©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mouse Interleukin-2 Receptor Gene Expression
INTERLEUKIN-1 AND INTERLEUKIN-2 CONTROL TRANSCRIPTION VIA DISTINCT cis-ACTING ELEMENTS (*)

Peter Sperisen(§)(¶) , San Ming Wang(§)(**) , Elisabetta Soldaini , Maria Pla (§§) , Corinne Rusterholz , Philipp Bucher , Patricia Corthésy , Patrick Reichenbach , Markus Nabholz (¶¶)

From the (1) Swiss Institute for Experimental Cancer Research (ISREC), 155 Chemin des Boveresses, CH-1066 Epalinges

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have shown that interleukin-1 (IL-1) and IL-2 control IL-2 receptor (IL-2R) gene transcription in CD4CD8 murine T lymphocyte precursors. Here we map the cis-acting elements that mediate interleukin responsiveness of the mouse IL-2R gene using a thymic lymphoma-derived hybridoma (PC60). The transcriptional response of the IL-2R gene to stimulation by IL-1 + IL-2 is biphasic. IL-1 induces a rapid, protein synthesis-independent appearance of IL-2R mRNA that is blocked by inhibitors of NF-B activation. It also primes cells to become IL-2 responsive and thereby prepares the second phase, in which IL-2 induces a 100-fold further increase in IL-2R transcripts.

Transient transfection experiments show that several elements in the promoter-proximal region of the IL-2R gene contribute to IL-1 responsiveness, most importantly an NF-B site conserved in the human and mouse gene. IL-2 responsiveness, on the other hand, depends on a 78-nucleotide segment 1.3 kilobases upstream of the major transcription start site. This segment functions as an IL-2-inducible enhancer and lies within a region that becomes DNase I hypersensitive in normal T cells in which IL-2R expression has been induced. IL-2 responsiveness requires three distinct elements within the enhancer. Two of these are potential binding sites for STAT proteins.


INTRODUCTION

Interleukin-2 (IL-2)() is a growth factor for antigen-activated T lymphocytes. IL-2 drives T cell proliferation by binding to a high affinity receptor that consists of three different transmembrane proteins, IL-2 receptors (IL-2R) , , and (1) . IL-2R expression is tightly regulated by extracellular stimuli (for review, see Ref. 2). It thereby controls, in part, IL-2 responsiveness.

In mature B and T lymphocytes, IL-2R synthesis is triggered by signals from the antigen receptor. In antigen-activated T cells, IL-2 itself is required for prolonged IL-2R expression (3, 4, 5, 6, 7, 8, 9) . In early CD4CD8 thymic T cell precursors that have been activated with phorbol 12-myristate 13-acetate and a calcium ionophore, IL-1 synergizes with IL-2 to increase IL-2R mRNA levels and surface expression (10) .

Regulation of IL-2R expression occurs mostly through changes in the rate of IL-2R gene transcription. This has been shown by run-on transcription experiments for stimulation by phytohemagglutinin, IL-2 and IL-1 (8, 9, 11, 12) , and confirmed by an analysis of mice carrying a mouse IL-2R 5`-flanking region/chloramphenicol acetyltransferase transgene (65) .()

In the human IL-2R gene, cis-acting regulatory elements have been identified by transient transfection of leukemia cell lines in which, however, the regulation of the resident gene had not been well characterized (13, 14, 15, 16, 17, 18, 19, 20) . Regulatory elements conferring responsiveness to a number of stimuli including phorbol 12-myristate 13-acetate and IL-1 were found in the promoter proximal segment of the 5`-flanking region (5`-FR) of the gene (3` of position -390, see below). The elements required for IL-2 responsiveness have not yet been mapped.

