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Section de Biologie, Institut Curie, F-91405 Orsay Cédex, France; Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; and || Génétique et Microbiologie, Centre Médical Universitaire, Universite de Genève, 1211 Genève 4, Switzerland
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gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2–responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4–CD8– cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4–CD8– thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti–Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.
IL-2 is a T cell–derived cytokine implicated in the regulation of growth and differentiation of a variety of cells expressing IL-2 receptors. The high affinity IL-2 receptor (IL-2R)1 consists of three distinct subunits, the IL-2R
The rodent thymic lymphoma-derived cell line PC60 is growth factor independent and resembles early thymic T cell precursors in that it expresses neither CD4 nor CD8 antigens. This line was the first in which induction of IL-2R, -β, and -
chains, which are assembled into a signaling complex after their interaction with IL-2 (for reviews see references 1–3). Lymphocytes control their level of IL-2 responsiveness by regulating the expression level of IL-2R. This is achieved via transcriptional regulation of the IL-2R gene, and in certain cells the IL-2Rβ gene; the IL-2R subunit is constitutively expressed (4–6). Resting lymphocytes do not express the IL-2R gene. Transcription of the gene is induced by signals from the antigen receptor, but in the absence of any other stimuli, these signals only trigger a transient wave of IL-2R synthesis in T lymphocytes. Maximal and sustained IL-2R transcription depends on IL-2 itself, which thus acts as a positive feedback regulator of IL-2R expression and IL-2 responsiveness. The level of IL-2R gene expression is also modulated by other extracellular stimuli, notably IL-1 and TNF (7–9). In CD4– CD8– thymocytes, IL-1 has a similar effect as antigen in mature T cells in that it acts synergistically with IL-2 to increase IL-2R transcription (6, 10). expression by IL-2 was reported (11) and is a well-characterized model system in which to analyze the IL-2 responsiveness of this gene (6, 12). As in normal CD4–CD8– thymocytes, IL-1 and IL-2 act synergistically to induce IL-2R transcription (9). In a manner similar to antigenic stimulation in mature T cells, in PC60 IL-1 induces only a transient wave of IL-2R expression, and it primes cells to become IL-2 responsive. Transcriptional control of the IL-2R gene is exerted by two separate regulatory regions in the 5' flanking region, one of which is promoter proximal and required for IL-1 inducibility and the other a more distal IL-2– responsive enhancer (IL-2rE) (6). Promoter-proximal positive regulatory regions (PRRs in the terminology proposed by John et al. [13]), located between positions –54 to –584 in the mouse, and –244 to –276 (PRRI) and –137 to –64 (PRRII) in the human IL-2R genes, respectively, are required for the rapid appearance of IL-2R mRNA after IL-1 exposure of PC60 or EL4 (14) cells, or PMA treatment of human T cell leukemias (12, 13, 15). Several DNA-binding factors were implicated in exerting effects on transcription via these elements, including NF-kB, Elf-1, and HMG-I(Y) (12–14, 16). Transcriptional stimulation by IL-2, on the other hand, operates via IL-2rE. The position of this enhancer in the mouse gene was mapped, by transient transfection experiments, to a 48-nucleotide stretch, 1.3 kb upstream of the transcriptional start site (12). It corresponds to a DNase I hypersensitive site (DH2), that appears in the chromatin of normal mouse T cells upon stimulation with Con A and IL-2 (10), and can increase transcription in response to IL-2 stimulation both in its normal context of 2.5 kb of IL-2R 5' flanking region as well as when inserted upstream of a non-orthologous promoter. Recently, the human homologue of the mouse IL-2rE has been identified in a region 
4 kb upstream of the transcription start site of the human IL-2R gene (17, 18). The mouse IL-2rE contains three separable cis-acting elements, named sites I, II, and III; mutations in any one of these three elements abolishes IL-2 responsiveness of reporter constructs containing the IL-2rE, demonstrating functional cooperativity between the sites (12) (see Fig. 1 A for summary). By identifying the proteins which bind to these DNA elements it will be possible to characterize the nature of some of the downstream transducers of the IL-2 signaling pathway.
