A regulatory element in the human interleukin 2 gene promoter is a binding site for the zinc finger proteins Sp1 and EGR-1.

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.

Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.
T cell activation by antigenic peptides in combination with antigen presenting cells induces a cascade of metabolic events which result in the transcriptional activation of a large number of different genes (1,2). Among the transcripts induced in the "immediate early phase," i.e. about 15 min following the activating stimulus in the absence of de novo protein synthesis, are a large number of transcription factors. These factors are considered to regulate the induction of genes which are transcribed in a second phase, i.e. the "early phase." A family of genes, termed "early growth response genes" (EGR), 1 which encode DNA-binding proteins with almost identical zinc finger domains is induced directly in the immediate early phase. The four EGR genes, termed EGR-1 (3), EGR-2 (4), EGR-3 (5,6), and EGR-4/pAT133 (5, 7) are coordinately regulated. No transcript is detected in quiescent, nonproliferating T lymphocytes, nor in resting, serum-deprived fibroblasts; however, all genes are transiently induced in a variety of cells upon mitogenic stimulation (8 -13). Although the four proteins are closely related within their zinc finger domains, their flanking regions are much less conserved. As already expected from the high amino acid homology of their zinc finger domains, in vitro experiments confirmed binding of all four proteins to the same target sequence, i.e. GCG G/TGG GCG (4 -6, 13-18). Despite binding to the identical sequence, the amino acid differences in the flanking regions suggest distinct biological functions of the four proteins.
As primary response factors, EGR-1 to EGR-4 are candidates for the regulation of distal gene expression. Based on their later time course of induction, cytokine genes are likely target genes for EGR regulation in T lymphocytes. In order to provide evidence for such a regulation, we searched for EGR binding motifs in the promoter region of human cytokine genes. This approach resulted in the identification of putative EGR binding motifs within the promoter of the human IL-2 and tumor necrosis factor ␣ genes. The EGR motif in the tumor necrosis factor ␣ promoter has recently been characterized as a binding site for the EGR-1 protein (19).
Transcriptional activation of the IL-2 gene and IL-2 secretion are essential steps for T cell proliferation, differentiation, and effector functions. IL-2 gene regulation is analyzed extensively, and several regulatory elements and their corresponding binding proteins have been identified in both the human and the mouse IL-2 promoter (1, 20 -24). In particular, a critical role for IL-2 gene expression has been demonstrated for the nuclear factor of activated T cells (NFAT). This complex factor assembles only in the nucleus of activated T cells and is essential for transcriptional induction of the IL-2 gene (25)(26)(27). The NFAT binding region includes a purine-rich stretch and an AP-1 site. Binding of members of the fos and jun gene family to the AP-1 site has been demonstrated (28), and two factors (termed NFATc and NFATp) which bind to the purine-rich region directly upstream of the AP-1 site have been cloned recently (25,29).
The putative EGR recognition element in the human IL-2 promoter is located directly upstream of the NFAT site. We asked whether this site serves as a binding site for nuclear proteins and whether it participates in transcriptional regulation of IL-2 gene expression. Here we demonstrate that this G-rich domain is a binding site for the two zinc finger proteins Sp1 and EGR-1. In stimulated Jurkat T cells, this EGR-1 binding element is a novel and important regulatory element for IL-2 gene activation.

Expression of Recombinant Proteins in the Baculovirus System-
Plaque-purified EGR-1 or AT133/EGR-4 baculoviruses 2 were prepared by standard methods (30) and were used to infect Sf9 insect cells, cultivated in Grace's medium. A multiplicity of infection of 5 was used.
