Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Interaction of STAT5 Dimers on Two Low Affinity Binding Sites Mediates Interleukin 2 (IL-2) Stimulation of IL-2 Receptor α Gene Transcription

Interaction of STAT5 Dimers on Two Low Affinity Binding Sites Mediates Interleukin 2 (IL-2)... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 50, Issue of December 12, pp. 31821–31828, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Interaction of STAT5 Dimers on Two Low Affinity Binding Sites Mediates Interleukin 2 (IL-2) Stimulation of IL-2 a Gene Transcription* Receptor (Received for publication, August 5, 1997, and in revised form, October 2, 1997) Wolfram K.-H. Meyer‡, Patrick Reichenbach‡, Ulrike Schindler§, Elisabetta Soldaini‡¶, and Markus Nabholz‡i From the ‡Lymphocyte Biology Unit, Swiss Institute for Experimental Cancer Research (ISREC), 155 Chemin des Boveresses, CH-1066 Epalinges, Switzerland and §Tularik Incorporate, Two Corporate Drive, South San Francisco, California 94080 Stimulation of the interleukin 2 receptor a (IL-2Ra) receptor (for review see Waldmann et al. (10)). The capacity of gene by IL-2 is important for the proliferation of anti- mature T cells to proliferate in response to IL-2 correlates with gen-activated T lymphocytes. IL-2 regulates IL-2R IL-2Ra expression (11). Mouse T cells that constitutively ex- transcription via a conserved 51-nucleotide IL-2 respon- press the human IL-2Ra chain can respond to IL-2 in the sive enhancer. Mouse enhancer function depends on co- absence of antigen stimulation (12). Thus, IL-2Ra expression operative activity of three distinct sites. Two of these controls, at least in part, IL-2 responsiveness. are weak binding sites for IL-2-activated STAT5 (signal IL-2Ra cell surface expression is regulated mainly through transducer and activator of transcription) proteins, and changes in IL-2Ra gene transcription (13–16). In mature T mutational analysis indicates that binding of STAT5 to cells, antigen induces a transient wave of IL-2Ra transcription. both sites is required for IL-2 responsiveness of the en- Prolonged, maximal expression of IL-2Ra depends on stimula- hancer. The STAT5 dimers interact to form a STAT5 tion by IL-2, which thus acts as a positive feedback regulator of tetramer. The efficiency of tetramerization depends on its own high affinity receptor (17, 18). the relative rotational orientation of the two STAT mo- We have shown that stimulation of the mouse IL-2Ra gene tifs on the DNA helix. STAT5 tetramerization on en- by IL-2 is controlled by an IL-2-responsive enhancer (IL-2rE) hancer mutants correlates well with the IL-2 responsive- 1.3 kilobase pairs upstream of the major transcription start site ness of these mutants. This provides strong evidence (19). The IL-2rE maps to the same position as a DNase I that interactions between STAT dimers binding to a pair hypersensitive site that appears in the chromatin of normal of weak binding sites play a biological role by control- mouse T cells upon stimulation with concanavalin A and IL-2 ling the activity of a well characterized, complex cyto- kine-responsive enhancer. (18). The enhancer is 51 nt long and contains three distinct elements (named sites I, II, and III, see Fig. 1), all of which are required for enhancer activity (19). As pointed out previously Interleukin-2 (IL-2) is a growth factor for antigen-activated (19), sites I and II resemble binding sites for transcription lymphocytes (1, 2). It stimulates T cells through a high affinity factors of the STAT family (20 –23). Site II also overlaps with a cell surface receptor (IL-2R) composed of three transmembrane consensus binding site for GATA factors. Site III includes a polypeptide chains designated IL-2Ra, IL-2Rb, and IL-2Rg or consensus site for Ets proteins and contributes to IL-2rE activ- g (3). The b and g chains, which are shared with other inter- ity by binding the constitutive Ets protein Elf-1 (24). The hu- leukin receptors (4, 5), belong to the family of hematopoietic man homologue of the mouse IL-2rE has recently been identi- cytokine receptors. Resting T cells constitutively express the g fied approximately 4 kilobase pairs upstream of the chain (6, 7) and basal levels of the b chain (8). IL-2Rb expres- transcription start site (25–27). The binding motifs in sites I, II, sion increases upon antigen stimulation (9), . IL-2Ra is not a and III are conserved in the human IL-2rE (25, 26). member of this family and does not participate in the formation STAT proteins are latent transcription factors that dimerize of other known receptors. IL-2Ra expression is undetectable on in response to cytokine receptor activation. Dimerization re- resting lymphocytes but is induced by signals from the antigen sults in activation of their specific DNA binding activity and accumulation in the nucleus (22). IL-2 predominantly activates STAT5a and STAT5b, two closely related proteins encoded by * This work was supported, in part, by grants from the Swiss Na- separate genes (28, 29). STAT5 is rapidly activated by IL-2 in tional Science Foundation, the Swiss Cancer League and the Copley- May Foundation as well as by a grant from the Swiss Federal Office of antigen-stimulated but not in quiescent T cells (28, 30). The Education and Science, awarded in conjunction with a project approved human IL-2rE site I does bind STAT5a and b (25, 26). Muta- by the Human Capital and Mobility program of the European Union. tions in this site that abolish STAT5 binding also destroy The costs of publication of this article were defrayed in part by the IL-2-inducible IL-2rE activity in the human IL-2-dependent payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to leukemia Kit225 cell line (25). Site II may also contribute to indicate this fact. STAT5 binding (26). To what extent STAT5a and STAT5b ¶ Present address: Laboratory of Molecular Immunology, NHLBI, functions overlap is not yet clear, but the phenotypes of STAT5 National Institutes of Health, Bldg. 10, Rm. 7N264, Bethesda MD 20892. (31)- and STAT5b (32)-deficient mice show that they are not i To whom correspondence should be addressed: Tel.: 141 21 692 58 34; Fax 141 21 652 69 33; E-mail: [email protected]. completely redundant. The abbreviations used are: IL-2, interleukin 2; IL-2R, IL-2 recep- Here we provide evidence that IL-2 responsiveness of the tor; IL-2rE, IL-2-responsive enhancer; nt, nucleotide(s); STAT, signal mouse IL-2rE depends on the binding of STAT5 to both sites I transducer and activator of transcription; DOC, deoxycholate; PCR, and II and that the synergistic effect of these two sites on polymerase chain reaction; bp, base pair(s). S. Barange ´ and M. Nabholz, unpublished data. enhancer activity is due to the interaction of the bound STAT5 This paper is available on line at http://www.jbc.org 31821 This is an Open Access article under the CC BY license. 31822 IL-2Ra Regulation by STAT5 44). Mutagenesis of pwt was performed as described by Ho et al. (40). dimers. Our data show that DNA-induced association of STAT For this purpose, an additional XbaI site was created by changing the dimers, which has been observed for STAT4 and STAT1 (33, adenine at position 21307 into a guanine. This substitution did not 34), plays an important role in cytokine-regulation of gene alter IL-2 responsiveness (data not shown). A unique BclI site and the expression. new XbaI site were used to subclone PCR products of the site-directed mutagenesis. Positive clones were sequenced. EXPERIMENTAL PROCEDURES Reporter Gene Assay—IL-1-primed cells were transiently transfected Cell Lines, Culture Conditions, and Cytokine Stimulation—The using DEAE dextran according to Queen and Baltimore (45) and cul- PC60.21.14 cell line (referred to as PC60) is a hybrid between a mouse tured with IL-1 only or with IL-1 and IL-2. Reporter gene expression cytolytic T cell line and a rat thymic lymphoma. It has inherited from its was measured 48 h later, as described previously (44). Briefly, cells lymphoma parent (35) the capacity to grow independently of IL-2 and a were cotransfected with a defined ratio of an IL-2Ra/rabbit b-globin 2 2 2 2 CD4 CD8 phenotype. Like normal CD4 CD8 thymocytes (36), it gene together with the reference plasmid (pGbAcbGlD), which is con- expresses IL-2Ra when stimulated with IL-1 and IL-2. IL-1 plays a stitutively expressed in PC60 cells and contains a 40-bp deletion in the similar role in these cells as antigen in mature T lymphocytes and second exon (46). Relative amounts of mRNA transcribed from the induces PC60 cells to become IL-2-responsive. This effect has been reference plasmid and the IL-2Ra/b-globin construct were determined described in detail in previous publications (16, 19, 35, 37–39). The cells by PCR (see Refs. 19 and 44 for details). were cultured for 2–3 days in 1 ng/ml human IL-1b before stimulation Proteolysis and Amino Acid Sequence Analysis—20 mg of recombi- with 100 units/ml human IL-2 in the continued presence of IL-1. Re- nant active STAT5a were digested with 3 mg of thermolysin (Boehringer combinant interleukins were gifts of Glaxo Institute for Molecular Mannheim) in a total volume of 400 ml of digestion buffer (50 mM Biology S.A. (Plan-les-Ouates, Geneva, Switzerland). Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol) at room temperature Probes—Oligonucleotides from MWG-Biotech (Duebendorf, Ger- for 10 and 30 min. Thermolysin was inactivated by adding 4 mlof500 many) or Microsynth (Balgach, Switzerland) were annealed, purified on mM EDTA, pH 8. Undigested protein was incubated in the same buffer acrylamide gels, quantified, and stored in 10 mM Tris-HCl, pH 7.5, 50 without thermolysin. Electromobility shift assays were performed as mM NaCl. As a nonspecific competitor (NS) for bandshift assays, a described above, with 1 ml of the thermolysin reaction. The remaining double-stranded oligonucleotide with the sequence 59-AGAGTTAGCT- reaction was diluted with SDS sample buffer (47), and the fragments TGCGGTTCCCAGG-39 was used. were separated on a 10% SDS gel. The proteins were blotted onto Probes were obtained either by annealing appropriate oligonucleo- polyvinylidene difluoride membrane (Millipore) and visualized by Coo- tides (FcgRI, single IL-2rE sites I or II, IL-2rE sites I and II, nonspecific massie staining. N-terminal protein sequence analysis was performed probe NS (for sequences see Fig. 1)) or by PCR amplification of different by the protein facility at Tularik, Inc. using an AB477A protein se- IL-2rE templates with primers spanning segments of the IL-2rE. For quencer and an AB120A analyzer. competition assays, probes extended from nt 21420 (primer A) to nt 21331 (primer B) of the IL-2Ra 59-flanking region, including sites I and RESULTS II. For affinity precipitation and the bandshift experiment in Fig. 7C, Sites I and II of the IL-2rE Are Weak Binding Sites for probes extended from nt 21420 (primer A) to nt 21286 (primer C), STAT5—Two of the functional sites in the IL-2rE (sites I and including site III. To generate probes carrying a mutation in a single enhancer site, we amplified the corresponding reporter plasmid (M4 for II) contain potential STAT binding motifs (see Fig. 1 and site I, M9 for site II, and M12 for site III; see Fig. 1 and Sperisen et al. Sperisen et al. (19)). To determine whether the IL-2rE could (19) for sequences). Probes with mutations in more than one site were bind IL-2-induced STAT5, we incubated PC60 extracts with a obtained in three steps, essentially