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The aim of this study is to elucidate which, if any, of the Ets-like proteins bind to site III and form part of the complex of proteins generating the response of the IL-2R gene to IL-2. Using several experimental approaches, we demonstrate that site III specifically binds the Elf-1 transcription factor. A direct correlation between the effect of site III point mutations on Elf-1 binding in vitro and on the in vivo activity of the corresponding IL-2rE forms, strongly implies Elf-1 as the transcription factor involved in the IL-2 inducibility of the IL-2R gene. Experiments designed to elucidate the molecular basis of the cooperativity observed between the three sites making up the IL-2rE suggest that this phenomenon does not rely on the formation of direct interactions between Elf-1 and the factors binding to sites I and II.
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Plasmids and Preparation of Mutant Constructs.
The reference plasmid for transient transfection experiments, pGβAcβG1D, the wild-type IL-2R/reporter construct, pwt1βG1, in which the segment from nt –2539 to +93 of the IL-2R 5' flanking region is fused to a β-globin reporter gene, and the mutants used for the experiments described in Fig. 1, B and D have been described previously (12, 32). Mutations within the IL-2rE sequence were introduced into the pwt1βG1 plasmid using the Chameleon Mutagenesis Kit (Stratagene, Zürich, Switzerland). Plasmids were prepared using Qiagen Plasmid kits, and the validity of all the constructs was confirmed by DNA sequencing using a T7 polymerase sequencing kit (Amersham Rahn, Zürich, Switzerland).
Transfection of PC60 Cells and Reporter Gene Assays.
PC60 cells were cultured in the presence of IL-1 for 3 d and then transiently transfected with DNA constructs using the DEAE-dextran method (33). Each transfection included a defined ratio between the reference plasmid and a IL-2R/reporter construct. After transfection, cells were split into two equal aliquots. One aliquot was grown for an additional two days in the presence of IL-1 and IL-2, while the other was cultured in the presence of IL-1 alone. The transcripts from the IL-2R/reporter gene and from the reference plasmid were then measured by RT PCR as described previously (12, 32). Results from this assay were quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunny Vale, CA). Signals due to the IL-2R/reporter gene constructs were normalized by comparing them to signals generated from the reference plasmid. To determine the level of IL-2 induction, transcriptional activity generated from the reporter construct in the presence of both IL-1 and IL-2 was divided by the activity observed in cells cultured in the presence of IL-1 alone.
Electrophoretic Mobility Shift Assays (Bandshifts).
Nuclear extracts were prepared essentially as described by Schreiber et al. (34): 1 x 106 cells were rinsed in PBS and lysed in 200 µl of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 2% NP-40). The lysate was centrifuged, the supernatant discarded, and the proteins extracted from the nuclear pellet in buffer B (high salt buffer: 20 mM Hepes, pH 7.9, 0.4 M KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT). Both buffers A and B contained protease (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF) and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO3). Binding reactions were performed in a final volume of 20 µl in binding buffer (10 mM Tris, pH 7.5, 100 mM KCl, 10% glycerol, 1 mM DTT, 1 µg/ml BSA, 1 µg dIdC, 0.5 µg sonicated salmon sperm DNA) containing 1 µg of cell nuclear extract or 1 µg of recombinant protein, 1 x 106 cpm of an end-labeled PCR fragment (spanning bases –1384 to –1290 of the 5' flanking region of the IL-2R gene, Fig. 1 B) or SIII oligonucleotide probe (see Table 1). Reactions were incubated on ice for 20 min and then separated on 4.5% non-denaturing polyacrylamide gels in 0.3x TBE. For competition experiments, unlabeled PCR fragments or oligonucleotides were premixed with the radiolabeled probe before the addition of the proteins; for antibody supershift experiments the antibodies were preincubated with the nuclear proteins for the specified time before the addition of the radiolabeled probe.
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DNaseI Footprint Analysis.
