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), ()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, 9, 10, 11, 12, 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, 5, 6, 13, 14, 15, 16, 17, 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, 21, 22, 23, 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.

EXPERIMENTAL PROCEDURES

Insect Cell Culture

Expression of Recombinant Proteins in the Baculovirus System

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IL-2 Reporter Constructs

Cell Culture and Transfection

Preparation of Nuclear Extracts and Mobility Shift Assay

For electrophoretic mobility shift assays, 0.2 ng (about 10 cpm) of P-end-labeled double-stranded oligonucleotides were incubated at 4 °C with 5-10 µg of nuclear proteins in the presence of 2 µg of poly(dI-dC) and 80 ng of salmon sperm DNA in 20 µl of 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 µM ZnCl, 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 ) 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

RESULTS

Binding of Nuclear Proteins to the ZIP Element of the Human IL-2 Gene

()

Figure 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 -250 to -300 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 -303 to -280 of the human IL-2 promoter, NFAT represents position -280 to -260 of the human IL-2 promoter, the NFAT and AP-1 binding sites are underlined, NFAT represents a mutated NFAT domain, the mutated nucleotides are underlined, and GC includes the boxed EGR consensus binding site(14) .

Figure 2: Protein complexes binding to the ZIP (position -303 to -280) oligonucleotide of the human IL-2 promoter. Electrophoretic mobility shift assay was performed with nuclear protein extracts prepared from Jurkat T cells stimulated with PHA (1 µg/ml) and PMA (25 ng/ml) for 2 h. Two specific complexes bound to the radiolabeled ZIP site (Upper and Lower complex). Competition with a 200-fold excess of unlabeled ZIP oligonucleotides showed specificity of both the upper and lower protein complexes (lane 3). Competition with an oligonucleotide representing the EGR consensus site (GC) showed specific competition of the lower band (lane 2), while an oligonucleotide with unrelated sequence (NFAT) had no effect (lane 4).

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. Competition of the lower, induced complex with EGR-specific oligonucleotides (GC) (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.

Figure 3: Identification of Sp1 and EGR-1 as proteins binding to the ZIP sequence of the human IL-2 promoter. In nuclear extract prepared from unstimulated human Jurkat T cells, the upper complex is specifically detected and the lower complex is absent (lane 1). However, in extract prepared from Jurkat cells stimulated for 2 h, two complexes are detected (lane 2). Specific antiserum identifies Sp1 in the upper complex, present in unstimulated and in stimulated Jurkat cells, and EGR-1 in the lower complex of extract prepared from stimulated Jurkat cells (lanes 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) did not affect binding, while competition with an oligonucleotide displaying an EGR consensus site (GC) specifically interfered with formation of the lower EGR-1-containing complex.

Regulation and Binding of the EGR-1 and Sp1 Proteins

Figure 4: Regulation of Sp1 and EGR-1 expression in the human helper T cell line Jurkat. A, Northern blot analyses demonstrates constitutive expression of Sp1 mRNA in the human helper T cell line Jurkat (upper panel). EGR-1 mRNA was not detected in unstimulated cells, but is detected upon stimulation with PHA (1 µg/ml) and PMA (25 ng/ml) (lower panel). Total cellular RNA was prepared from unstimulated Jurkat cells (0 h) or from cells stimulated for the indicated times in the absence or presence of cycloheximide (Chx; 10 µg/ml). Rehybridization of the filters with an actin cDNA probe showed that equal amounts of RNA were loaded (data not shown). B, the upper Sp1-containing complex is constitutively expressed in Jurkat T cells, and the lower EGR-1-containing complex is transiently induced upon stimulation with PHA and PMA. Binding assays were performed using 6 µg of nuclear extract prepared from untreated Jurkat cells or from cells treated for the indicated time with PHA and PMA. A P-labeled ZIP oligonucleotide was used for electrophoretic mobility shift assays.

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 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

Figure 5: Recombinant EGR-1 protein binds to the ZIP oligonucleotide (position -303 to -280) of the IL-2 promoter. Binding of recombinant EGR-1, expressed in the baculovirus expression system to the ZIP oligonucleotide (position -303 to -280 of the human IL-2 promoter). Binding was assessed by electrophoretic mobility shift assay in the absence (lane 1) or the presence of a 200-fold excess of unlabeled, unspecific (lane 2, NFAT) or specific (lane 3, ZIP) competitor DNA. Antiserum raised against EGR-1 affected formation of the complex (lane 4), while antiserum raised against the related zinc finger protein EGR-3 showed no effect (lane 5). In contrast to EGR-1, recombinant AT133/EGR-4 protein, also prepared in baculovirus-infected insect cells, did not bind to the ZIP oligonucleotide (lane 6). However, recombinant AT133/EGR-4 protein bound to the GC oligonucleotide, which includes a consensus site for the EGR proteins (lane 7).

Regulation of Gene Expression

()

Figure 6: The ZIP and NFAT sites regulate gene expression. A, schematic representation of the IL-2/luciferase gene hybrids used for transient transfections. 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 -253 to +51), while constructs pMILuc1-4 have the tested regulatory elements linked to a minimal IL-2 promoter (-63 to +51). 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 µg/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.

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 -63, the transcriptional role of the NFAT or ZIP site was also tested in front of a minimal IL-2 promoter (pMILuc4; -63 to +51). 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 1). 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 -298 to -290) 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.

Figure 7: Conservation of ZIP, NFAT, and AP-1 sites in the human and mouse IL-2 promoter region. A, the nucleotide sequences of the human IL-2 promoter region, position -303 to -250 bp upstream the transcriptional start site is aligned with the mouse IL-2 promoter region. The ZIP, NFAT, and AP-1 sites are boxed, the region inserted in the mouse promoter is indicated by colons. B, alignment of the complementary G-rich zinc finger binding elements of the human and mouse IL-2 gene promoters.

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 (35-38), 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 -63 were ruled out by analyzing the NFAT or ZIP sites in front of a minimal IL-2 promoter (pMILuc4; -63 to +51). 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. 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.

FOOTNOTES

*

This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant Zi 432/2-1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 0049-40-3-11-82-472; Fax: 0049-40-3-11-82-400.

()

The abbreviations used are: EGR, early growth response genes; NFAT, nuclear factor of activated T cells; ZIP, zinc finger protein binding region; AP-1, activator protein 1; IL-2, interleukin 2; bp, base pair(s); PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate.

()

C. Skerka and P. F. Zipfel, manuscript in preparation.

()

C. Skerka and P. F. Zipfel, unpublished data.

()

E. Decker, C. Skerka, and P. F. Zipfel, unpublished data.

ACKNOWLEDGEMENTS

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