as described in Ho et al. (40) and biotinylated, double-stranded oligonucleotide comprising the explained here for a probe in which all three enhancer sites are de- entire enhancer. Bound proteins were recovered by incubating stroyed. (a) The plasmid carrying the mutation in site I (M4) was the probe with streptavidin-coated beads and characterized by amplified with oligonucleotide A and a 39 oligonucleotide D9 covering site II, with changes destroying this site. Simultaneously the plasmid Western blotting. The complete IL-2rE binds proteins of about with a mutation in site III (M12) was amplified with a 59 primer (D) 90 kDa, which are present in extracts from IL-2-stimulated complementary to oligonucleotide D9 and oligonucleotide C. (b) The PC60 cells and react with anti-STAT5a and anti-STAT5b an- resulting PCR products were gel-purified, denatured, annealed, and tisera (Fig. 2A) as well as with an anti-phosphotyrosine anti- used as template for an extension reaction. The resulting full-length body (data not shown). No STAT5 proteins could be recovered IL-2rE fragments were amplified with primers A and C. The final PCR from extracts of cells that had not been treated with IL-2. As products were quantified and analyzed by sequencing. Recombinant Proteins—Expression, activation, and purification of expected, IL-2 induces rapid activation of STAT5. The amount recombinant STAT5a have been described previously (41, 42). of IL-2rE-bound STAT5 continues to increase and reaches a Antibodies—The anti-STAT5 antibody was purchased from Santa plateau only after 24 – 48 h of culture in IL-2 (Fig. 2B). This Cruz Biotechnology, Inc., STAT5a and b antibodies are fromR&D correlates with the kinetics of IL-2-induced accumulation of Systems, and horseradish peroxidase-coupled goat-anti-rabbit antibod- IL-2Ra message and IL-2rE-driven reporter gene expression ies were from Amersham. (19). We did not detect any IL-2-inducible proteins that reacted Affinity Precipitation of IL-2rE-bound Proteins—Affinity precipita- tion of IL-2rE-bound proteins was performed as described in Ref. 30. with antisera against STAT1 and STAT3 (data not shown). Electrophoretic Mobility Shift Assays (Bandshifts)—Nuclear extracts To determine which segment of the IL-2rE is responsible for were prepared as described by Schreiber et al. (43). Lysis and extraction STAT5 binding, we used biotinylated IL-2rE oligonucleotides buffers contained protease (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM that carry mutations in one or more of the sites required for phenylmethylsulfonyl fluoride) and phosphatase inhibitors (10 mM enhancer function. Fig. 2A shows that a probe containing an NaF, 1 mM Na VO ). Binding reactions were performed in a final 3 4 intact site I and mutations in the other sites bind STAT5a and volume of 20 ml in binding buffer (10 mM Tris-HCl, pH 7.5, 60 mM KCl, 10% glycerol, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 1 mg b. Probes containing an intact site II and mutations in the of poly(dIcdC), 0.5 mg of sonicated salmon sperm DNA) containing 4 mg other sites bind a trace of STAT5, whereas probes with muta- of cell nuclear extract or the indicated quantity of recombinant STAT5a tions in both sites I and II fail to bind STAT5. Incubation of the protein and 2–3 3 10 cpm (30 fmol) of radiolabeled PCR fragments or same filters with an antibody against Elf-1 had shown that oligonucleotides (see Fig. 1 for sequences). only oligonucleotides with an intact site III bind this transcrip- Reactions were incubated on ice for 20 min and separated on 4% tion factor (24). nondenaturing polyacrylamide gels in 0.3 3 Tris/borate/EDTA. For competition experiments, unlabeled PCR fragments or oligonucleotides These results demonstrate that STAT5a and b can specifi- were mixed with the labeled probe before addition of the proteins. When cally bind to site I and, albeit very weakly, to site II. To confirm deoxycholate (DOC) was used, appropriate dilutions were added to the these data, we carried out bandshift experiments with recom- binding reaction at the same time as STAT5 and the probe. The mixture binant STAT5a protein (Fig. 3A) and PC60 nuclear extracts was incubated for 30 min at room temperature before loading on the gel. (Fig. 3B). Fig. 3 shows that both site I and site II can compete Plasmids—The reference plasmid pGbAcbGlD and the plasmid pwt, specifically for the binding of STAT5a to a probe that contains in which the mouse IL-2Ra 59-flanking region and promoter has been joined to the rabbit b-globin gene, have been described previously (19, the STAT binding site of the FcgRI gene (see Fig. 1). Titration IL-2Ra Regulation by STAT5 31823 FIG.1. Nucleotide sequence of site I and site II of IL-2rE and probes. Gray boxes indicate the previously defined IL-2rE regions required for IL-2 responsiveness of the IL-2Ra gene. Putative STAT binding sites have been underlined. Below, the sequences of the different probes used have been listed together with their designation in the text and the symbols used in the figures. A blank box indicates a wild type element, whereas a crossed box indicates a substitution mutation that destroys IL-2rE enhancer activity (19). F stands for the high affinity FcgRI STAT binding site. of various competitors indicates that the affinities of sites I and II for STAT5a are, respectively, 10-fold and 50-fold lower than that of the FcgRI STAT binding site. Extracts from IL-2-stim- ulated but not from unstimulated PC60 cells form a complex with the FcgRI probe that is due to IL-2-induced STAT5, as shown by supershift experiments with STAT5-specific antibod- ies (data not shown). The competition experiment shown in Fig. 3 demonstrates that PC60 STAT5 and recombinant STAT5a have the same relative affinity for site I and site II. In certain experiments, IL-2 also stimulated the appearance of a small amount of STAT1 (visible as a faint specific band below the STAT5 complex in Fig. 3B), but it is unlikely that STAT1 plays a role in IL-2rE activity. Interferon-g that induces STAT1 but not STAT5 does not stimulate IL-2Ra expression, nor does it affect the response to IL-2 (data not shown). Furthermore, IL-2-dependent IL2Ra expression in T lymphocytes from inter- feron-g receptor-deficient mice is normal despite the fact that stimulation with concanavalin A and IL-2 fails to induce STAT1 activation. The IL-2rE STAT Motifs Induce Association of STAT5a Dimers—IL-2 responsiveness of the IL-2rE depends on synergy between the individual IL-2rE sites. This could reflect cooper- ative binding of the transcription factors controlling enhancer activity. Cooperative binding to pairs of weak binding sites has indeed been observed for STAT4 (33) and STAT1 (34). The experiments described so far were carried out with a large excess of binding sites and were therefore unlikely to reveal any cooperativity between sites I and II. To look for synergistic binding, we resolved the complexes formed by IL-2rE probes with increasing concentrations of STAT5 in bandshift assays. Since STAT5 complexes formed by crude PC60 extracts with these probes are obscured by other, nonspecific complexes, we resorted to recombinant STAT5 for these experiments. Fig. 4A shows that STAT5a forms two retarded complexes (referred to as C1 and C2) with a 39-bp probe that contains intact sites I FIG.2. STAT5a and STAT5b from PC60 extracts bind to IL-2rE and II (probe I/II). Complex C2 comigrates with the specific oligonucleotide probes containing intact sites I and II. A, whole band formed by the short (20 bp) FcgRI probe used in Fig. 3 (see cell extracts were prepared from unstimulated PC60 cells or from cells also Fig. 4C) and is presumably due to STAT5a dimers. Quan- incubated for 30 min with IL-2. Extracts were incubated with the indicated biotinylated probes, which were then recovered by incubation titative analysis (by PhosphorImager) of the amount of probe with streptavidin-coated-agarose beads. Bound proteins were fraction- retained in the two complexes showed that the ratio of C1 to C2 ated by SDS-PAGE together with molecular mass markers and crude increased with rising concentrations of STAT5 protein, indicat- nuclear extracts (NE). Gels were blotted onto membranes that were ing that C1 represents a trimolecular complex between the incubated with different anti-STAT5 antibodies. B, extracts from PC60 cells stimulated for the indicated times with IL-2 were incubated with a biotinylated wild type IL-2rE probe, and bound, recovered proteins C. Rusterholz, unpublished data. were immunoblotted with anti-STAT5 antiserum. 31824 IL-2Ra Regulation by STAT5 This interpretation is supported by experiments with a 39-bp probe in which site I was replaced by the high affinity FcgRI STAT consensus site I, and site II was inactivated (probe F/2). Fig. 4C shows that, at low protein concentrations, this probe forms a single complex C2 that comigrates with the complex formed by STAT5a binding to the short FcgRI probe (F) used in Fig. 3. At higher STAT5 concentrations the F/2 probe, but not the short F probe, gives rise to an increasing proportion of C1 complexes. As expected, the ratio between C1 and C2 at a given STAT5 concentration is higher when the FcgRI STAT consen- sus site is combined with an intact site II (probe F/II). Thus, F/2,I/2, and 2/II probes, but not the short F probe, induce STAT tetramerization, indicating that binding of STAT5 to a single specific binding site induces the formation of a STAT5 tetramer, provided that the probe contains sufficient flanking DNA. STAT5 tetramers are expected to dissociate with a lower off-rate than dimers, as has indeed been demonstrated for STAT1 (34). This explains why the only complex detected in bandshifts with probes containing a single very weak binding site (2/II) is C1, whereas probes with a single high affinity binding site form predominantly complex C2. As expected, C1 complex formation is enhanced by the inclusion of two STAT5 binding sites in the same probe (I/II, F/II), resulting in a syn- ergistic increase of STAT binding to such probes. Thus, in the presence of 1–2 mg of STAT5a, the total amount of STAT5a bound to probe I/II is 2.5–3 times higher than the sum of protein bound to probe I/2 and to probe 2/II. Tetramer Formation Depends on Specific STAT5 Protein Segments—Formation of STAT4 and STAT1 tetramers de- pends on their N termini (33, 34). To determine whether STAT5 tetramer formation also requires interaction between specific protein domains, we investigated the effect of proteo- FIG.3. STAT5 binds to site I and site II of the IL-2rE with lytic clipping on the capacity of STAT5a to form tetrameric different affinities. Recombinant STAT5a (A) or nuclear extract of complexes. Treatment of STAT5a with thermolysin yields two PC60 cells stimulated for 15 min with IL-2 (B) was incubated for 15 min with 30 fmol of FcgRI probe mixed with different amounts of the proteolytic fragments with apparent molecular masses of about following unlabeled competitor oligonucleotides: nonspecific NS, FcgRI, 75 and 60 kDa (Fig. 5A). Western blotting with an antibody site I or site II of the IL-2rE (see Fig. 1; see “Experimental Procedures”). against the C-terminal domain of STAT5a revealed that both Reactions were analyzed by gel retardation assays. proteolytic fragments lack the C terminus (data not shown). N-terminal amino acid sequencing of the 60-kDa fragment IL-2rE probe and two STAT5 dimers binding to sites I and II, yielded the sequence ILVDAMSQK, indicating