The probe for in vitro footprinting assays was obtained by subcloning the 188-bp SauIIIA–HindII fragment of the IL-2R 5' flanking region into Sma I–BamHI– digested pUC18. To label the non-coding strand this plasmid was linearized with EcoRI and the resulting sticky end filled in with a nucleotide mixture containing [32P]ATP. After digestion with PstI the labeled IL-2R fragment was purified on a non-denaturing 8% polyacrylamide gel and recovered by electroelution. For each footprint reaction 2 ng of probe and, where indicated, a 100-fold excess of competitor was incubated with 35 µg of nuclear extract in 27 µl of buffer (33 mM Hepes, pH 6, 0.1 mM EDTA, 50 mM KCl, 0.6 mM DTT, 4 mM MgCl2, 0.4 mM 2-ME, 0.1 mg/ml poly-dIdC, containing several protease inhibitors, listed above) for 30 min on ice. The samples were prewarmed for 1 min at 20°C and 0.1 unit of Dnase I (Pharmacia, Duebendorf, Switzerland) in 2 µl 25 mM CaCl2 and 10 mM Hepes, pH 7.6, was added. After 1 min at 20°C the reaction was stopped and the DNA extracted, denatured, and fractionated on a 6% sequencing gel.
Recombinant Proteins and Antibodies.
Recombinant AcNPV baculoviruses expressing rElf-1 and rEts-1 proteins (35, 36), and anti– Ets-1/2 (no. 8 [37]), anti–Pu-1 (no. 65 [38]) and anti–Fli-1 (no. 61 [39]) antisera have been described previously. Anti–Elf-1 polyclonal antibodies (C-20) were purchased from Santa Cruz Biotechnology (CA), and HRP-coupled goat anti–rabbit antibodies from Biorad Laboratories (CA).
Affinity Precipitation of IL-2rE–bound Proteins.
Biotinylated IL-2rE probes were obtained as follows: (a) wild type and probes carrying mutations that abolish the activity of a single enhancer site were made by amplifying the corresponding reporter plasmid (M4 for site I, M9 for site II, M12 for III, see Table 1 and reference 12 for sequences) with primers spanning the segment between nucleotides –1402 (oligonucleotide A) and –1286 (biotinylated oligonucleotide B) of the IL-2R 5' flanking region. (b) Probes in which more than one site was inactivated were obtained in three steps essentially as described in Ho et al. (40) and explained here for the example of a probe in which all three enhancer sites are destroyed. (i) The plasmid carrying the mutation in site I (M4) was amplified with oligonucleotide A and a 3' oligonucleotide C', covering site II with the changes destroying this site. Simultaneously the plasmid with a mutation in site III (M12) was amplified with a 5' primer (C) complementary to oligonucleotide C' and oligonucleotide B. (ii) The resulting PCR products were gel-purified, denatured, and annealed with each other. (c) The annealing mixture was used as template for an extension reaction, and the resulting full-length IL-2rE fragments amplified with primers A and B. PCR fragments were purified and quantified by densitometric analysis of the corresponding bands on agarose gels.
IL-2rE–binding proteins recovered from whole PC60 cell extracts were isolated after lysis of 2.0 x 106 cells/ml in buffer C (50 mM Tris-HCl, pH 8.0, 0.5% NP-40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM Na3VO3, 1 mM PMSF, 1 mM DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin). Aliquots of extract from 2.0 x 106 cells were incubated with 1 µg biotinylated probe in buffer C at 4°C for 1 h. The probe and proteins bound were recovered by incubation with 30 µl streptavidin-agarose beads (Sigma Chemie, Buchs, Switzerland). Beads were washed twice in buffer C and then boiled in gel loading buffer (2% SDS, 10% glycerol, 80 mM Tris-HCl, pH 6.8, 0.01% bromophenol blue, 5% 2-mercaptoethanol). Eluted proteins were separated by SDSPAGE on 10% gels and transferred to Immobilon-P membranes (Millipore, Volketswil, Switzerland). Elf-1–specific bands were revealed with a 1/500 dilution of anti–Elf-1 antibody by ECL (according to the manufacturer's instructions, Amersham Rahn, Zürich, Switzerland).