After 70 h of incubation, cells were lysed by three freeze-thaw cycles, and whole cell extracts were prepared as described (31). The lysis buffer (10 mM sodium phosphate, pH 7.4, 0.5 mM dithiothreitol, 400 mM KCl, 10 M ZnCl 2 , 10% glycerol) contained proteinase inhibitors phenylmethylsulfonyl fluoride (1 mM), leupeptin (5 g/ml), and aprotinin (5 g/ml). IL-2 Reporter Constructs-Specific primers were used for polymerase chain reaction amplification of the indicated regulatory elements using a 417-bp fragment of the human IL-2 promoter region. Amplified fragments were ligated to the complete (Ϫ253 to ϩ51) or to a minimal IL-2 promoter region (Ϫ63 to ϩ51) linked to the luciferase reporter gene. Primers eu5 (tttggtaccCCACAATATGCTATTCACATGTTCA, position Ϫ366 to Ϫ342) and eu9 (tttgagctcGCAGGAGTTGAGGTTACT-GTGAGTAG, ϩ51 to ϩ26) (capital letters refer to the IL-2 promoter sequence, and introduced KpnI and SacI restriction sites are underlined) were digested with KpnI and SacI, respectively and ligated with KpnI-and SacI-treated luciferase reporter plasmid pGL2-Basic Vector (Promega Corp.) to yield plasmid pCILuc1. Plasmid pCILuc2 was constructed in the same way using primers eu9 and eu6 (tttggatccAGGA-GGAAAAACTGTTTCATACAGA, Ϫ279 to Ϫ255). Additional IL-2 promoter fragments generated with primers eu9 and eu4 (tttggatccG-GCGTTAATT GCATGAATTAGAGC, Ϫ253 to Ϫ230) and with primers eu9 and eu8 (tttggatccCATTTTGACACCCCCATAATA, Ϫ63 to Ϫ42; BamHI sites are underlined) were subcloned into pCRII vector (Invitrogen) to yield plasmids p4TA and p0TA, respectively. Inserts were excised from the two plasmids by KpnI and SacI restriction and were ligated into vector pGL2-Basic to yield pCILuc4 (Ϫ253 to ϩ51 of IL-2) and pMILuc4 (Ϫ63 to ϩ51; i.e. minimal IL-2 promoter fragment), respectively. For construction of pCILuc3 and pMILuc3, a doublestranded oligonucleotide corresponding to the ZIP site of the IL-2 promoter sequence (tttggtaccTGTATCCCCACCCCCTTAAAGAAggatcc, Ϫ302 to Ϫ280, introduced KpnI and BamHI restriction sites are underlined) was cleaved and inserted into KpnI-BamHI-digested vectors p4TA and p0TA, respectively. The resulting fragments were excised by KpnI and SacI and ligated into pGL2-Basic vectors. To engineer a construct with ZIP-NFAT sites upstream of the minimal IL-2 promoter region, a restricted double-stranded oligonucleotide covering the ZIP-NFAT sites of the IL-2 promoter (Ϫ302 to Ϫ257) was inserted into vector p0TA. The complete insert was excised and cloned into a KpnI-, SacI-treated pGL2-Basic vector to obtain pMILuc1. For plasmid pMI-LUC2, containing the NFAT region upstream of the minimal IL-2 promoter sequence, plasmid pMILuc1 was used as template for polymerase chain reaction-amplification with primers eu6 and eu9. The KpnI-, SacI-cleaved polymerase chain reaction product was ligated into the vector pGL2-Basic. The identity of all constructs was confirmed by restriction and DNA sequence analysis.
Cell Culture and Transfection-The human helper T cell line Jurkat was maintained in RPMI 1640 supplemented with 10% heat-inacti-vated fetal bovine serum, penicillin, streptomycin, and fungizone at a cell density of 5 ϫ 10 5 cells/ml. For transient transfection experiments, 1 ϫ 10 7 cells were transfected by electroporation (300 V, 960 microfarads) (Bio-Rad) with 10 g of CsCl-banded reporter plasmid constructs. After 24 h, transfected cells were stimulated with 1 g/ml PHA (phytohemagglutinin; Burroughs Wellcome) and 20 ng/ml PMA (phorbol 12-myristate 13-acetate; Sigma). Cells were harvested by washing three times with phosphate-buffered saline and lysed by incubation in 250 l of lysis reagent (Promega Corp.). A 50-l sample of cell lysate was mixed with 100 l of luciferase assay reagent and measured immediately at 25°C using a luminometer (Berthold Biolumat LB9500C). The initial 30-s integral of light emission was recorded. All assays were performed in triplicate.
Preparation of Nuclear Extracts and Mobility Shift Assay-Nuclear extracts of Jurkat cells were prepared as described (22) with minor modifications. Briefly, 2 ϫ 10 7 Jurkat T cells stimulated with PHA (1 g/ml) and PMA (25 ng/ml) were lysed in a buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10% glycerol, 0.15% Nonidet P-40, 0.5 g/ml leupeptin, 5 g/ml aprotinin, 10 M ZnCl 2 . Nuclei were centrifuged at 1,000 ϫ g for 5 min, washed, and resuspended in the above solution without Nonidet P-40. 3 M KCl was added drop by drop to reach 0.39 M, and the extract was incubated for 1 h at 4°C. Extracts were clarified by centrifugation at 100,000 ϫ g for 30 min and stored at Ϫ70°C. Protein concentrations were determined using the Bradford method.