that this clip- respectively. Surprisingly, a 39-bp IL-2rE probe containing a ping product starts at amino acid 130. The molecular mass of single cognate STAT5 binding site (probe I/2) also gave rise to this fragment is consistent with the absence of both N- and complexes C1 and C2, but both bands were weaker, and the C-terminal domains of STAT5a. No N-terminal sequence could proportion of the C2 complex was higher. Nevertheless, in- be obtained from the 75-kDa fragment. Its molecular mass creasing STAT5 concentrations resulted in a shift toward com- suggests that this fragment still retains the N terminus, which plex C1. A probe with an intact site II and a mutated site I is probably blocked. (probe 2/II) formed a small amount of complex C1 and virtually We compared the complexes formed by full-length STAT5a no detectable complex C2. Competition experiments confirmed and by the proteolytic digests with the IL-2rE probe I/II and the that the mutations in site I and site II abolish detectable short FcgRI probe F (Fig. 5B). As described above (Fig. 4A), binding of STAT5 to these sites (data not shown). intact STAT5 predominantly forms the slower migrating com- The observation that the IL-2rE probes containing a single plex C1 with the probe I/II (in this experiment C2 is barely binding site (probes I/2 and 2/II) form C1 complexes suggested visible), whereas probe F gives rise to the faster migrating that a STAT dimer bound to one site could interact with an- complex C2. The proteolytic fragments, on the other hand, form other dimer to form what we will refer to as STAT5 tetramers. with either probe a complex C2* that migrates faster than C2. Alternatively, complex C1 formed by these probes might be due A small amount of an additional complex C1* is observed with to preferential binding of a small amount of pre-existing tet- probe I/II but not with probe F. These data suggest that com- ramers in the STAT preparation. To distinguish between these plex C2* is formed by the 60-kDa fragment that lacks the possibilities, we compared the capacity of different IL-2rE probes to compete for the C1 and C2 complexes formed by the domain necessary for protein-protein interaction, whereas C1* is due to the 75-kDa fragment that is still capable of forming I/2 probe (Fig. 4B). If formation of C1 by the probes I/2 and 2/II is due to the higher affinity of preformed STAT5 tetramers tetramers. Thus, as observed for STAT1, proteolytic cleavage of STAT5 strongly reduced its propensity to form trimolecular for these oligonucleotides, then they should compete better for C1 than for C2. However, the IL-2rE oligonucleotides compete complexes with a probe containing two weak binding sites, with the same relative efficiency for the two complexes as the most likely because proteolysis has removed the N-terminal short, high affinity FcgRI probe (F). This indicates that forma- domain required for tetramerization. Alternatively, the small tion of STAT5 tetramers is DNA-dependent. amount of the putative trimolecular complex C1* could be due IL-2Ra Regulation by STAT5 31825 FIG.4. Cooperative binding of STAT5 to IL-2rE. A, increasing amounts of recombinant STAT5a were incubated with a wild type IL-2rE probe or with probes in which site I or site II contained a mutation that destroys STAT5 binding and enhancer activity. C1 and C2, specific STAT5 complexes. Empty arrowhead, free probe. B, 1.6 mg of recombinant STAT5a was incubated with the IL-2rE probe containing a mutation in site II (I/2) together with increasing amounts of the following unlabeled competitors: a 20-nt stretch containing the FcgRI high affinity STAT binding site (F) or IL-2rE segments harboring a mutation in site II (I/2)orinsiteI(2/II), respectively. C, increasing amounts of STAT5a were added to a 20-bp FcgRI probe (F) or to 39-bp probes in which the FcgR STAT motif was combined with a mutated (F/2) or wild type (F/II) site II of the IL-2rE. to simultaneous occupation of both STAT5 binding sites in severely reduced by 0.03% detergent (data not shown). This is probe I/II at high protein concentrations, even in the absence of further evidence that C1 complexes depend on an interaction protein-protein interactions. In either case, our data indicate between the bound STAT5 dimers and are not simply due to that, as reported for STAT4 and STAT1, the capacity of STAT5 occupancy of both IL-2rE binding sites by independently bind- to cooperatively bind to a probe containing two binding sites ing STAT5 molecules. depends on interactions involving modular protein domains. IL-2rE Activity Depends on Intact STAT5 Motifs in Sites I We also investigated the effect of deoxycholate, which is and II—Previously we defined the limits of site I and of site II known to disrupt protein-protein interactions (48), on the for- by substitution mutations. The finding that they coincided to mation of C1 and C2 complexes. Fig. 6 shows that 0.25% DOC within one nucleotide with the borders of STAT motifs provided is required to substantially reduce formation of C2 complexes the first evidence suggesting an involvement of STAT proteins with the wild type IL-2rE probe as well as the high affinity, in IL-2rE function (19). The binding site for STAT5 in site II single site FcgRI probe. Formation of C1 complexes, on the overlaps with a binding motif for GATA factors. The previously other hand, is almost completely prevented by 0.06% DOC and described mutants did not exclude that the contribution of site 31826 IL-2Ra Regulation by STAT5 FIG.6. Sensitivity of STAT5 complexes to deoxycholate. Bind- FIG.5. The effect of proteolytic cleavage on STAT5 tetramer ing reactions between STAT5a and the wild type IL-2rE probe or the formation. A, analysis of proteolytic cleavage products of STAT5. high affinity FcgRI probe were carried out in presence of the indicated Recombinant STAT5a was digested with thermolysin for the indicated concentrations of DOC. Complexes were separated on a nondenaturing times. Aliquots of the reaction were resolved on a SDS-polyacrylamide gel. gel, and proteins were visualized by Coomassie staining. B, analysis of DNA binding complexes formed by thermolysin-treated STAT5a with with regard to the helical axis of the DNA is important for an the IL-2rE probe I/II or the short FcgRI probe F. efficient association of the STAT5 dimers into tetramers and thereby affects IL-2rE enhancer activity. II to IL-2rE activity depends on a GATA protein. Fig. 7 shows DISCUSSION that a mutation in site II that leaves the GATA binding motif intact but destroys the STAT site (mutant S-II.1) abolishes Previously we showed that the response of the IL-2Ra gene enhancer activity. This indicates that the binding of STAT5 to to IL-2 is mediated by a 51-nt IL-2-responsive enhancer (IL- site II is required. In addition, a mutation that leaves the STAT 2rE). The IL-2rE consists of three sites, I, II,and III, that motif intact and destroys the GATA motif (mutant S-II.2) con- functionally cooperate to activate transcription (19). The func- serves IL-2rE activity. In fact, this mutation, which transforms tion of site III most likely depends on Elf-1, constitutively site II into a copy of site I, gives an enhancer that responds present in PC60 cells and normal T lymphocytes (Serdobova et slightly better than the wild type IL-2rE. Probably this reflects al. (24)). This paper provides strong evidence that cooperative the stronger affinity of STAT5 for site I compared with site II. binding of IL-2-activated STAT5 to sites I and II plays a crucial The inverse change, resulting in a replacement of site I with role in IL-2rE activity. another copy of site II (S-I.1) abolishes IL-2 responsiveness and Stimulation of IL-2Ra Gene Transcription by IL-2 Depends greatly diminishes STAT5 binding (data not shown), indicating on STAT5 Activation—IL-2 stimulates IL-2Ra transcription that two copies of site II do not bind STAT5 with sufficient via binding of induced STAT5 to the IL-2rE. The onset of affinity to allow the enhancer to function. STAT5 activation is as rapid as expected for STAT factors, but The Topological Relationship between Sites I and II Affects the DNA binding activity of STAT5 continues to increase for STAT5 Binding and IL-2rE Function—The center-to-center 48 h. This is parallel with increasing IL-2Ra expression and distance between the STAT binding motifs in sites I and II is 20 IL-2rE-driven reporter gene activity (19). IL-2rE sites I and II bp or two turns of the DNA helix in both man and mouse (25, bind STAT5a as well as STAT5b with a weak affinity. Their 26). Thus, the STAT molecules binding to the two sites face the relative affinity for STAT5 in nuclear extracts is the same as same side of the helix, and it appeared likely that tetramer that for recombinant STAT5a, indicating that no accessory formation depends on this topological relationship between the proteins are required for the binding of the IL-2rE sites to interacting STAT5 molecules. To test this, we changed the STAT5. spacing between the two sites by inserting 5 or 10 bp (Fig. 7A) Previous mapping of site II had left open the question of and compared the complexes that such probes formed with whether the contribution of this site was due to a STAT motif STAT5 with those formed by the wild type probe. The addition or to an overlapping GATA binding site. The data presented of 5 bp or half a helical turn (mutant I-5-II) clearly reduces the here show that the STAT motif in site II is required for en- formation of complex C1 but has a lesser effect on complex C2 hancer activity and argue against a role of GATA factors. (Fig. 7B). PhosphorImager analysis showed that probe I-5-II The affinity of site II for STAT5 is approximately 5 times binds approximately half the number of STAT5a molecules as lower than that of site I. A point mutation that changes the the wild type probe. This mutation reduces the IL-2 response of STAT motif in site I to that in site II abolishes the IL-2 re- the enhancer from 8- to 3.5-fold (Fig. 7C). Insertion of 10 bp sponse of the IL-2rE. Together these results support a predom- (I-10-II) or one helical turn has no significant effect on either inant role of STAT5 in the regulation of IL-2rE activity. As has STAT5 complex formation or enhancer response. These results been pointed out by others (49, 50), STAT binding motifs in suggest that the rotational orientation of the STAT5 molecules natural regulatory elements are often sites with relatively low IL-2Ra Regulation by STAT5 31827 FIG.7. Effect of IL-2rE mutations on IL-2 responsiveness and STAT5 binding. The effects of different mutations (A) on STAT5 binding (B) and the IL-2 inducibility of the IL-2rE (C) were investigated. B, analysis of DNA binding complexes formed by different IL-2rE probes spanning the segment between nt 21286 and 21420. In the last two lanes a 100-fold excess of competitor was included in the reaction. Competitors were: F,FcgRI STAT motif; NS, nonspecific oligonucleotide. C, 2.5-kilobase pairs of the mouse IL-2Ra gene 59-flanking region containing either the wild type or the indicated mutant form of the IL-2rE were fused to the rabbit b-globin gene. These plasmids were transiently transfected into PC60 cells together with a reference plasmid. The transfected cells were cultured either with or without IL-2. Two days later, the relative amounts of reporter gene mRNA were measured, and the IL-2 response of each plasmid was determined (see “Experimental Procedures”). Transfections