. 272:21137-21141.
46. Santini, M. P., C. Talora, T. Seki, L. Bolgan, and G. P. Dotto. 2001. Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21(WAF1/CIP1) expression in keratinocyte differentiation. Proc. Natl. Acad. Sci. USA 98:9575-9580.
47. Strengell, M., S. Matikainen, J. Siren, A. Lehtonen, D. Foster, I. Julkunen, and T. Sareneva. 2003. IL-21 in synergy with IL-15 or IL-18 enhances IFN-gamma production in human NK and T cells. J. Immunol. 170:5464-5469.
48. Suto, A., H. Nakajima, K. Hirose, K. Suzuki, S. Kagami, Y. Seto, A. Hoshimoto, Y. Saito, D. C. Foster, and I. Iwamoto. 2002. Interleukin 21 prevents antigen-induced IgE production by inhibiting germ line C(epsilon) transcription of IL-4-stimulated B cells. Blood 100:4565-4573.
49. Thomas, R. S., M. J. Tymms, L. H. McKinlay, M. F. Shannon, A. Seth, and I. Kola. 1997. ETS1, NFkappaB and AP1 synergistically transactivate the human GM-CSF promoter. Oncogene 14:2845-2855.[CrossRef][Medline]
50. Trejo, S. R., W. E. Fahl, and L. Ratner. 1997. The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet-derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1. J. Biol. Chem. 272:27411-27421.
51. Turner, J., and M. Crossley. 1999. Mammalian Kruppel-like transcription factors: more than just a pretty finger. Trends Biochem. Sci. 24:236-240.[CrossRef][Medline]
52. Wang, G., M. Tschoi, R. Spolski, Y. Lou, K. Ozaki, C. Feng, G. Kim, W. J. Leonard, and P. Hwu. 2003. In vivo antitumor activity of interleukin 21 mediated by natural killer cells. Cancer Res. 63:9016-9022.
53. Weinmann, A. S., S. E. Plevy, and S. T. Smale. 1999. Rapid and selective remodeling of a positioned nucleosome during the induction of IL-12 p40 transcription. Immunity 11:665-675.[CrossRef][Medline]
54. Xiao, S., K. Matsui, A. Fine, B. Zhu, A. Marshak-Rothstein, R. L. Widom, and S. T. Ju. 1999. FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3. Eur. J. Immunol. 29:3456-3465.[CrossRef][Medline]
55. Xue, H. H., J. Bollenbacher, V. Rovella, R. Tripuraneni, Y. B. Du, C. Y. Liu, A. Williams, J. P. McCoy, and W. J. Leonard. 2004. GA binding protein regulates interleukin 7 receptor alpha-chain gene expression in T cells. Nat. Immunol. 5:1036-1044.[CrossRef][Medline]
56. Ye, X., and S. F. Liu. 2002. Lipopolysaccharide down-regulates Sp1 binding activity by promoting Sp1 protein dephosphorylation and degradation. J. Biol. Chem. 277:31863-31870.
57. Zeng, R., R. Spolski, S. E. Finkelstein, S. Oh, P. E. Kovanen, C. S. Hinrichs, C. A. Pise-Masison, M. F. Radonovich, J. N. Brady, N. P. Restifo, J. A. Berzofsky, and W. J. Leonard. 2005. Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function. J. Exp. Med. 201:1-10.
58. Zhu, Q., and K. Liao. 2000. Differential expression of the adipocyte amino acid transporter is transactivated by SP1 and SP3 during the 3T3-L1 preadipocyte differentiation process. Biochem. Biophys. Res. Commun. 271:100-106.[CrossRef][Medline]
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