Work in our laboratory has focused on the identification of regulatory elements in the mouse IL-2R gene. We have shown (65) that in transgenic mice the segment between bp -2539 and +93 of the mouse IL-2R gene confers on a reporter gene correct cell type specificity and inducible expression in T cells and their early thymic precursors. In the experiments described in this paper, we have used a rodent thymic lymphoma-derived cell line, PC60 (21, 22) to define the regions in the 5`-FR that are required for IL-1- and IL-2-inducible expression. PC60 cells express neither CD4 nor CD8 surface markers (22) , and, like normal CD4CD8 thymocytes (10) , they respond to IL-1 and IL-2 by a strong increase in IL-2R gene transcription. Previous work had shown that in this cell line the 2.5-kb 5`-FR of the mouse IL-2R gene conferred a response pattern on a reporter gene that was very similar to that of the genomic IL-2R genes (11) . Interleukin responsiveness clearly depended on sequences upstream of the promoter proximal region analyzed in the human gene. Experiments using other cell lines, including an antigen specific IL-2-dependent T cell clone confirmed that the upstream segment was crucial for correct expression of mouse IL-2R gene recombinants.

Here we show that the response of the IL-2R gene to IL-1 + IL-2 is biphasic, and that this reflects a bipartite distribution of the interleukin response elements. IL-1 responsiveness is mediated by several elements in the promoter proximal region, which includes an NF-B site conserved in both human and mouse genes, whereas IL-2 responsiveness depends on three separable 8-12-nucleotide elements within 48 nucleotides of an IL-2-responsive enhancer 1.3 kb upstream of the transcription start site. This segment overlaps with a DNase I hypersensitive site detected only in normal T cells in which IL-2R expression has been induced (65) .


MATERIALS AND METHODS

Cell Lines and Culture Conditions: Cytokines and Inhibitors

The PC60.21.14 cell line (here referred to as PC60) has been described in previous publications (11, 21, 22, 23) . PC60(G3) are stable transfectants that constitutively express a cDNA clone encoding the human IL-2R chain (24) . PC60 has inherited from its lymphoma parent the CD4CD8 phenotype (22) and the capacity to grow independently of IL-2. The culture conditions used, as well as the stimulation with IL-1 and IL-2, have been described previously (11, 23, 25) .

Human recombinant interleukins were gifts of GLAXO IMB S.A. (Plan-les-Ouates, Geneva, Switzerland). IL-1 (IL-1) was used at a final concentration of 1 ng/ml; IL-2 was used at 100 units/ml.

Dithiocarbamate (Sigma, Buchs, Switzerland) compounds were used at final concentrations of 10 and 100 µM.

RNA and Nuclear Extract Preparations: RNase Protection and Band Shift Assays

RNA was extracted according to the method of Gough (26) . Nuclear extracts were prepared by a modification of the same method (27) . Methods used for RNase protection and band shift assays are modifications of techniques developed by others and have been described in previous publications (23, 28) . The probes used have been described in detail elsewhere (10) . The mouse IL-2R probe corresponds to a PstI cDNA segment (bp 89-503). Experiments with rat T cell blast RNA showed that this probe also protects defined fragments of rat IL-2R mRNA (data not shown).

RNase protection reactions were fractionated on sequencing gels. Signals were quantified with a PhosphorImager, model SF (Molecular Dynamics, Sunnyvale CA 94086). IL-2R signals were normalized to the -actin signal, which was reduced to comparable levels by labeling the -actin probe with [-P]CTP of 10-fold lower specific activity.

Sequence Analysis

Parts of the sequence of the 5`-FR of the mouse IL-2R gene have been published previously (29) . The sequence from bp -2539 to +93 has been determined on both strands by the dideoxy method and entered into the Genebank/EMBL data bases as modification of the original sequence (EMBL accession M16398, version 10).