were repeated at least twice with all constructs. For each mutant, the mean response 6 S.D. is given. Shading of the histogram bars distinguishes different classes of mutations: black, wild type (wt) IL-2rE; white, point mutations; hatched, insertions between sites I and II. affinity. In contrast to high affinity binding sites, low affinity in the mouse IL-2rE depends on the DNA-induced interaction sites preferentially bind a subset of STAT factors. This may between two STAT5 dimers. Efficient formation of STAT5 tet- contribute to the selective responsiveness of a gene to particu- ramers appears to require that both STAT5 molecules bind to lar STAT proteins and cytokines. the same side of the DNA helix, since insertion of a half-turn Similarities and Differences between Human and Mouse IL-2 between the two binding sites reduces association, whereas Responsive Enhancers—The human homologue of the mouse insertion of a complete turn does not result in a significant IL-2rE is located approximately 4 kilobase pairs upstream of change. The correlation of these effects with those on IL-2 the transcription start site (25–27). The two enhancers share responsiveness of the IL-2rE strongly indicates that the pair of the consensus motifs first identified in the mouse gene (19), STAT motifs in the IL-2rE affects enhancer activity by induc- and the spacing between these motifs is conserved. Partial ing STAT5 association. This is the first evidence that STAT functional analysis of the human IL-2rE results in a picture tetramer formation plays an important role in the function of a that is similar but not identical to that of the mouse IL-2rE (25, well characterized cytokine-responsive enhancer. Thus, our re- 26). The most striking difference between the human and sults demonstrate that the phenomenon of STAT tetrameriza- mouse IL-2rE is a base pair inversion, which creates an addi- tion that has been described by others for STAT4 and STAT1 tional Ets motif that overlaps with the STAT motif in site I in (33, 34) is indeed biologically important. the human IL-2rE. The effect of different mutations in this site It is very likely that cooperative binding of STAT5 to the two as well as cotransfection experiments with Elf-1 expression IL-2rE sites reflects a reduction in the off-rate of the DNA from vectors suggest that the Ets motif in the human site I acts as a tetramers compared with that from dimers, as has been dem- negative regulator of IL-2rE activity in unstimulated cells (25, onstrated for STAT1 (34). Our data suggest that, like STAT4 26). Since STAT5 binds more avidly to the human site I than to and STAT1, STAT5 requires the N-terminal domain for tet- the mouse homologue, such an element may be required to ramerization. But our results also point to differences between prevent inappropriate IL-2Ra expression. the interactions of different STAT dimers. Unlike STAT4, STAT5 Controls IL-2rE Activity by Cooperative Binding to STAT5 does not form tetramers with short (20 nt) probes. Two Adjacent Sites—Our experiments indicate that the func- However, STAT5 tetramers are formed on a 39-nt probe that tional cooperativity between the two weak STAT5 binding sites contains a single cognate binding site between nt 6 and 15. This suggests that tetramerization depends on simultaneous con- P. Le ´ cine and J. Imbert, personal communication. tacts of both participating dimers with the same DNA molecule 31828 IL-2Ra Regulation by STAT5 14. Ondek, B., Shepard, A., and Herr, W. (1987) EMBO J. 6, 1017–1025 but that nonspecific interactions are sufficient to induce asso- 15. Depper, J. M., Leonard, W. J., Drogula, C., Kro ¨ nke, M., Waldmann, T. A., and ciation. Unlike that of STAT5, STAT1 tetramerization does not Greene, W. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4230 – 4234 16. Plaetinck, G., Combe, M.-C., Corthe ´ sy, P., Sperisen, P., Kanamori, H., Honjo, appear to be significantly affected by the rotational orientation T., and Nabholz, M. (1990) J. Immunol. 145, 3340 –3347 of the two STAT motifs on the DNA (34). Regardless of these 17. Pimentel-Muinos, F. X., Munoz-Fernandez, M. A., and Fresno, M. (1994) differences, our data indicate that tetramerization of STAT J. Immunol. 152, 5714 –5722 18. Soldaini, E., Pla, M., Beermann, F., Espel, E., Corthe ´ sy, P., Barange ´ , S., proteins (33, 34) is a general mechanism through which these Waanders, G. A., MacDonald, H. R., and Nabholz, M. (1995) J. Biol. Chem. factors control transcription via weak pairs of binding sites, 270, 10733–10742 which have been observed in several other putative regulatory 19. Sperisen, P., Wang, S. M., Soldaini, E., Pla, M., Rusterholz, C., Bucher, P., Corthe ´ sy, P., Reichenbach, P., and Nabholz, M. (1995) J. Biol. Chem. 270, elements (50 –53). 10743–10753 Tetramer formation may result in functional cooperativity 20. Ihle, J. N., and Kerr, I. M. (1995) Trends Genet. 11, 69 –74 21. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415–1421 between the two weak STAT5 binding sites simply by increas- 22. Schindler, C., and Darnell, J. E., Jr. (1995) Annu. Rev. Biochem. 64, 621– 651 ing the level of occupancy of sites I and II to a threshold 23. Ivashkiv, L. B. (1995) Immunity 3, 1– 4 required for enhancer activity. But protein-protein interaction 24. Serdobova, I., Pla, M., Reichenbach, P., Sperisen, P., Ghysdael, J., Wilson, A., Freeman, J., and Nabholz, M. (1997) J. Exp. Med. 185, 1211–1221 between STAT5 molecules may also lead to the formation of a 25. Le ´ cine, P., Algarte ´ , M., Rameil, P., Beadling, C., Bucher, P., Nabholz, M., and new molecular surface that binds a coactivator required for Imbert, J. (1996) Mol. Cell. Biol. 16, 6829 – 6840 IL-2rE activity. If tetramer formation is restricted to certain 26. John, S., Robbins, C. M., and Leonard, W. J. (1996) EMBO J. 15, 5627–5635 27. Bucher, P., Corthe ´ sy, P., Imbert, J., and Nabholz, M. (1997) Immunobiology STAT combinations, it may contribute to cytokine response 197, 133–140 specificity. For example, STAT1, which also binds to site I and 28. Hou, J., Schindler, U., Henzel, W. J., Wong, S. C., and McKnight, S. L. (1995) Immunity 2, 321–329 site II, does not contribute to IL-2Ra regulation, perhaps 29. Lin, J.-X., Mietz, J., Modi, W. S., John, S., and Leonard, W. J. (1996) J. Biol. because it does not form transactivating tetramers. If tetramer- Chem. 271, 10738 –10744 ization is required for binding to coactivators or basic transcrip- 30. Beadling, C., Guschin, D., Witthuhn, B. A., Ziemiecki, A., Ihle, J. N., Kerr, I. M., and Cantrell, D. A. (1994) EMBO J. 13, 5605–5615 tion factors, it may serve as a mechanism to select particular 31. Liu, X., Robinson, G. W., Wagner, K.-W., Garrett, L., Wynshaw-Boris, A., and mediators of transactivation. We are planning to explore these Hennighausen, L. (1997) Genes Dev. 11, 179 –186 32. Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S.-H., Ram, P. A., questions, taking advantage of the IL-2rE as a good model for Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, a natural STAT-controlled transcriptional enhancer. 7239 –7244 33. Xu, X., Sun, Y.-L., and Hoey, T. (1996) Science 273, 794 –797 Acknowledgments—We are grateful to Glaxo, Geneva, for generous 34. Vinkemeier, U., Cohen, S. L., Moarefi, I., Chait, B. T., Kuriyan, J., and Darnell, gifts of interleukins. Doreen Cantrell and Carol Beadling of the Impe- J. J. E. (1996) EMBO J. 15, 5616 –5626 35. Silva, A., MacDonald, H. R., Conzelmann, A., Corthe ´ sy, P., and Nabholz, M. rial Cancer Research Fund provided advice and help in setting up the (1983) Immunol. Rev. 76, 105–129 system for affinity precipitation of STAT proteins. Claudine Ravussin, 36. Wilson, A., Corthe ´ sy, P., Reichenbach, P., MacDonald, H. R., and Nabholz, M. Pierre Dubied, and Marcel Allegrini helped with the preparation of the (1994) Eur. J. Immunol. 24, 1729 –1735 figures. We thank Dean Scott for amino acid sequence analysis as well 37. Espel, E., Fromental, C., Reichenbach, P., and Nabholz, M. (1990) EMBO J. 9, as Sonia Barange ´ and Corinne Rusterholz for letting us quote their 929 –937 unpublished results. Michel Aguet, Stefan Ryter, and Corinne Ruster- 38. Geering, G., Old, L. J., and Boyse, E. (1966) J. Exp. Med. 124, 753–772 holz made many helpful suggestions concerning the manuscript. 39. Conzelmann, A., Corthe ´ sy, P., Cianfriglia, M., Silva, A., and Nabholz, M. (1982) Nature 298, 170 –172 REFERENCES 40. Ho, S. N., Hunt, H. D., Horton, R. M., Puller, J. K., and Pease, L. R. (1989) Gene 77, 51–59 1. Coffman, R. L., Lebman, D. A., and Shrader, B. (1989) J. Exp. Med. 170, 41. Schindler, U., Wu, P., Rothe, M., Brasseur, M., and McKnight, S. L. (1995) 1039 –1044 Immunity 2, 689 – 697 2. Waldmann, T. A. (1996) Immunol. Today 14, 264 –270 42. Mikita, T., Campbell, D., Wu, P., Williamson, K., and Schindler, U. (1996) Mol. 3. Asao, H., Takeshita, T., Ishii, N., Kumaki, S., Nakamura, M., and Sugamura, Cell. Biol. 16, 5811–5820 K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4127– 4131 43. Schreiber, E., Matthias, P., Mu ¨ ller, M. M., and Schaffner, W. (1989) Nucleic 4. Taga, T., and Kishimoto, T. (1995) Curr. Opin. Immunol. 7, 17–23 Acids Res. 17, 6419 5. The ` ze, J., Alzari, P. M., and Bertoglio, J. (1996) Immunol. Today 17, 481– 486 44. Sperisen, P., Wang, S. M., Reichenbach, P., and Nabholz, M. (1992) PCR 6. Nakarai, T., Robertson, M. J., Streuli, M., Wu, Z., Ciardelli, T. L., Smith, K. A., Methods Applications 1, 164 –170 and Ritz, J. (1994) J. Exp. Med. 180, 241–251 45. Queen, C., and Baltimore, D. (1983) Cell 33, 741–748 7. Kondo, M., Ohashi, Y., Tada, K., Nakamura, M., and Sugamura, K. (1994) Eur. 46. Kost, T. A., Theodorakis, N., and Hughes, S. H. (1983) Nucleic Acids Res. 11, J. Immunol. 24, 2026 –2030 8287– 8301 8. Takeshita, T., Ohtani, K., Asao, H., Kumaki, S., Nakamura, M., and 47. Sambrook, J., Fritsch, F. E., and Maniatis, T. (1989) Molecular Cloning: A Sugamura, K. (1992) J. Immunol. 148, 2154 –2158 Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring 9. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T., Harbor, NY Miyasaka, M., and Taniguchi, T. (1989) Science 244, 551–556 48. Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211–217 10. Waldmann, T. A., White, J. D., Goldman, C. K., Top, L., Grant, A., Bamford, R., 49. Horvath, C. M., Wen, Z., and Darnell, J. E., Jr. (1995) Genes Dev. 9, 984 –994 Roessler, E., Horak, I. D., Zaknoen, S., Kasten-Sportes, C., England, R., 50. Bergad, P. L., Shih, H.-M., Towle, C. T., Schwarzenberg, S. J., and Berry, S. A. Horak, E., Mishra, B., Dipse, M., Hale, P., Fleisher, T., Junghans, E., Jaffe, (1995) J. Biol. Chem. 270, 24903–24910 E., and Nelson, D. L. (1993) Blood 82, 1701–1712 11. Lowenthal, J. W., Tougne, C., MacDonald, H. R., Smith, K. A., and Nabholz, M. 51. Yan, R., Small, S., Desplan, C., Dearolf, C. R., and Darnell, J. E. J. (1996) Cell 84, 421– 430 (1985) J. Immunol. 134, 931–939 12. Nishi, M., Ishida, Y., and Honjo, T. (1988) Nature 331, 267–269 52. Hou, X. S., Melnick, M. B., and Perrimon, N. (1996) Cell 84, 411– 419 53. Guyer, N. B., Severns, C. W., Wong, P., Feghali, C. A., and Wright, T. M. (1995) 13. Kro ¨ nke, M., Leonard, W. J., Depper, J. M., and Greene, W. C. (1985) J. Exp. Med. 161, 1593–1598 J. Immunol. 155, 3472–3480 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Interaction of STAT5 Dimers on Two Low Affinity Binding Sites Mediates Interleukin 2 (IL-2) Stimulation of IL-2 Receptor α Gene Transcription

Download PDF
8 pages

Loading next page...