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enhancer (nucleotides –1384 to –1290). This probe forms a number of complexes with proteins extracted from IL-1–primed and IL-2–induced PC60 nuclei (Fig. 1 B). To determine which of these complexes is due to specific DNA-binding proteins recognizing functionally important sites within the IL-2rE, we used a DNA competition-based assay. In this assay DNA fragments harboring specific mutations defining the sites required for IL-2rE enhancer activity are used as competitors to disrupt the formation of the IL-2rE complexes (Fig. 1 B). Three of the IL-2rE complexes were inhibited by the addition of an excess of unlabeled DNA, but only the complex with the lowest mobility (complex 1) results from an interaction with a protein specifically binding a discrete region of the IL-2rE. Mutations M12 and M13 strongly impair the capacity of the corresponding DNA fragments to compete for the formation of this specific complex. The flanking mutations M11 and M14 result in a relatively lower, but still significant, reduction in the competitive efficiency of the IL-2rE DNA. M12 and M13 abolish IL-2rE activity and define site III; the striking correlation of this in vivo effect with the effect on competitor efficiency in vitro suggests that the specific bandshift complex is due to one or several proteins participating in the control of the IL-2rE activity. On a probe spanning the entire IL-2rE (nucleotides –1472 to –1268), PC60 extracts produce a specific footprint extending from the 5' end of site III to several bases downstream, essentially covering the Ets-like protein consensus binding motif (Fig. 1 C, lanes 2 and 3). Detection of this footprint is abolished by the addition of unlabeled probe (Fig. 1 C, lanes 1 and 4). The protein complex that gives rise to the footprint is present in nuclear extracts from both unstimulated and IL-1–primed and IL-2–stimulated cells (compare Fig. 1 C, lanes 2 and 3). The same is true for the protein(s) forming the site III–specific complex detected in bandshift assays (data not shown). (Note that under the conditions used here we could not detect PC60 proteins binding specifically to site I or II. However, using other techniques we have shown that IL-2–induced STAT5 proteins bind to these sites but not to site III [Meyer, M., P. Reichenbach, V. Schindler, E. Soldaini, M. Nabholz, manuscript submitted for publication].)
SIII, a shorter IL-2rE probe spanning nucleotides –1336 to –1302, contains all the necessary sequence information to allow the formation of the site III–specific complex with extracts from PC60 (Fig. 1 D) or normal CD4–CD8– thymocytes (Fig. 1 E). The single, specific SIII complex demonstrates the same sensitivity to mutant IL-2rE fragments in competition assays as that of the complete IL-2rE probe– site III–specific complex (compare Fig. 1, B, D, and E).
The Ets-like Transcription Factor Elf-1 Is Present in the Site III–specific Complex.
Site III contains a consensus binding sequence for members of the Ets-like family of transcription factors. The two mutations, M12 and M13, which define site III and abolish competition for the site III–specific complex, destroy this consensus binding site. Mutation M14, which has no significant effect on enhancer activity and reduces competition in band shifts only to a minor extent (compare Fig. 1, A and B), results in a sequence that can still bind some Ets-like proteins. To determine which, if any, of the numerous Ets family members participates in the formation of the site III–specific bandshift complex several approaches were employed.
First, using recombinant Elf-1, an Ets-like protein, and Ets-1 itself in bandshift assays, we found that Elf-1 forms a complex with the SIII probe that migrates with a mobility identical to that of the complex generated in PC60 extracts. Recombinant Ets-1 binds to the SIII probe but results in the formation of a faster migrating complex (Fig. 2 A). The observation that the complex generated by PC60 nuclear extracts comigrates with that formed by recombinant Elf-1 suggests that there is no requirement for additional proteins.