Human and mouse IL-2R 5`-flanking sequences were aligned with the program BestFit from the GCG sequence analysis software package (30) using the default scoring system (match = 1.0, mismatch = -0.9, gap weight = 5.0 + 0.3 gap length). TATA box (31) and GC box (31) motifs were identified using program EukProm from PC/Gene (release 6.7 (1992), IntelliGenetics Inc., Mountain View, CA). Consensus recognition sequences for transcription factors were located with the GCG program FindPattern (30) . Searches for local sequence similarities were performed using the BLAST server at the National Institute for Biotechnology Information in Bethesda, MD (32) . Optimal local alignments between selected sequence regions were generated with the aid of GCG program BestFit (30) using the default scoring system (match = 1.0, mismatch = -0.9, gap weight = 5.0 + 0.3 gap length). Statistical significance of optimal alignment scores was assessed by calculating Z scores based on 100 shuffled sequence pairs. Type and orientation of repetitive elements in the mouse IL-2R 5`-FR were identified by computing optimal alignment scores against a representative set of mammalian LINE and SINE sequences, including the sequences with the following EMBL accession numbers: X52235 (version 10), M30839 (version 1), M30839 (version 4), J00628 (version 1, position 93-222), M87890 (version 2), K02592 (version 2, position 250-660), J01879 (version 1, position 441-522), X02215 (version 3, position 587-849). The number and approximate location of elements sharing significant sequence similarities with one of the promoter regions was determined by matrix comparisons using GCG programs Compare and DotPlot (30) . The exact delineation of the repetitive regions as shown (see Fig. 4) is based on optimal local sequence alignments between the regions highlighted by the DotPlot method and the corresponding repetitive element sequence. Internal direct repeats were searched for by the DotPlot method and by generating multiple locally optimal sequence alignments with the aid of program LAlign from the FastA package (33) . The scoring system for multiple local sequence alignment was as follows: match = 4, mismatch = -3, gap weight = 8 + 4 gap length. Statistical significance of direct repeats was assessed by Z score calculations using program RSS from the same package.


Figure 4: The IL-2R 5`-FR controls transcriptional responses to IL-1 and IL-2. A,mouse IL-2R 5`-FR and IL-2R/rabbit -globin reporter gene recombinants. Note that nucleotide numbering is changed with respect to previous publications (11, 29) to start at the major cap site (+1) of IL-2R transcripts in T cell lines (61) as well as normal mature T cells and thymocytes. In the top representation are also indicated the positions of repetitive elements. Plasmid pwt contains the -2539/+93 ( SphI ( S) to PstI ( P)) segment of the IL-2R gene. The segment is subdivided into regions A, B, C and D, separated by BglII sites ( B). Plasmid pR is identical to pwt except for restriction sites that have been inserted into the BglII sites ( H, HindIII; X, XbaI; see ``Materials and Methods''). In pRC, the entire C segment is deleted. B, interleukin response pattern of IL-2R/rabbit -globin reporter gene recombinants. PC60 cells were cultured in normal medium or in the presence of IL-1 for 3 days prior to transfection. Nonstimulated or IL-1-primed cells were transfected with a mixture of the indicated IL-2R construct, and a reference plasmid carrying a modified version of the same reporter gene joined to the constitutive rabbit -actin promoter (pGAcGlD or ref). Transfected cells were split into aliquots that were cultured in the presence or the absence of the indicated interleukins for 48 h. Reporter gene transcripts were revealed by PCR amplification of -globin cDNA according to Ref. 34. The ratio of IL-2R ( i) to reference signal ( r), determined with a PhosphorImager, is a measure of the promoter activity of the corresponding IL-2R construct. By comparing this normalized promoter activity in unstimulated and interleukin-stimulated cells, one can quantify the interleukin response of the IL-2R recombinant. The values shown represent means of three independent experiments with ranges below each column. C and D, kinetics of the IL-2 response of IL-2R 5`-FR/-globin reporter gene recombinants. Primed or unstimulated PC60 cells were transfected with 0.4 pmol of the reference plasmid and 2 pmol of IL-2R 5`-FR/-globin recombinants pwt or pRC3 (see Fig. 5 A). Aliquots of the transfected cells were cultured either in normal medium or in the presence of IL-1 for 48 h. IL-2 was added to individual cultures to be present for the indicated period prior to harvest and RNA extraction and assay of reporter gene expression. Note that the IL-2R mRNA levels were measured in the same samples (see Fig. 3). The gel shown ( D) represents the results obtained with primed cells.