 
/lp/unpaywall/interaction-of-stat5-dimers-on-two-low-affinity-binding-sites-mediates-BkWLfoEIda

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.272.50.31821
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 50, Issue of December 12, pp. 31821–31828, 1997 © 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Interaction of STAT5 Dimers on Two Low Affinity Binding Sites Mediates Interleukin 2 (IL-2) Stimulation of IL-2 a Gene Transcription* Receptor (Received for publication, August 5, 1997, and in revised form, October 2, 1997) Wolfram K.-H. Meyer‡, Patrick Reichenbach‡, Ulrike Schindler§, Elisabetta Soldaini‡¶, and Markus Nabholz‡i From the ‡Lymphocyte Biology Unit, Swiss Institute for Experimental Cancer Research (ISREC), 155 Chemin des Boveresses, CH-1066 Epalinges, Switzerland and §Tularik Incorporate, Two Corporate Drive, South San Francisco, California 94080 Stimulation of the interleukin 2 receptor a (IL-2Ra) receptor (for review see Waldmann et al. (10)). The capacity of gene by IL-2 is important for the proliferation of anti- mature T cells to proliferate in response to IL-2 correlates with gen-activated T lymphocytes. IL-2 regulates IL-2R IL-2Ra expression (11). Mouse T cells that constitutively ex- transcription via a conserved 51-nucleotide IL-2 respon- press the human IL-2Ra chain can respond to IL-2 in the sive enhancer. Mouse enhancer function depends on co- absence of antigen stimulation (12). Thus, IL-2Ra expression operative activity of three distinct sites. Two of these controls, at least in part, IL-2 responsiveness. are weak binding sites for IL-2-activated STAT5 (signal IL-2Ra cell surface expression is regulated mainly through transducer and activator of transcription) proteins, and changes in IL-2Ra gene transcription (13–16). In mature T mutational analysis indicates that binding of STAT5 to cells, antigen induces a transient wave of IL-2Ra transcription. both sites is required for IL-2 responsiveness of the en- Prolonged, maximal expression of IL-2Ra depends on stimula- hancer. The STAT5 dimers interact to form a STAT5 tion by IL-2, which thus acts as a positive feedback regulator of tetramer. The efficiency of tetramerization depends on its own high affinity receptor (17, 18). the relative rotational orientation of the two STAT mo- We have shown that stimulation of the mouse IL-2Ra gene tifs on the DNA helix. STAT5 tetramerization on en- by IL-2 is controlled by an IL-2-responsive enhancer (IL-2rE) hancer mutants correlates well with the IL-2 responsive- 1.3 kilobase pairs upstream of the major transcription start site ness of these mutants. This provides strong evidence (19). The IL-2rE maps to the same position as a DNase I that interactions between STAT dimers binding to a pair hypersensitive site that appears in the chromatin of normal of weak binding sites play a biological role by control- mouse T cells upon stimulation with concanavalin A and IL-2 ling the activity of a well characterized, complex cyto- kine-responsive enhancer. (18). The enhancer is 51 nt long and contains three distinct elements (named sites I, II, and III, see Fig. 1), all of which are required for enhancer activity (19). As pointed out previously Interleukin-2 (IL-2) is a growth factor for antigen-activated (19), sites I and II resemble binding sites for transcription lymphocytes (1, 2). It stimulates T cells through a high affinity factors of the STAT family (20 –23). Site II also overlaps with a cell surface receptor (IL-2R) composed of three transmembrane consensus binding site for GATA factors. Site III includes a polypeptide chains designated IL-2Ra, IL-2Rb, and IL-2Rg or consensus site for Ets proteins and contributes to IL-2rE activ- g (3). The b and g chains, which are shared with other inter- ity by binding the constitutive Ets protein Elf-1 (24). The hu- leukin receptors (4, 5), belong to the family of hematopoietic man homologue of the mouse IL-2rE has recently been identi- cytokine receptors. Resting T cells constitutively express the g fied approximately 4 kilobase pairs upstream of the chain (6, 7) and basal levels of the b chain (8). IL-2Rb expres- transcription start site (25–27). The binding motifs in sites I, II, sion increases upon antigen stimulation (9), . IL-2Ra is not a and III are conserved in the human IL-2rE (25, 26). member of this family and does not participate in the formation STAT proteins are latent transcription factors that dimerize of other known receptors. IL-2Ra expression is undetectable on in response to cytokine receptor activation. Dimerization re- resting lymphocytes but is induced by signals from the antigen sults in activation of their specific DNA binding activity and accumulation in the nucleus (22). IL-2 predominantly activates STAT5a and STAT5b, two closely related proteins encoded by * This work was supported, in part, by grants from the Swiss Na- separate genes (28, 29). STAT5 is rapidly activated by IL-2 in tional Science Foundation, the Swiss Cancer League and the Copley- May Foundation as well as by a grant from the Swiss Federal Office of antigen-stimulated but not in quiescent T cells (28, 30). The Education and Science, awarded in conjunction with a project approved human IL-2rE site I does bind STAT5a and b (25, 26). Muta- by the Human Capital and Mobility program of the European Union. tions in this site that abolish STAT5 binding also destroy The costs of publication of this article were defrayed in part by the IL-2-inducible IL-2rE activity in the human IL-2-dependent payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to leukemia Kit225 cell line (25). Site II may also contribute to indicate this fact. STAT5 binding (26). To what extent STAT5a and STAT5b ¶ Present address: Laboratory of Molecular Immunology, NHLBI, functions overlap is not yet clear, but the phenotypes of STAT5 National Institutes of Health, Bldg. 10, Rm. 7N264, Bethesda MD 20892. (31)- and STAT5b (32)-deficient mice show that they are not i To whom correspondence should be addressed: Tel.: 141 21 692 58 34; Fax 141 21 652 69 33; E-mail: [email protected]. completely redundant. The abbreviations used are: IL-2, interleukin 2; IL-2R, IL-2 recep- Here we provide evidence that IL-2 responsiveness of the tor; IL-2rE, IL-2-responsive enhancer; nt, nucleotide(s); STAT, signal mouse IL-2rE depends on the binding of STAT5 to both sites I transducer and activator of transcription; DOC, deoxycholate; PCR, and II and that the synergistic effect of these two sites on polymerase chain reaction; bp, base pair(s). S. Barange ´ and M. Nabholz, unpublished data. enhancer activity is due to the interaction of the bound STAT5 This paper is available on line at http://www.jbc.org 31821 This is an Open Access article under the CC BY license. 31822 IL-2Ra Regulation by STAT5 44). Mutagenesis of pwt was performed as described by Ho et al. (40). dimers. Our data show that DNA-induced association of STAT For this purpose, an additional XbaI site was created by changing the dimers, which has been observed for STAT4 and STAT1 (33, adenine at position 21307 into a guanine. This substitution did not 34), plays an important role in cytokine-regulation of gene alter IL-2 responsiveness (data not shown). A unique BclI site and the expression. new XbaI site were used to subclone PCR products of the site-directed mutagenesis. Positive clones were sequenced. EXPERIMENTAL PROCEDURES Reporter Gene Assay—IL-1-primed cells were transiently transfected Cell Lines, Culture Conditions, and Cytokine Stimulation—The using DEAE dextran according to Queen and Baltimore (45) and cul- PC60.21.14 cell line (referred to as PC60) is a hybrid between a mouse tured with IL-1 only or with IL-1 and IL-2. Reporter gene expression cytolytic T cell line and a rat thymic lymphoma. It has inherited from its was measured 48 h later, as described previously (44). Briefly, cells lymphoma parent (35) the capacity to grow independently of IL-2 and a were cotransfected with a defined ratio of an IL-2Ra/rabbit b-globin 2 2 2 2 CD4 CD8 phenotype. Like normal CD4 CD8 thymocytes (36), it gene together with the reference plasmid (pGbAcbGlD), which is con- expresses IL-2Ra when stimulated with IL-1 and IL-2. IL-1 plays a stitutively expressed in PC60 cells and contains a 40-bp deletion in the similar role in these cells as antigen in mature T lymphocytes and second exon (46). Relative amounts of mRNA transcribed from the induces PC60 cells to become IL-2-responsive. This effect has been reference plasmid and the IL-2Ra/b-globin construct were determined described in detail in previous publications (16, 19, 35, 37–39). The cells by PCR (see Refs. 19 and 44 for details). were cultured for 2–3 days in 1 ng/ml human IL-1b before stimulation Proteolysis and Amino Acid Sequence Analysis—20 mg of recombi- with 100 units/ml human IL-2 in the continued presence of IL-1. Re- nant active STAT5a were digested with 3 mg of thermolysin (Boehringer combinant interleukins were gifts of Glaxo Institute for Molecular Mannheim) in a total volume of 400 ml of digestion buffer (50 mM Biology S.A. (Plan-les-Ouates, Geneva, Switzerland). Tris-HCl, pH 8, 1 mM EDTA, 1 mM dithiothreitol) at room temperature Probes—Oligonucleotides from MWG-Biotech (Duebendorf, Ger- for 10 and 30 min. Thermolysin was inactivated by adding 4 mlof500 many) or Microsynth (Balgach, Switzerland) were annealed, purified on mM EDTA, pH 8. Undigested protein was incubated in the same buffer acrylamide gels, quantified, and stored in 10 mM Tris-HCl, pH 7.5, 50 without thermolysin. Electromobility shift assays were performed as mM NaCl. As a nonspecific competitor (NS) for bandshift assays, a described above, with 1 ml of the thermolysin reaction. The remaining double-stranded oligonucleotide with the sequence 59-AGAGTTAGCT- reaction was diluted with SDS sample buffer (47), and the fragments TGCGGTTCCCAGG-39 was used. were separated on a 10% SDS gel. The proteins were blotted onto Probes were obtained either by annealing appropriate oligonucleo- polyvinylidene difluoride membrane (Millipore) and visualized by Coo- tides (FcgRI, single IL-2rE sites I or II, IL-2rE sites I and II, nonspecific massie staining. N-terminal protein sequence analysis was performed probe NS (for sequences see Fig. 1)) or by PCR amplification of different by the protein facility at Tularik, Inc. using an AB477A protein se- IL-2rE templates with primers spanning segments of the IL-2rE. For quencer and an AB120A analyzer. competition assays, probes extended from nt 21420 (primer A) to nt 21331 (primer B) of the IL-2Ra 59-flanking region, including sites I and RESULTS II. For affinity precipitation and the bandshift experiment in Fig. 7C, Sites I and II of the IL-2rE Are Weak Binding Sites for probes extended from nt 21420 (primer A) to nt 21286 (primer C), STAT5—Two of the functional sites in the IL-2rE (sites I and including site III. To generate probes carrying a mutation in a single enhancer site, we amplified the corresponding reporter plasmid (M4 for II) contain potential STAT binding motifs (see Fig. 1 and site I, M9 for site II, and M12 for site III; see Fig. 1 and Sperisen et al. Sperisen et al. (19)). To determine whether the IL-2rE could (19) for sequences). Probes with mutations in more than one site were bind IL-2-induced STAT5, we incubated PC60 extracts with a obtained in three steps, essentially as described in Ho et al. (40) and biotinylated, double-stranded