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Third, the nature of the protein(s) which could be precipitated from PC60-derived nuclear extracts by biotinylated IL-2rE oligonucleotide probes was investigated. IL-2rE probes containing all three sites precipitate a protein recognized by the anti–Elf-1 antiserum and whose molecular mass, 91 kD, corresponds to that of Elf-1 (Fig. 3 A). The Elf-1 doublet observed on Western blots has been reported previously (41), and the relative levels of the two protein species remain unchanged after IL-1 priming or IL-2 induction (data not shown). As expected from the bandshift experiments, the presence in PC60 nuclei of Elf-1 active for DNA binding does not depend on IL-2 stimulation, although sometimes a small increase is seen in extracts from IL-2–treated cells (Fig. 3, A and C). Experiments with biotinylated IL-2rE probes containing mutations in sites I, II, or III clearly show that Elf-1 binding to IL-2rE is abolished by a mutation in site III, whereas mutations in sites I or II have no effect on the amount of Elf-1 protein precipitated (Fig. 3, B and C). The human IL-2rE contains an additional Ets-like protein-binding site overlapping a STAT consensus binding motif in site I (17, 18). Fig. 3 B demonstrates that the murine site I does not bind Elf-1, in agreement with the inability of site I to compete for the site III– specific complex (Fig. 1 B). Transfection experiments in human lymphoid cell lines suggest that Elf-1 binding to site I may produce a negative effect on the activity of the IL-2rE enhancer (17). If this effect is important in the regulation of the human IL-2R gene expression in normal lymphocytes, it suggests the presence of subtle differences in the transcriptional control of the homologous mouse and human genes.
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gene is interesting as this gene contains two (or, in man, three) Elf-1 sites, forming part of both the promoter proximal and distal control elements. Elf-1 DNA-binding activity in PC60 cells is not affected by IL-2 (Fig. 3 A). Thus, the simplest model for the molecular basis of the IL-2rE's IL-2 inducibility is that enhancer activity depends on the binding of other IL-2–activated proteins (most likely STAT5 [Meyer, W., P. Reichenbach, V. Schindler, E. Soldaini, M. Nabholz, manuscript submitted for publication]) to sites I and II and that Elf-1 remains constitutively present on site III. Constitutive binding of Elf-1 to regulatory elements implicated in stimulation of transcription has been described previously in studies of the PHA and PMA inducible IL-3 gene (23). Experiments to detect possible posttranslational modifications of Elf-1 controlling its capacity to contribute to enhancer activity have so far been unsuccessful. Elf-1 activity may also depend on interactions with other inducible factors binding in its immediate vicinity. In this context it is interesting that mutations M11 and 
15 flanking the Elf-1 binding site result in a moderate reduction of IL-2rE activity; these regions contain potential binding sites for NFIL-6 and AP-1. Note, however, that the site III specific complex formed by PC60 extracts has the same electrophoretic mobility as that formed with recombinant Elf-1, indicating that the PC60 complex does not contain additional proteins.
The Nature of the Functional Cooperativity between the Three Sites Making Up the IL-2rE Remains Unresolved.
One of the striking features resulting from the study of the mouse IL-2rE is that mutations in any one of the three sites that make up this enhancer abrogate IL-2 responsiveness. The observation that the distances between the three IL-2rE sites (20 nucleotides or two helical turns from center to center) are conserved between mouse and man suggests that evolutionary conservation of the relative orientations of proteins binding to them is important, possibly because of a requirement for direct interactions between the factors binding to the three sites. However, the insertion of five nucleotides in mutant I5, which changes the relative orientation of site III with respect to sites I and II, results in only a minor effect on IL-2 inducibility, arguing against direct protein– protein interactions (Fig. 5). Using biotinylated IL-2rE oligonucleotide probes, we demonstrated that the presence or absence of sites I and II has no affect on the amount of Elf-1 protein precipitated from PC60-derived nuclear extracts (Fig. 3 C; probing the same membranes with anti-STAT5 antiserum reveals that STAT5 binds to site I and II [Reichenbach, P., manuscript in preparation]) again arguing against direct protein–protein interactions. However, this possibility needs to be explored further using additional approaches.
The simplest model consistent with our findings is that the different IL-2rE binding proteins cooperate by interacting independently with components of the transcription initiation complex, as has been described previously in in vitro studies of transcriptional regulation by the Drosophila proteins Bicoid and Hunchback (46) and in the control of the c-fos gene (47). But our data do not rule out more complex models involving additional proteins or cofactors needed for an efficient stimulation of transcription by the IL-2rE.
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Acknowledgments |
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Submitted: 15 October 1996
Revised: 31 January 1997
1 Abbreviations used in this paper: IL-2R, IL-2 receptor; IL-2rE, IL-2–responsive enhancer; PRR, promoter-proximal positive regulatory region; STAT, signal transducers and activators of transcription.
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