Plasmids

Plasmids were prepared according to Ref. 23. Unless mentioned otherwise, plasmid constructions were made in pGEM3Zf(+) (Promega). The reference plasmid pGAcGlD (hereafter referred to as ref) has been described previously (34) . In plasmid pwt1Gl (hereafter referred to as pwt, see Fig. 4A) the SphI- PstI fragment from pUC18 mIL-2Rp (29) was joined to the 1664-bp PvuII rabbit -globin gene fragment from Z-pCRI/pRChrG-1 (35) . To facilitate the introduction of deletions in segment B and C, we constructed plasmid pBC in which B and C, separated by a PstI site, are cloned between the XbaI and HindIII sites of pGEM3Zf(+). In plasmids pBC1Gl (pBC) and pBC2Gl, (pBC2) segments B and C are replaced by a double-stranded oligonucleotide (5`-gatcAAGCTTaagTCTA 3`, 3` TTCGAAttcAGATCTag 5`) containing two unique restriction sites, HindIII (boldface) and XbaI (underlined), in either one or the other orientation. pR1Gl (pR) and pRiGl (pRi) were obtained by introducing the XbaI- HindIII fragment of pBC into pBC or pBC2. All constructs were checked by DNA sequencing.

Mutations in Segment C

Deletions C1-C5 (see Fig. 5 A) were introduced into pBC using existing restriction sites. The modified XbaI- HindIII fragments were used to replace the XbaI- HindIII fragment in pR. Deletions and substitution mutations within segment C3 (see Fig. 5, B and D) were introduced into pBC with the in vitro mutagenesis system described in Ref. 36. The modified NsiI fragments were used to replace the corresponding fragment of pwt.


Figure 5: Mapping of IL-2 responsive elements in segment C. A, the sequences required for IL-2 responsiveness lie between bp -1389 and -1228. The indicated IL-2R/-globin recombinants were transfected into IL-1 primed PC60 cells together with reference plasmid at a ratio of 5:1. The transfected cells were cultured for 48 h in IL-1 with or without IL-2. Promoter activity of the IL-2R constructs is determined by normalizing the corresponding mRNA signal to that from the cotransfected reference plasmid. The IL-2 response is the ratio between promoter activity after culture in IL-1 + IL-2 and the activity after culture in IL-1 alone. Indicated is the mean of two independent experiments, with ranges given below each bar. Base-line promoter activity (in IL-1 only) of the different IL-2R constructs, normalized to that of pwt (=1), varied within the following ranges: pBC, 1.4-1.5; pR, 0.6-0.8; pRi, 0.3-0.5; pRC1, 0.2-0.3; pRC2, 0.3-0.5; pRC3, 1.6-1.7; pRC4, 0.4-0.5; pRC5, 1.0-1.5. B, IL-2 responsiveness is controlled by 75 bp in segment C3. IL-2 responses of the different plasmids were determined as in A. Base-line expressions, normalized to that of pwt, ranged as follows: pRC3, 1.5-2.0; pRC3.1, 1.1-1.8; pRC3.2, 0.6-1.0; pRC3.3, 0.9-1.6; pRC3.4, 0.9-1.3; pRC3.5, 1.0; pRC3.6, 1.9-3.0. C, segment C3 acts as an IL-2 responsive enhancer. Single copies of different portions of the IL-2R 5`-flanking region were inserted upstream of the minimal -globin promoter of pG1. IL-2 responses of the different plasmids were determined as in A. The results shown are means of two experiments. The responses varied by less than 20%. Mean base-line expressions, normalized to that of pGAcGlD (reference plasmid), were 0.06 ( pG1), 0.06 ( pG1. BC), 0.15 ( pG1. C2+C3) and 0.12 ( pG1.C3). D, three separate elements in the enhancer are required for IL-2 responsiveness. Below the sequence of segments C3.1, C3.2 and C3.3 are indicated the 14 mutations that were introduced into pwt ( dash, deletion; lowercaseletter, substitution) and the IL-2 responses of the corresponding plasmids. The mean of two experiments is given, and the result of a single experiment is shown below. IL-2 inducibility varied by less than 20% between experiments. I, II, and III indicate the separate elements mutations in which abolish IL-2 inducibility.



Deletions in Segment D

To construct deletions in region D of pwt, this segment was subcloned as a BglII (-802) to PstI (+93) fragment. Segments D1-D6 (see Fig. 6 B) were removed using suitable restriction sites. The modified D segments were used to replace the XbaI- BsmI fragment of plasmid pR, to give rise to plasmids pRD1-pRD6. From each of these plasmids, segment C was removed by digestion with PstI and XbaI to give rise to pRCD1, etc. (see Fig. 6 B).