oligonucleotide comprising the explained here for a probe in which all three enhancer sites are de- entire enhancer. Bound proteins were recovered by incubating stroyed. (a) The plasmid carrying the mutation in site I (M4) was the probe with streptavidin-coated beads and characterized by amplified with oligonucleotide A and a 39 oligonucleotide D9 covering site II, with changes destroying this site. Simultaneously the plasmid Western blotting. The complete IL-2rE binds proteins of about with a mutation in site III (M12) was amplified with a 59 primer (D) 90 kDa, which are present in extracts from IL-2-stimulated complementary to oligonucleotide D9 and oligonucleotide C. (b) The PC60 cells and react with anti-STAT5a and anti-STAT5b an- resulting PCR products were gel-purified, denatured, annealed, and tisera (Fig. 2A) as well as with an anti-phosphotyrosine anti- used as template for an extension reaction. The resulting full-length body (data not shown). No STAT5 proteins could be recovered IL-2rE fragments were amplified with primers A and C. The final PCR from extracts of cells that had not been treated with IL-2. As products were quantified and analyzed by sequencing. Recombinant Proteins—Expression, activation, and purification of expected, IL-2 induces rapid activation of STAT5. The amount recombinant STAT5a have been described previously (41, 42). of IL-2rE-bound STAT5 continues to increase and reaches a Antibodies—The anti-STAT5 antibody was purchased from Santa plateau only after 24 – 48 h of culture in IL-2 (Fig. 2B). This Cruz Biotechnology, Inc., STAT5a and b antibodies are fromR&D correlates with the kinetics of IL-2-induced accumulation of Systems, and horseradish peroxidase-coupled goat-anti-rabbit antibod- IL-2Ra message and IL-2rE-driven reporter gene expression ies were from Amersham. (19). We did not detect any IL-2-inducible proteins that reacted Affinity Precipitation of IL-2rE-bound Proteins—Affinity precipita- tion of IL-2rE-bound proteins was performed as described in Ref. 30. with antisera against STAT1 and STAT3 (data not shown). Electrophoretic Mobility Shift Assays (Bandshifts)—Nuclear extracts To determine which segment of the IL-2rE is responsible for were prepared as described by Schreiber et al. (43). Lysis and extraction STAT5 binding, we used biotinylated IL-2rE oligonucleotides buffers contained protease (1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mM that carry mutations in one or more of the sites required for phenylmethylsulfonyl fluoride) and phosphatase inhibitors (10 mM enhancer function. Fig. 2A shows that a probe containing an NaF, 1 mM Na VO ). Binding reactions were performed in a final 3 4 intact site I and mutations in the other sites bind STAT5a and volume of 20 ml in binding buffer (10 mM Tris-HCl, pH 7.5, 60 mM KCl, 10% glycerol, 1 mM dithiothreitol, 1 mg/ml bovine serum albumin, 1 mg b. Probes containing an intact site II and mutations in the of poly(dIcdC), 0.5 mg of sonicated salmon sperm DNA) containing 4 mg other sites bind a trace of STAT5, whereas probes with muta- of cell nuclear extract or the indicated quantity of recombinant STAT5a tions in both sites I and II fail to bind STAT5. Incubation of the protein and 2–3 3 10 cpm (30 fmol) of radiolabeled PCR fragments or same filters with an antibody against Elf-1 had shown that oligonucleotides (see Fig. 1 for sequences). only oligonucleotides with an intact site III bind this transcrip- Reactions were incubated on ice for 20 min and separated on 4% tion factor (24). nondenaturing polyacrylamide gels in 0.3 3 Tris/borate/EDTA. For competition experiments, unlabeled PCR fragments or oligonucleotides These results demonstrate that STAT5a and b can specifi- were mixed with the labeled probe before addition of the proteins. When cally bind to site I and, albeit very weakly, to site II. To confirm deoxycholate (DOC) was used, appropriate dilutions were added to the these data, we carried out bandshift experiments with recom- binding reaction at the same time as STAT5 and the probe. The mixture binant STAT5a protein (Fig. 3A) and PC60 nuclear extracts was incubated for 30 min at room temperature before loading on the gel. (Fig. 3B). Fig. 3 shows that both site I and site II can compete Plasmids—The reference plasmid pGbAcbGlD and the plasmid pwt, specifically for the binding of STAT5a to a probe that contains in which the mouse IL-2Ra 59-flanking region and promoter has been joined to the rabbit b-globin gene, have been described previously (19, the STAT binding site of the FcgRI gene (see Fig. 1). Titration IL-2Ra Regulation by STAT5 31823 FIG.1. Nucleotide sequence of site I and site II of IL-2rE and probes. Gray boxes indicate the previously defined IL-2rE regions required for IL-2 responsiveness of the IL-2Ra gene. Putative STAT binding sites have been underlined. Below, the sequences of the different probes used have been listed together with their designation in the text and the symbols used in the figures. A blank box indicates a wild type element, whereas a crossed box indicates a substitution mutation that destroys IL-2rE enhancer activity (19). F stands for the high affinity FcgRI STAT binding site. of various competitors indicates that the affinities of sites I and II for STAT5a are, respectively, 10-fold and 50-fold lower than that of the FcgRI STAT binding site. Extracts from IL-2-stim- ulated but not from unstimulated PC60 cells form a complex with the FcgRI probe that is due to IL-2-induced STAT5, as shown by supershift experiments with STAT5-specific antibod- ies (data not shown). The competition experiment shown in Fig. 3 demonstrates that PC60 STAT5 and recombinant STAT5a have the same relative affinity for site I and site II. In certain experiments, IL-2 also stimulated the appearance of a small amount of STAT1 (visible as a faint specific band below the STAT5 complex in Fig. 3B), but it is unlikely that STAT1 plays a role in IL-2rE activity. Interferon-g that induces STAT1 but not STAT5 does not stimulate IL-2Ra expression, nor does it affect the response to IL-2 (data not shown). Furthermore, IL-2-dependent IL2Ra expression in T lymphocytes from inter- feron-g receptor-deficient mice is normal despite the fact that stimulation with concanavalin A and IL-2 fails to induce STAT1 activation. The IL-2rE STAT Motifs Induce Association of STAT5a Dimers—IL-2 responsiveness of the IL-2rE depends on synergy between the individual IL-2rE sites. This could reflect cooper- ative binding of the transcription factors controlling enhancer activity. Cooperative binding to pairs of weak binding sites has indeed been observed for STAT4 (33) and STAT1 (34). The experiments described so far were carried out with a large excess of binding sites and were therefore unlikely to reveal any cooperativity between sites I and II. To look for synergistic binding, we resolved the complexes formed by IL-2rE probes with increasing concentrations of STAT5 in bandshift assays. Since STAT5 complexes formed by crude PC60 extracts with these probes are obscured by other, nonspecific complexes, we resorted to recombinant STAT5 for these experiments. Fig. 4A shows that STAT5a forms two retarded complexes (referred to as C1 and C2) with a 39-bp probe that contains intact sites I FIG.2. STAT5a and STAT5b from PC60 extracts bind to IL-2rE and II (probe I/II). Complex C2 comigrates with the specific oligonucleotide probes containing intact sites I and II. A, whole band formed by the short (20 bp) FcgRI probe used in Fig. 3 (see cell extracts were prepared from unstimulated PC60 cells or from cells also Fig. 4C) and is presumably due to STAT5a dimers. Quan- incubated for 30 min with IL-2. Extracts were incubated with the indicated biotinylated probes, which were then recovered by incubation titative analysis (by PhosphorImager) of the amount of probe with streptavidin-coated-agarose beads. Bound proteins were fraction- retained in the two complexes showed that the ratio of C1 to C2 ated by SDS-PAGE together with molecular mass markers and crude increased with rising concentrations of STAT5 protein, indicat- nuclear extracts (NE). Gels were blotted onto membranes that were ing that C1 represents a trimolecular complex between the incubated with different anti-STAT5 antibodies. B, extracts from PC60 cells stimulated for the indicated times with IL-2 were incubated with a biotinylated wild type IL-2rE probe, and bound, recovered proteins C. Rusterholz, unpublished data. were immunoblotted with anti-STAT5 antiserum. 31824 IL-2Ra Regulation by STAT5 This interpretation is supported by experiments with a 39-bp probe in which site I was replaced by the high affinity FcgRI STAT consensus site I, and site II was inactivated (probe F/2). Fig. 4C shows that, at low protein concentrations, this probe forms a single complex C2 that comigrates with the complex formed by STAT5a binding to the short FcgRI probe (F) used in Fig. 3. At higher STAT5 concentrations the F/2 probe, but not the short F probe, gives rise to an increasing proportion of C1 complexes. As expected, the ratio between C1 and C2 at a given STAT5 concentration is higher when the FcgRI STAT consen- sus site is combined with an intact site II (probe F/II). Thus, F/2,I/2, and 2/II probes, but not the short F probe, induce STAT tetramerization, indicating that binding of STAT5 to a single specific binding site induces the formation of a STAT5 tetramer, provided that the probe contains sufficient flanking DNA. STAT5 tetramers are expected to dissociate with a lower off-rate than dimers, as has indeed been demonstrated for STAT1 (34). This explains why the only complex detected in bandshifts with probes containing a single very weak binding site (2/II) is C1, whereas probes with a single high affinity binding site form predominantly complex C2. As expected, C1 complex formation is enhanced by the inclusion of two STAT5 binding sites in the same probe (I/II, F/II), resulting in a syn- ergistic increase of STAT binding to such probes. Thus, in the presence of 1–2 mg of STAT5a, the total amount of STAT5a bound to probe I/II is 2.5–3 times higher than the sum of protein bound to probe I/2 and to probe 2/II. Tetramer Formation Depends on Specific STAT5 Protein Segments—Formation of STAT4 and STAT1 tetramers de- pends on their N termini (33, 34). To determine whether STAT5 tetramer formation also requires interaction between specific protein domains, we investigated the effect of proteo- FIG.3. STAT5 binds to site I and site II of the IL-2rE with lytic clipping on the capacity of STAT5a to form tetrameric different affinities. Recombinant STAT5a (A) or nuclear extract of complexes. Treatment of STAT5a with thermolysin yields two PC60 cells stimulated for 15 min with IL-2 (B) was incubated for 15 min with 30 fmol of FcgRI probe mixed with different amounts of the proteolytic fragments with apparent molecular masses of about following unlabeled competitor oligonucleotides: nonspecific NS, FcgRI, 75 and 60 kDa (Fig. 5A). Western blotting with an antibody site I or site II of the IL-2rE (see Fig. 1; see “Experimental Procedures”). against the C-terminal domain of STAT5a revealed that both Reactions were analyzed by gel retardation assays. proteolytic fragments lack the C terminus (data not shown). N-terminal amino acid sequencing of the 60-kDa fragment IL-2rE probe and two STAT5 dimers binding to sites I and II, yielded the sequence ILVDAMSQK, indicating that this