For electrophoretic mobility shift assays, 0.2 ng (about 10 4 cpm) of 32 P-end-labeled double-stranded oligonucleotides were incubated at 4°C with 5-10 g of nuclear proteins in the presence of 2 g of poly(dI-dC) and 80 ng of salmon sperm DNA in 20 l of 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 M ZnCl 2 , and 5% glycerol. For some experiments, EGR-1-and Sp1-specific antisera (Santa Cruz Biotechnology) were added after 10 min, and the reaction mixture was incubated for another 20 min at 4°C. Protein-DNA complexes were separated from free probes on 4 -6% polyacrylamide gels in (0.25 ϫ) TBE at 4°C. The gels were dried and exposed to x-ray films. All binding assays were repeated at least five times.
RNA Isolation and Northern Blotting-Total cellular RNA was extracted with guanidinium isothiocyanate and isolated by centrifugation (32). RNA electrophoresis and transfer to nylon membranes were performed as described (7). For hybridization, the following 32 P-labeled fragments were used: (i) a 2.1-kilobase cDNA insert from plasmid pPacSp1 covering the 696 C-terminal amino acids of Sp1 (kindly provided by R. Tjian) and a pAT 225/EGR-1 cDNA probe representing a 429-bp SphI-PvuII fragment of the C-terminal part (position 1388 -1817).

Binding of Nuclear Proteins to the ZIP Element of the Human
IL-2 Gene-In order to prove whether the G-rich element in the human IL-2 gene promoter (Fig. 1A) serves as binding site for nuclear proteins, a corresponding oligonucleotide was used for 2 C. Skerka and P. F. Zipfel, manuscript in preparation.
FIG. 1. Protein binding sites in the human IL-2 promoter between position ؊250 and ؊303. A, nucleotide sequence of the human IL-2 promoter region Ϫ250 to Ϫ300 bp upstream of the transcriptional start site. The protein binding sites required for NFAT and AP-1 binding are boxed. The ZIP site is described in this paper. B, sequence of oligonucleotides used in the gel shift assays. ZIP represents the nucleotide sequences Ϫ303 to Ϫ280 of the human IL-2 promoter, NFAT represents position Ϫ280 to Ϫ260 of the human IL-2 promoter, the NFAT and AP-1 binding sites are underlined, NFAT M represents a mutated NFAT domain, the mutated nucleotides are underlined, and GC 7 includes the boxed EGR consensus binding site (14). electrophoretic mobility shift assays (Fig. 1B, ZIP). Two distinct DNA-protein complexes were detected in nuclear extracts prepared from PMA/PHA-stimulated Jurkat T cells (Fig. 2, lane 1, Upper and Lower complex). Both complexes were specific as they were competed by unlabeled ZIP oligonucleotides (lane 3, ZIP) but not by unrelated oligonucleotides (lane 4, NFAT m ). An oligonucleotide representing an EGR consensus site (termed GC 7 ) blocked formation of the lower complex (Fig.  2, lane 2, GC 7 ), indicating that a member of the EGR gene family participates in binding. Sensitivity of both complexes to the metal chelator 1,10-phenanthroline and restoration of binding by Zn 2ϩ ions 3 indicated that zinc finger proteins are forming the two complexes.
As de novo protein biosynthesis is critical for IL-2 gene induction, we investigated whether both protein complexes were pre-existing in unstimulated cells. In nuclear extracts prepared from unstimulated Jurkat T cells, only the upper complex was observed (Fig. 3, lane 1), while both complexes were detected in extracts prepared from stimulated cells (lane 2). Specific antiserum was used for further differentiation and identification of the two protein complexes. Sp1 and EGR-1 antiserum specifically interfered with formation of the two complexes. Antiserum directed against EGR-1 specifically altered migration of the lower complex (Fig. 3, lane 3), indicating that EGR-1 gives rise to the inducible protein complex. The effect of this antiserum was specific as equivalent concentrations of related rabbit antisera did not interfere with binding. 3 Competition of the lower, induced complex with EGR-specific oligonucleotides (GC 7 ) (Fig. 3, lane 7) are in agreement with the supershift observed with the EGR-1-specific antiserum (lane 3) and confirmed that EGR-1 is part of the lower complex. The observation that the pre-existing complex required zinc ions for binding suggested that the zinc finger protein and transcription factor Sp1 was part of this complex. Antiserum directed against the Sp1 protein specifically altered migration of the upper complex (Fig. 3, lane 4), indicating that Sp1 gives rise to the inducible protein complex. These results indicated that Sp1 binds to the ZIP site of the human IL-2 promoter. Previously, specific and exclusive binding to G-rich consensus sequences of EGR-1 (GCG GGG GCG) and Sp1 (GGG GCG GGG) has been demonstrated for EGR-1 and the mouse homolog Zif 268, for Krox 20, the mouse homolog of EGR-2 as well as for Sp1 (14,15,18). Taken together the results suggest that the G-rich region of the human IL-2 promoter serves as a binding site for the zinc finger proteins Sp1 and EGR-1. Therefore, this region was termed the zinc finger protein (ZIP) binding site.