Figure 6: Mapping of IL-1 responsive elements in segment D. A, sequence comparison of the promoter-proximal segments of human and mouse IL-2R genes. The sequences upstream of positions -339 in the human (EMBL accession number M15864, version 2) and -335 in the mouse gene do not share significant similarities according to shuffling tests (see ``Materials and Methods''). Cap sites are indicated by arrows. Apart from minor differences transcription start sites in normal mouse lymphocyte (S. M. Wang and M. Nabholz, unpublished results) correspond to the sites determined by Froussard et al. (61). TATA boxes and consensus binding sites for transcription factors were identified by computer analysis. TATA boxes were searched for on the + strand only. TATA and GC boxes conform to the weight matrix descriptions by Bucher (31). The score (-8.80) of the TATA box at position -30 in the human gene falls slightly below the recommended cut-off value of -8.16. A SRF binding site is defined as a perfect match with the sequence CCWWWWWWGG (62). An NF-B site is defined as occurrence of the consensus sequence GGGRNNNYYCC (63) with one mismatch allowed in the YYC segment. An AP-1 site is defined as the consensus sequence TGASTCA (62), with one mismatch allowed in any position. Studies on the human gene have provided evidence for a regulatory role of negative regulatory elements (NRE)-1 (64) and -2 (17) and for NF IL-2RA or UE-1 (19, 44), as well as for the SRF (19), Sp1 (GC-box) (43, 44), and NF-B (15, 16, 18) binding sites upstream of bp -230. B, delineation of IL-1 response elements. Nonstimulated PC60 cells were transfected with a mixture of the reference plasmid, and the indicated IL-2R 5`-FR/-globin recombinants, which lack region C as well as a portion of region D. B1 and B2 indicate the positions of the two NF-B sites shown in Fig. 6 A. After transfection cells were cultured for 48 h with or without IL-1. IL-1 responses are plotted as fold stimulation of expression over base-line expression. For each plasmid, the mean and range of values obtained in three independent transfection experiments are indicated. C, amplified mRNA signals from one transfection experiment. The ratios between IL-2R constructs and reference plasmid were 10:1 (pRC, pRCD2, pRCD4); 2.5:1 (pRCD1); 25:1 (pRCD3, pRCD6); and 100:1 (pRCD5). i, mRNA signals from IL-2R 5`-flanking region/-globin reporter gene recombinants; r, mRNA signals from reference plasmid.



pG1 Recombinants

pG1.BC was obtained by subcloning the XbaI- HindIII fragment of pBC into pG1 (37) . The resulting plasmid contains a single copy of segment B+C of the IL-2R 5`-flanking region in the reverse orientation.

To introduce fragments C3 or C2+C3 into pG1, these segments were amplified from pwt with the proofreading Pfu polymerase (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The primers used contained an XbaI site at the 5` and a HindIII site at the 3` end. The PCR products were digested with these enzymes and ligated with HindIII- XbaI-digested pG1. The resulting plasmids contain single copies of segment -1635 to -1301 (pG1.C2+C3) or segment -1403 to -1301 (pG1.C3) of the IL-2R 5`-flanking region in their normal orientation.

Reporter Gene Assay

Cells were transiently transfected using DEAE-dextran according to Ref. 38. Reporter gene expression was measured as described previously (34) . Briefly, cells were transfected with a defined ratio of an IL-2R/rabbit -globin gene construct and the reference plasmid. This contains a rabbit -globin gene with a 40-bp deletion in the second exon and is transcribed under the control of the constitutive chicken -actin promoter (39) . Cytoplasmic RNA of the transfected cells was treated with DNase I. After cDNA synthesis from a poly(dT) primer with avian myoblastosis virus reverse transcriptase, -globin cDNA was amplified in the presence of [-P]dATP with primers spanning the deletion in the reference plasmid and the first intron. In some experiments we used, instead of the previously described 3` primer (34) , the oligonucleotide 5` ACGTTGCCCAGGAGGAAGT 3`, which spans the second intron of the -globin gene ( marks exon boundaries). As expected, this reduces signals due to residual plasmid contaminating mRNA preparations. PCR products were denatured and separated on a sequencing gel. mRNA derived signals were quantified by PhosphorImager.