clip- respectively. Surprisingly, a 39-bp IL-2rE probe containing a ping product starts at amino acid 130. The molecular mass of single cognate STAT5 binding site (probe I/2) also gave rise to this fragment is consistent with the absence of both N- and complexes C1 and C2, but both bands were weaker, and the C-terminal domains of STAT5a. No N-terminal sequence could proportion of the C2 complex was higher. Nevertheless, in- be obtained from the 75-kDa fragment. Its molecular mass creasing STAT5 concentrations resulted in a shift toward com- suggests that this fragment still retains the N terminus, which plex C1. A probe with an intact site II and a mutated site I is probably blocked. (probe 2/II) formed a small amount of complex C1 and virtually We compared the complexes formed by full-length STAT5a no detectable complex C2. Competition experiments confirmed and by the proteolytic digests with the IL-2rE probe I/II and the that the mutations in site I and site II abolish detectable short FcgRI probe F (Fig. 5B). As described above (Fig. 4A), binding of STAT5 to these sites (data not shown). intact STAT5 predominantly forms the slower migrating com- The observation that the IL-2rE probes containing a single plex C1 with the probe I/II (in this experiment C2 is barely binding site (probes I/2 and 2/II) form C1 complexes suggested visible), whereas probe F gives rise to the faster migrating that a STAT dimer bound to one site could interact with an- complex C2. The proteolytic fragments, on the other hand, form other dimer to form what we will refer to as STAT5 tetramers. with either probe a complex C2* that migrates faster than C2. Alternatively, complex C1 formed by these probes might be due A small amount of an additional complex C1* is observed with to preferential binding of a small amount of pre-existing tet- probe I/II but not with probe F. These data suggest that com- ramers in the STAT preparation. To distinguish between these plex C2* is formed by the 60-kDa fragment that lacks the possibilities, we compared the capacity of different IL-2rE probes to compete for the C1 and C2 complexes formed by the domain necessary for protein-protein interaction, whereas C1* is due to the 75-kDa fragment that is still capable of forming I/2 probe (Fig. 4B). If formation of C1 by the probes I/2 and 2/II is due to the higher affinity of preformed STAT5 tetramers tetramers. Thus, as observed for STAT1, proteolytic cleavage of STAT5 strongly reduced its propensity to form trimolecular for these oligonucleotides, then they should compete better for C1 than for C2. However, the IL-2rE oligonucleotides compete complexes with a probe containing two weak binding sites, with the same relative efficiency for the two complexes as the most likely because proteolysis has removed the N-terminal short, high affinity FcgRI probe (F). This indicates that forma- domain required for tetramerization. Alternatively, the small tion of STAT5 tetramers is DNA-dependent. amount of the putative trimolecular complex C1* could be due IL-2Ra Regulation by STAT5 31825 FIG.4. Cooperative binding of STAT5 to IL-2rE. A, increasing amounts of recombinant STAT5a were incubated with a wild type IL-2rE probe or with probes in which site I or site II contained a mutation that destroys STAT5 binding and enhancer activity. C1 and C2, specific STAT5 complexes. Empty arrowhead, free probe. B, 1.6 mg of recombinant STAT5a was incubated with the IL-2rE probe containing a mutation in site II (I/2) together with increasing amounts of the following unlabeled competitors: a 20-nt stretch containing the FcgRI high affinity STAT binding site (F) or IL-2rE segments harboring a mutation in site II (I/2)orinsiteI(2/II), respectively. C, increasing amounts of STAT5a were added to a 20-bp FcgRI probe (F) or to 39-bp probes in which the FcgR STAT motif was combined with a mutated (F/2) or wild type (F/II) site II of the IL-2rE. to simultaneous occupation of both STAT5 binding sites in severely reduced by 0.03% detergent (data not shown). This is probe I/II at high protein concentrations, even in the absence of further evidence that C1 complexes depend on an interaction protein-protein interactions. In either case, our data indicate between the bound STAT5 dimers and are not simply due to that, as reported for STAT4 and STAT1, the capacity of STAT5 occupancy of both IL-2rE binding sites by independently bind- to cooperatively bind to a probe containing two binding sites ing STAT5 molecules. depends on interactions involving modular protein domains. IL-2rE Activity Depends on Intact STAT5 Motifs in Sites I We also investigated the effect of deoxycholate, which is and II—Previously we defined the limits of site I and of site II known to disrupt protein-protein interactions (48), on the for- by substitution mutations. The finding that they coincided to mation of C1 and C2 complexes. Fig. 6 shows that 0.25% DOC within one nucleotide with the borders of STAT motifs provided is required to substantially reduce formation of C2 complexes the first evidence suggesting an involvement of STAT proteins with the wild type IL-2rE probe as well as the high affinity, in IL-2rE function (19). The binding site for STAT5 in site II single site FcgRI probe. Formation of C1 complexes, on the overlaps with a binding motif for GATA factors. The previously other hand, is almost completely prevented by 0.06% DOC and described mutants did not exclude that the contribution of site 31826 IL-2Ra Regulation by STAT5 FIG.6. Sensitivity of STAT5 complexes to deoxycholate. Bind- FIG.5. The effect of proteolytic cleavage on STAT5 tetramer ing reactions between STAT5a and the wild type IL-2rE probe or the formation. A, analysis of proteolytic cleavage products of STAT5. high affinity FcgRI probe were carried out in presence of the indicated Recombinant STAT5a was digested with thermolysin for the indicated concentrations of DOC. Complexes were separated on a nondenaturing times. Aliquots of the reaction were resolved on a SDS-polyacrylamide gel. gel, and proteins were visualized by Coomassie staining. B, analysis of DNA binding complexes formed by thermolysin-treated STAT5a with with regard to the helical axis of the DNA is important for an the IL-2rE probe I/II or the short FcgRI probe F. efficient association of the STAT5 dimers into tetramers and thereby affects IL-2rE enhancer activity. II to IL-2rE activity depends on a GATA protein. Fig. 7 shows DISCUSSION that a mutation in site II that leaves the GATA binding motif intact but destroys the STAT site (mutant S-II.1) abolishes Previously we showed that the response of the IL-2Ra gene enhancer activity. This indicates that the binding of STAT5 to to IL-2 is mediated by a 51-nt IL-2-responsive enhancer (IL- site II is required. In addition, a mutation that leaves the STAT 2rE). The IL-2rE consists of three sites, I, II,and III, that motif intact and destroys the GATA motif (mutant S-II.2) con- functionally cooperate to activate transcription (19). The func- serves IL-2rE activity. In fact, this mutation, which transforms tion of site III most likely depends on Elf-1, constitutively site II into a copy of site I, gives an enhancer that responds present in PC60 cells and normal T lymphocytes (Serdobova et slightly better than the wild type IL-2rE. Probably this reflects al. (24)). This paper provides strong evidence that cooperative the stronger affinity of STAT5 for site I compared with site II. binding of IL-2-activated STAT5 to sites I and II plays a crucial The inverse change, resulting in a replacement of site I with role in IL-2rE activity. another copy of site II (S-I.1) abolishes IL-2 responsiveness and Stimulation of IL-2Ra Gene Transcription by IL-2 Depends greatly diminishes STAT5 binding (data not shown), indicating on STAT5 Activation—IL-2 stimulates IL-2Ra transcription that two copies of site II do not bind STAT5 with sufficient via binding of induced STAT5 to the IL-2rE. The onset of affinity to allow the enhancer to function. STAT5 activation is as rapid as expected for STAT factors, but The Topological Relationship between Sites I and II Affects the DNA binding activity of STAT5 continues to increase for STAT5 Binding and IL-2rE Function—The center-to-center 48 h. This is parallel with increasing IL-2Ra expression and distance between the STAT binding motifs in sites I and II is 20 IL-2rE-driven reporter gene activity (19). IL-2rE sites I and II bp or two turns of the DNA helix in both man and mouse (25, bind STAT5a as well as STAT5b with a weak affinity. Their 26). Thus, the STAT molecules binding to the two sites face the relative affinity for STAT5 in nuclear extracts is the same as same side of the helix, and it appeared likely that tetramer that for recombinant STAT5a, indicating that no accessory formation depends on this topological relationship between the proteins are required for the binding of the IL-2rE sites to interacting STAT5 molecules. To test this, we changed the STAT5. spacing between the two sites by inserting 5 or 10 bp (Fig. 7A) Previous mapping of site II had left open the question of and compared the complexes that such probes formed with whether the contribution of this site was due to a STAT motif STAT5 with those formed by the wild type probe. The addition or to an overlapping GATA binding site. The data presented of 5 bp or half a helical turn (mutant I-5-II) clearly reduces the here show that the STAT motif in site II is required for en- formation of complex C1 but has a lesser effect on complex C2 hancer activity and argue against a role of GATA factors. (Fig. 7B). PhosphorImager analysis showed that probe I-5-II The affinity of site II for STAT5 is approximately 5 times binds approximately half the number of STAT5a molecules as lower than that of site I. A point mutation that changes the the wild type probe. This mutation reduces the IL-2 response of STAT motif in site I to that in site II abolishes the IL-2 re- the enhancer from 8- to 3.5-fold (Fig. 7C). Insertion of 10 bp sponse of the IL-2rE. Together these results support a predom- (I-10-II) or one helical turn has no significant effect on either inant role of STAT5 in the regulation of IL-2rE activity. As has STAT5 complex formation or enhancer response. These results been pointed out by others (49, 50), STAT binding motifs in suggest that the rotational orientation of the STAT5 molecules natural regulatory elements are often sites with relatively low IL-2Ra Regulation by STAT5 31827 FIG.7. Effect of IL-2rE mutations on IL-2 responsiveness and STAT5 binding. The effects of different mutations (A) on STAT5 binding (B) and the IL-2 inducibility of the IL-2rE (C) were investigated. B, analysis of DNA binding complexes formed by different IL-2rE probes spanning the segment between nt 21286 and 21420. In the last two lanes a 100-fold excess of competitor was included in the reaction. Competitors were: F,FcgRI STAT motif; NS, nonspecific oligonucleotide. C, 2.5-kilobase pairs of the mouse IL-2Ra gene 59-flanking region containing either the wild type or the indicated mutant form of the IL-2rE were fused to the rabbit b-globin gene. These plasmids were transiently transfected into PC60 cells together with a reference plasmid. The transfected cells were cultured either with or without IL-2. Two days later, the relative amounts of reporter gene mRNA were measured, and the IL-2 response of each plasmid was determined (see “Experimental