Regulation and Binding of the EGR-1 and Sp1 Proteins-To examine the expression of the ZIP binding factors in more detail, a time course of induction was determined on the mRNA and on the protein level. Sp1 mRNA was found synthesized in unstimulated (Fig. 4A, upper panel, lane 1) as well as in stimulated (lanes 2-6) Jurkat T cells, without significant modulation in expression levels. In contrast, EGR-1 messenger RNA was not expressed in unstimulated cells, but was induced 30 min after stimulation (Fig. 4A, lower panel). mRNA transcription was independent of protein biosynthesis (cycloheximide (Chx), lanes 7-9) and confirmed EGR-1 as an immediate early gene in T cell activation (2,33).
Similarly, binding assays of the two proteins paralleled the results obtained by Northern blotting. Sp1 protein binding to the ZIP site was constitutive, equal levels were detected in unstimulated cells and during the course of stimulation. Again the product of the immediate early response gene EGR-1 was 3 C. Skerka and P. F. Zipfel, unpublished data.   3 and 4). Antibody binding to the protein complexes results in a "supershift" and in a reduction in mobility. Competition experiments confirm different binding specificities of the two complexes. Competition with unrelated oligonucleotides (NFAT and NFAT m ) did not affect binding, while competition with an oligonucleotide displaying an EGR consensus site (GC 7 ) specifically interfered with formation of the lower EGR-1-containing complex. transiently expressed, and binding was detected 1 and 2 h after stimulation (Fig. 4B). Later time points did not show any EGR-1 protein complex formation (lanes 5 and 6). Thus, constitutive expression of Sp1 and transient synthesis of EGR-1 following cell stimulation was detected on the RNA level and in the protein binding assays. The fact that no additional band was detected when both proteins Sp1 and EGR-1 were present in the same nuclear extract suggests that EGR-1 competes with Sp1 for binding to the ZIP site and replaces Sp1.
Binding Specificities of EGR-1 and pAT133/EGR-4 -We asked whether the lower complex was represented by a single EGR-1 protein or by a complex of proteins including EGR-1. Determining the binding of recombinant, baculovirus-expressed EGR-1 protein to the ZIP oligonucleotide resulted in one specific shifted band (Fig. 5, lane 1). This complex showed the same binding specificities as the induced complex detected in Jurkat cells, as it was competed with unlabeled ZIP oligonucleotides (compare Fig. 5, lane 3, with Fig. 2, lane 3), but not with an unrelated NFAT oligonucleotide (compare Fig. 5, lane 2, with Fig. 3, lane 5). In addition, antiserum raised against EGR-1, but not EGR-3 antiserum, abolished binding of the recombinant protein (Fig. 5, lanes 4 and 5). Thus, binding of recombinant EGR-1 seems to be independent of additional T cell-specific transcription factors. Upon activation of peripheral blood T lymphocytes and Jurkat T cells, transcription of the four known EGR genes is simultaneously and coordinately induced (7), and binding to the consensus site (GC 7 ) has been demonstrated in vitro for the corresponding proteins (4 -6, 13-18). As the ZIP sequence differs from the known EGR consensus site, we addressed the question of whether other EGR proteins could bind to this promoter element. No binding of baculovirus-expressed AT133/EGR-4 protein was detected (Fig. 5, lane 6), although the recombinant AT133/EGR-4 protein recognized the EGR consensus site (lane 7; GC 7 ). This result demonstrates different binding specificities of the highly related zinc finger proteins EGR-1 and AT133/EGR-4. Preferential binding of EGR-1 to the ZIP region was also strengthened by the use of antisera. In Jurkat cells, antisera against EGR-1, but not antisera specific for EGR-2, EGR-3, and AT133/ EGR-4, did interfere with formation of the induced complex. 3 Regulation of Gene Expression-The in vivo significance of the ZIP site on IL-2 gene expression was analyzed by transfection experiments. Various hybrid plasmid constructs containing the ZIP and NFAT sites with a regular or minimal IL-2 promoter were linked to the firefly luciferase (Luc) gene (Fig.  6A). Upon transient transfection into Jurkat T cells, the individual constructs responded differently to T lymphocyte activation signals (Fig. 6B). The complete IL-2 reporter gene construct pCILuc1 (Ϫ366 to ϩ51) (Fig. 6A) displayed regulatory activities characteristic for the endogenous IL-2 gene: it was inactive in unstimulated cells, but luciferase activity was induced 96-fold by stimulation with PHA and PMA. This induction was abrogated in the presence of cyclosporin A. 4 The minimal activity of construct pCILuc4 (Ϫ253 to ϩ51), which includes the previously identified binding sites for Oct-1, NF-B, AP-1, CD28RE, NRE, and Ets (for a review, see Ref. 24), shows the importance of a combination of the ZIP and the NFAT sites for IL-2 gene induction (Fig. 6B). The high activity obtained with the NFAT-containing construct (pCILuc2) is in agreement with previous results (34) and underlines the critical role of NFAT for IL-2 gene induction.