RESULTS

Induction of IL-2R Expression in PC60 Occurs in Two Phases

PC60 is a somatic cell hybrid between the mouse cytolytic T lymphocyte line B6.1 and the rat thymic lymphoma C58 (21) . It has inherited from its lymphoma parent the CD4CD8 phenotype (22) and the capacity to grow independently of IL-2. In unstimulated PC60 cells, IL-2R mRNA is barely detectable. IL-1 and IL-2 induce appearance of IL-2R mRNA and synergize to control its level (11) . The interleukin response pattern of the rat IL-2R gene in the parental C58 lymphoma and in PC60 is identical to that of the mouse gene in PC60 (data not shown). This excludes the possibility that the interleukin response of the mouse IL-2R gene is an artifact due to a rearrangement of the mouse gene in the parental mouse T cell line or in PC60 itself. A very similar response is observed in normal CD4CD8 thymocytes (10) . Previously reported run-on experiments showed that IL-1 and IL-2 regulate IL-2R gene expression in PC60 cells by changing its rate of transcription, and we have shown (65) that the same is true for normal thymocytes. Detailed analysis by RNase protection experiments (Fig. 1) reveals that the interleukin-induced increase in IL-2R mRNA levels in PC60 cells is biphasic. Stimulation with IL-1 + IL-2 induces detectable transcripts within 1 h. The rate of increase in transcript levels slows down between 4 and 5 h after interleukin addition, but accelerates again between 10 and 18 h. This biphasic increase in IL-2R mRNA has been observed in three other experiments. Maximal transcript levels, more than 1000-fold above background, are reached by about 48 h (Ref. 11 and data not shown).


Figure 1: Kinetics of IL-1 + IL-2-induced IL-2R mRNA increase in PC60. A, PC60 cells were cultured for the indicated times in saturating concentrations of IL-1 and IL-2. Total cytoplasmic RNA was assayed by RNase protection with a mixture of antisense RNA probes for mouse IL-2R and -actin ( emptyarrowheads on left side of gel, see ``Material and Methods''). Note that the specific activity of the -actin probe is 10 times lower than that of the IL-2R probe. B, mouse IL-2R signals were quantified by PhosphorImager and normalized to the mouse -actin signal in the same lane.



The Initial, IL-1-induced Phase of IL-2R Induction Can Be Blocked by Inhibitors of NF-B Activation

We previously showed that the early response of the IL-2R gene can be induced by IL-1 alone and is protein synthesis-independent (11) . IL-2 has no or very little effect during this stage. The immediate responses of many genes to IL-1 are due to activation of NF-B (40) . In PC60 cells, IL-1 induces appearance of NF-B in the nucleus and activates transcription from promoters with NF-B binding sites (23, 41) . We therefore tested whether inhibitors of NF-B activation (42) block the early response of the IL-2R gene to IL-1. Fig. 2shows that the addition of 100 µM pyrrolidinedithiocarbamate (PDTC) prior to and during a 3-h stimulation with IL-1 inhibits appearance of NF-B in the nucleus and completely blocks the rise in IL-2R mRNA. 10 µM PDTC has no inhibitory effect. Longer incubation at inhibitory doses results in significant loss of viability. Diethyl-DTC has the same effects (results not shown). The similar sensitivity of NF-B activation and IL-2R mRNA appearance to PDTC suggests that the two phenomena are linked and that the early phase of IL-2R gene induction by IL-1 is mediated by NF-B.


Figure 2: Effect of an inhibitor of NF-B activation on the early response of the IL-2R gene to IL-1. PC60 cells were cultured with or without IL-1, in the presence of the indicated concentrations of PDTC. The inhibitor was added 30 min prior to IL-1. Three h after IL-1 addition, cytoplasmic RNA and nuclear extracts were prepared. A, IL-2R and -actin mRNA levels were determined by RNase protection. Emptyarrowheads on the left, undigested probes; filledarrowheads on the right, mRNA signals. The signals just below the band corresponding to the IL-2R probe are due to a interferon- probe also present in the reaction. B, NF-B was detected by band shift assay with a probe containing the TC-II element of the SV40 enhancer. The identity of the protein giving rise to the complex labeled as NF-B has been verified previously (41). F, free probe.