Procedures”). Transfections were repeated at least twice with all constructs. For each mutant, the mean response 6 S.D. is given. Shading of the histogram bars distinguishes different classes of mutations: black, wild type (wt) IL-2rE; white, point mutations; hatched, insertions between sites I and II. affinity. In contrast to high affinity binding sites, low affinity in the mouse IL-2rE depends on the DNA-induced interaction sites preferentially bind a subset of STAT factors. This may between two STAT5 dimers. Efficient formation of STAT5 tet- contribute to the selective responsiveness of a gene to particu- ramers appears to require that both STAT5 molecules bind to lar STAT proteins and cytokines. the same side of the DNA helix, since insertion of a half-turn Similarities and Differences between Human and Mouse IL-2 between the two binding sites reduces association, whereas Responsive Enhancers—The human homologue of the mouse insertion of a complete turn does not result in a significant IL-2rE is located approximately 4 kilobase pairs upstream of change. The correlation of these effects with those on IL-2 the transcription start site (25–27). The two enhancers share responsiveness of the IL-2rE strongly indicates that the pair of the consensus motifs first identified in the mouse gene (19), STAT motifs in the IL-2rE affects enhancer activity by induc- and the spacing between these motifs is conserved. Partial ing STAT5 association. This is the first evidence that STAT functional analysis of the human IL-2rE results in a picture tetramer formation plays an important role in the function of a that is similar but not identical to that of the mouse IL-2rE (25, well characterized cytokine-responsive enhancer. Thus, our re- 26). The most striking difference between the human and sults demonstrate that the phenomenon of STAT tetrameriza- mouse IL-2rE is a base pair inversion, which creates an addi- tion that has been described by others for STAT4 and STAT1 tional Ets motif that overlaps with the STAT motif in site I in (33, 34) is indeed biologically important. the human IL-2rE. The effect of different mutations in this site It is very likely that cooperative binding of STAT5 to the two as well as cotransfection experiments with Elf-1 expression IL-2rE sites reflects a reduction in the off-rate of the DNA from vectors suggest that the Ets motif in the human site I acts as a tetramers compared with that from dimers, as has been dem- negative regulator of IL-2rE activity in unstimulated cells (25, onstrated for STAT1 (34). Our data suggest that, like STAT4 26). Since STAT5 binds more avidly to the human site I than to and STAT1, STAT5 requires the N-terminal domain for tet- the mouse homologue, such an element may be required to ramerization. But our results also point to differences between prevent inappropriate IL-2Ra expression. the interactions of different STAT dimers. Unlike STAT4, STAT5 Controls IL-2rE Activity by Cooperative Binding to STAT5 does not form tetramers with short (20 nt) probes. Two Adjacent Sites—Our experiments indicate that the func- However, STAT5 tetramers are formed on a 39-nt probe that tional cooperativity between the two weak STAT5 binding sites contains a single cognate binding site between nt 6 and 15. This suggests that tetramerization depends on simultaneous con- P. Le ´ cine and J. Imbert, personal communication. tacts of both participating dimers with the same DNA molecule 31828 IL-2Ra Regulation by STAT5 14. Ondek, B., Shepard, A., and Herr, W. (1987) EMBO J. 6, 1017–1025 but that nonspecific interactions are sufficient to induce asso- 15. Depper, J. M., Leonard, W. J., Drogula, C., Kro ¨ nke, M., Waldmann, T. A., and ciation. Unlike that of STAT5, STAT1 tetramerization does not Greene, W. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4230 – 4234 16. Plaetinck, G., Combe, M.-C., Corthe ´ sy, P., Sperisen, P., Kanamori, H., Honjo, appear to be significantly affected by the rotational orientation T., and Nabholz, M. (1990) J. Immunol. 145, 3340 –3347 of the two STAT motifs on the DNA (34). Regardless of these 17. Pimentel-Muinos, F. X., Munoz-Fernandez, M. A., and Fresno, M. (1994) differences, our data indicate that tetramerization of STAT J. Immunol. 152, 5714 –5722 18. Soldaini, E., Pla, M., Beermann, F., Espel, E., Corthe ´ sy, P., Barange ´ , S., proteins (33, 34) is a general mechanism through which these Waanders, G. A., MacDonald, H. R., and Nabholz, M. (1995) J. Biol. Chem. factors control transcription via weak pairs of binding sites, 270, 10733–10742 which have been observed in several other putative regulatory 19. Sperisen, P., Wang, S. M., Soldaini, E., Pla, M., Rusterholz, C., Bucher, P., Corthe ´ sy, P., Reichenbach, P., and Nabholz, M. (1995) J. Biol. Chem. 270, elements (50 –53). 10743–10753 Tetramer formation may result in functional cooperativity 20. Ihle, J. N., and Kerr, I. M. (1995) Trends Genet. 11, 69 –74 21. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Science 264, 1415–1421 between the two weak STAT5 binding sites simply by increas- 22. Schindler, C., and Darnell, J. E., Jr. (1995) Annu. Rev. Biochem. 64, 621– 651 ing the level of occupancy of sites I and II to a threshold 23. Ivashkiv, L. B. (1995) Immunity 3, 1– 4 required for enhancer activity. But protein-protein interaction 24. Serdobova, I., Pla, M., Reichenbach, P., Sperisen, P., Ghysdael, J., Wilson, A., Freeman, J., and Nabholz, M. (1997) J. Exp. Med. 185, 1211–1221 between STAT5 molecules may also lead to the formation of a 25. Le ´ cine, P., Algarte ´ , M., Rameil, P., Beadling, C., Bucher, P., Nabholz, M., and new molecular surface that binds a coactivator required for Imbert, J. (1996) Mol. Cell. Biol. 16, 6829 – 6840 IL-2rE activity. If tetramer formation is restricted to certain 26. John, S., Robbins, C. M., and Leonard, W. J. (1996) EMBO J. 15, 5627–5635 27. Bucher, P., Corthe ´ sy, P., Imbert, J., and Nabholz, M. (1997) Immunobiology STAT combinations, it may contribute to cytokine response 197, 133–140 specificity. For example, STAT1, which also binds to site I and 28. Hou, J., Schindler, U., Henzel, W. J., Wong, S. C., and McKnight, S. L. (1995) Immunity 2, 321–329 site II, does not contribute to IL-2Ra regulation, perhaps 29. Lin, J.-X., Mietz, J., Modi, W. S., John, S., and Leonard, W. J. (1996) J. Biol. because it does not form transactivating tetramers. If tetramer- Chem. 271, 10738 –10744 ization is required for binding to coactivators or basic transcrip- 30. Beadling, C., Guschin, D., Witthuhn, B. A., Ziemiecki, A., Ihle, J. N., Kerr, I. M., and Cantrell, D. A. (1994) EMBO J. 13, 5605–5615 tion factors, it may serve as a mechanism to select particular 31. Liu, X., Robinson, G. W., Wagner, K.-W., Garrett, L., Wynshaw-Boris, A., and mediators of transactivation. We are planning to explore these Hennighausen, L. (1997) Genes Dev. 11, 179 –186 32. Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S.-H., Ram, P. A., questions, taking advantage of the IL-2rE as a good model for Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, a natural STAT-controlled transcriptional enhancer. 7239 –7244 33. Xu, X., Sun, Y.-L., and Hoey, T. (1996) Science 273, 794 –797 Acknowledgments—We are grateful to Glaxo, Geneva, for generous 34. Vinkemeier, U., Cohen, S. L., Moarefi, I., Chait, B. T., Kuriyan, J., and Darnell, gifts of interleukins. Doreen Cantrell and Carol Beadling of the Impe- J. J. E. (1996) EMBO J. 15, 5616 –5626 35. Silva, A., MacDonald, H. R., Conzelmann, A., Corthe ´ sy, P., and Nabholz, M. rial Cancer Research Fund provided advice and help in setting up the (1983) Immunol. Rev. 76, 105–129 system for affinity precipitation of STAT proteins. Claudine Ravussin, 36. Wilson, A., Corthe ´ sy, P., Reichenbach, P., MacDonald, H. R., and Nabholz, M. Pierre Dubied, and Marcel Allegrini helped with the preparation of the (1994) Eur. J. Immunol. 24, 1729 –1735 figures. We thank Dean Scott for amino acid sequence analysis as well 37. Espel, E., Fromental, C., Reichenbach, P., and Nabholz, M. (1990) EMBO J. 9, as Sonia Barange ´ and Corinne Rusterholz for letting us quote their 929 –937 unpublished results. Michel Aguet, Stefan Ryter, and Corinne Ruster- 38. Geering, G., Old, L. J., and Boyse, E. (1966) J. Exp. Med. 124, 753–772 holz made many helpful suggestions concerning the manuscript. 39. Conzelmann, A., Corthe ´ sy, P., Cianfriglia, M., Silva, A., and Nabholz, M. (1982) Nature 298, 170 –172 REFERENCES 40. Ho, S. N., Hunt, H. D., Horton, R. M., Puller, J. K., and Pease, L. R. (1989) Gene 77, 51–59 1. Coffman, R. L., Lebman, D. A., and Shrader, B. (1989) J. Exp. Med. 170, 41. Schindler, U., Wu, P., Rothe, M., Brasseur, M., and McKnight, S. L. (1995) 1039 –1044 Immunity 2, 689 – 697 2. Waldmann, T. A. (1996) Immunol. Today 14, 264 –270 42. Mikita, T., Campbell, D., Wu, P., Williamson, K., and Schindler, U. (1996) Mol. 3. Asao, H., Takeshita, T., Ishii, N., Kumaki, S., Nakamura, M., and Sugamura, Cell. Biol. 16, 5811–5820 K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4127– 4131 43. Schreiber, E., Matthias, P., Mu ¨ ller, M. M., and Schaffner, W. (1989) Nucleic 4. Taga, T., and Kishimoto, T. (1995) Curr. Opin. Immunol. 7, 17–23 Acids Res. 17, 6419 5. The ` ze, J., Alzari, P. M., and Bertoglio, J. (1996) Immunol. Today 17, 481– 486 44. Sperisen, P., Wang, S. M., Reichenbach, P., and Nabholz, M. (1992) PCR 6. Nakarai, T., Robertson, M. J., Streuli, M., Wu, Z., Ciardelli, T. L., Smith, K. A., Methods Applications 1, 164 –170 and Ritz, J. (1994) J. Exp. Med. 180, 241–251 45. Queen, C., and Baltimore, D. (1983) Cell 33, 741–748 7. Kondo, M., Ohashi, Y., Tada, K., Nakamura, M., and Sugamura, K. (1994) Eur. 46. Kost, T. A., Theodorakis, N., and Hughes, S. H. (1983) Nucleic Acids Res. 11, J. Immunol. 24, 2026 –2030 8287– 8301 8. Takeshita, T., Ohtani, K., Asao, H., Kumaki, S., Nakamura, M., and 47. Sambrook, J., Fritsch, F. E., and Maniatis, T. (1989) Molecular Cloning: A Sugamura, K. (1992) J. Immunol. 148, 2154 –2158 Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring 9. Hatakeyama, M., Tsudo, M., Minamoto, S., Kono, T., Doi, T., Miyata, T., Harbor, NY Miyasaka, M., and Taniguchi, T. (1989) Science 244, 551–556 48. Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211–217 10. Waldmann, T. A., White, J. D., Goldman, C. K., Top, L., Grant, A., Bamford, R., 49. Horvath, C. M., Wen, Z., and Darnell, J. E., Jr. (1995) Genes Dev. 9, 984 –994 Roessler, E., Horak, I. D., Zaknoen, S., Kasten-Sportes, C., England, R., 50. Bergad, P. L., Shih, H.-M., Towle, C. T., Schwarzenberg, S. J., and Berry, S. A. Horak, E., Mishra, B., Dipse, M., Hale, P., Fleisher, T., Junghans, E., Jaffe, (1995) J. Biol. Chem. 270, 24903–24910 E., and Nelson, D. L. (1993) Blood 82, 1701–1712 11. Lowenthal, J. W., Tougne, C., MacDonald, H. R., Smith, K. A., and Nabholz, M. 51. Yan, R., Small, S., Desplan, C., Dearolf, C. R., and Darnell, J. E. J. (1996) Cell 84, 421– 430 (1985) J. Immunol. 134, 931–939 12. Nishi, M., Ishida, Y., and Honjo, T. (1988) Nature 331, 267–269 52. Hou, X. S., Melnick, M. B., and Perrimon, N. (1996) Cell 84, 411– 419 53. Guyer, N. B., Severns, C. W., Wong, P., Feghali, C. A., and Wright, T. M. (1995) 13. Kro ¨ nke, M., Leonard, W. J., Depper, J. M., and Greene, W. C. (1985) J. Exp. Med. 161, 1593–1598 J. Immunol. 155, 3472–3480

Journal

Journal of Biological Chemistry – Unpaywall

Published: Dec 1, 1997

There are no references for this article.