A single ZIP site (pCILuc3) increased promoter activity 3-fold and enhanced promoter activity in combination with the NFAT site (pCILuc1). To rule out regulatory effects by transcription factors binding between the NFAT site and position Ϫ63, the transcriptional role of the NFAT or ZIP site was also tested in front of a minimal IL-2 promoter (pMILuc4; Ϫ63 to ϩ51). A single NFAT or ZIP element (pMILuc2 and pMILuc3) increased activity of the minimal promoter pMILuc4, 2.2-and 4.2-fold, respectively ( Fig. 6B and Table I). The 20-fold increase in activity obtained by a combination of one ZIP with a single NFAT site highlights the role of the ZIP and NFAT binding factors in IL-2 gene expression and suggests synergism of action.

DISCUSSION
The current study identified a novel protein binding region in the human IL-2 gene promoter, which has a regulatory function in the early phase of T lymphocyte activation. Two zinc finger proteins Sp1 and EGR-1 were shown to bind to this element. In unstimulated and in stimulated Jurkat T cells, the ubiquitous transcription factor Sp1 binds to this region, while in activated cells the transiently induced EGR-1 protein binds this element. Due to binding of two differently regulated zinc finger proteins, this binding site was termed the zinc finger protein binding region (ZIP). By transfection experiments, a regulatory function of the ZIP site for IL-2 gene induction was demonstrated in Jurkat T cells. In PHA/PMA-stimulated cells, the ZIP site activated gene expression, particularly in combination with the NFAT region.
The ZIP site seems conserved in evolution as a highly related element is located in the same orientation in the mouse IL-2 gene promoter (position Ϫ298 to Ϫ290) immediately upstream of the NFAT binding site (Fig. 7). The conservation of the ZIP and NFAT domains in sequence and in location in both the human and the mouse IL-2 gene promoter is in agreement with the proposed role of this element in IL-2 gene regulation.
Although the ZIP site of the human IL-2 promoter does not display a typical binding site for Sp1 or EGR-1 (Fig. 1), both zinc finger proteins bind to this site. However, it has been demonstrated that Sp1 does not bind to the EGR consensus sequence (35)(36)(37)(38), and, similarly, no binding of EGR-1 to the Sp1 consensus sequence was observed (14,15). Despite this specificity of binding to the corresponding consensus sequences, binding to a range of different sites has been described for Sp1 as well as for EGR-1 (14,39,40). We conclude that there is exclusive binding of either Sp1 or EGR-1 to the ZIP site, as the mobilities of the factors remained unchanged, when both proteins were present in the nuclear extract at the same time (Fig. 3). The unchanged mobility of both complexes are also in agreement with this explanation. Overlapping EGR-1 and Sp1 binding sites as described here for the IL-2 gene promoter seem to be of general physiological relevance, as similar regions were identified recently in the promoters of the EGR-1 (15), the tumor necrosis factor ␣ (19), the mouse adenosine/deaminase (39), and the mouse thrombospondin 1 gene (40). Thus, competitive binding of these two distinct zinc finger proteins seems to play a critical role in gene regulation. Further experiments will clarify whether other Sp1 elements, found in a variety of gene promoters, can serve as binding sites and regulatory domains for inducible EGR proteins.
For the ZIP element of the IL-2 promoter, we could demonstrate preferential binding of EGR-1 but not of AT133/EGR-4 (Fig. 3). This is the first time that different binding specificities are reported for two members of this protein family. Although all four members of the EGR zinc finger gene family are coordinately induced upon mitogenic stimulation of T cells, binding of the individual proteins to their target sequences seems tightly regulated. Specificity of binding may be regulated by binding to slightly modified target sequences or by interactions with factors binding in close proximity of the identified binding region.
The significance of the ZIP site on IL-2 gene expression was demonstrated in vivo by transfection experiments using plasmid constructs containing the ZIP and NFAT site with a regular or minimal IL-2 promoter (Fig. 6A). The complete IL-2 reporter gene construct pCILuc1 displayed regulatory activities characteristic of the endogenous IL-2 gene, and induction was completely abrogated in the presence of cyclosporin A. The previously identified binding sites for Oct-1, NF-B, AP-1, CD28RE, NRE, and Ets are all included in construct pCILuc4. However, the low activity obtained with this construct demonstrates that these sites alone have a minimal effect on transcription. The high activity obtained with the NFAT-containing construct (pCILuc2) demonstrates the important role of this regulatory element for IL-2 gene induction in T cells. A single ZIP element (pCILuc3) increased promoter activity; however, in context with the regular IL-2 promoter, the activating effect of a single NFAT region was more pronounced than that of a single ZIP site. The increase in activity observed with a combination of the two sites (pCILuc1) suggests an additive effect of the binding proteins.
A regulatory activity of the ZIP and NFAT elements and a possible interaction of the binding factors are confirmed from the minimal IL-2 promoter constructs. Regulatory effects by transcription factors binding to the identified sites located between the NFAT site and position Ϫ63 were ruled out by analyzing the NFAT or ZIP sites in front of a minimal IL-2 promoter (pMILuc4; Ϫ63 to ϩ51). A single NFAT or ZIP element (pMILuc2 and pMILuc3) increased activity of the minimal promoter pMILuc4, 2.2-and 4.0-fold, respectively (Fig.  6B). The 20-fold increase in activity obtained by an IL-2 promoter element including the NFAT and the ZIP site, highlights the role of the ZIP and NFAT binding factors in IL-2 gene expression and suggests synergism of action.
Binding of the zinc finger protein EGR-1 to the ZIP site was demonstrated in stimulated Jurkat cells, suggesting that EGR-1 protein functions as a positive regulator of IL-2 gene transcription. Previous studies have shown that EGR-1 can function equally well as an activator and repressor of gene expression (5,14,15,19,39,40), and different functional domains have been localized within the protein (41). The close proximity of the ZIP and NFAT sites in the IL-2 promoter in combination with the experimental results suggests an interaction between their binding factors. Several proteins which are regulated differently have been shown to bind to the NFAT element (42,43). NFAT is composed of a pre-existing cytoplasmic component (NFATc/p) that translocates into the nucleus upon T cell activation (25,29,44). There it interacts with the ubiquitous, newly synthesized AP-1 component to form a protein complex. Neither the two recently cloned NFAT proteins (NFATp and NFATc) nor EGR-1 display a common motif indicative for this kind of interaction. Additional experiments aimed at describing the interaction of these transcription factors will contribute to an understanding of their synergism of action. A 366-bp fragment of the IL-2 gene promoter and deletions thereof were fused to the firefly luciferase gene (Luc). Constructs pCILuc1-4 include a regular IL-2 promoter (position Ϫ253 to ϩ51), while constructs pMILuc1-4 have the tested regulatory elements linked to a minimal IL-2 promoter (Ϫ63 to ϩ51). The ZIP and NFAT binding regions (compare Fig. 1A) are indicated. For construction details, see "Experimental Procedures." B, Jurkat T cells were transfected with the indicated plasmids by electroporation. After 24 h, the transfected cells were stimulated with PHA (1 g/ml) and PMA (25 ng/ml), and, 24 h later, cells were lysed and luciferase activity of the cell lysate was determined. Luciferase activity obtained with constructs (pCILuc1 and pMILuc1), which include a single ZIP and a single NFAT site, was set at 100%. Luciferase activity of each set of constructs is shown relative to the activity of construct pCILuc1 or pMILuc1. Each column represents the mean of five independent experiments, and standard deviations are indicated by bars.
Our data suggest that the zinc finger proteins Sp1 and EGR-1 are part of this transcriptional machinery which regulate the induction of the T cell growth factor IL-2.