IL-1 Primes PC60 Cells to Become IL-2 Responsive

Stimulation of PC60 with IL-1 alone induces, beyond the first protein synthesis-independent phase of the response, a significant further increase in IL-2R expression (11) . This is due to the fact that IL-1 induces PC60 cells to secrete IL-2, so that treatment with IL-1 alone amounts to stimulation with both interleukins. IL-2 secretion peaks around 24 h, and after 72 h IL-2 is no longer detectable in culture supernatants. This explains why IL-2R mRNA levels remain maximal in cells stimulated for 72 h with both IL-1 and exogenously supplied IL-2 (11) , while they have returned to very low levels in cells stimulated with IL-1 only. However, the addition of IL-2 to such IL-1-prestimulated cells induces a rapid rise in IL-2R mRNA levels, whereas in cells that have not been exposed to IL-1, IL-2 induces a barely detectable increase (Fig. 3). The initial kinetics of the response of cells precultured in IL-1 do not depend on the continued presence of IL-1 (data not shown). Thus, preculture in IL-1 increases the capacity of PC60 cells to respond to IL-2. We refer to this effect as IL-1 priming. Priming of PC60 cells presumably depends on a low level of IL-2R expression that persists after 72 h in IL-1, necessary for IL-2 responsiveness. This is consistent with the finding that the IL-2 response of primed PC60(G3) cells, which constitutively express a human IL-2R cDNA clone (24) , is much stronger than that of PC60 itself. But the fact that priming can be observed also in PC60(G3) indicates that it requires other IL-1-induced changes. PC60 cells express the IL-2R gene constitutively but prolonged exposure to IL-1 induces a 10-30-fold increase in IL-2R mRNA and a 10-fold increase in high affinity IL-2 receptors (data not shown). This suggests that priming also depends on increased IL-2R chain expression. There may yet be other factors that control responsiveness. This might explain the considerable variation in the maximal inducible level of IL-2R mRNA in primed PC60 cells, which is 3-10 times weaker than that in unprimed cells stimulated with IL-1 + IL-2 in the same experiment (data not shown).


Figure 3: Priming with IL-1 increases the responsiveness of the IL-2R gene to stimulation with IL-2. PC60 cells were cultured for 3 days in normal medium or in the presence of IL-1. Both IL-1-primed and nonprimed cells were aliquoted into fresh cultures (0 h), and cells were harvested 48 h later. During this time, IL-2 was added to individual cultures to be present for the indicated period prior to RNA extraction. IL-2R mRNA was detected by RNase protection. Signals were quantified and normalized to -actin mRNA signals (A). The gel shown in B shows the results obtained with primed cells.



Thus, IL-1 has two effects on PC60 cells. First, it rapidly induces IL-2R gene transcription, at least in part via protein synthesis-independent pathways that lead to activation of NF-B. Second, it increases the capacity of PC60 cells to respond to IL-2, partly by increasing IL-2R and IL-2R expression. This priming effect permitted us to use PC60 cells for the mapping of the cis-acting elements that control IL-2 responsiveness of the IL-2R gene.

Mapping of Interleukin Responsive cis-Acting Elements in the IL-2R Gene

Previously, we had shown that in PC60 cells a segment of the mouse IL-2R gene comprising the promoter, the transcription start site, and a 2.5-kb 5`-FR (position -2539 to +93, segment A-D, see Fig. 4 A) conferred on a chloramphenicol acetyltransferase reporter gene a response pattern to IL-1 and IL-2 that resembled closely that of the genomic IL-2R gene (11) . In normal CD4CD8 thymocytes of transgenic mice carrying the same construct, chloramphenicol acetyltransferase expression responds to IL-1 and IL-2 with the same pattern as in PC60 (65) .

For the mapping of interleukin response elements in the IL-2R 5`-FR, we switched from the chloramphenicol acetyltransferase to a rabbit -globin reporter gene in order to be able to measure reporter gene mRNA levels with a previously described PCR-based assay (34) . This method allows very accurate quantification of promoter responses to stimulation, e.g. with interleukins (41) . Fig. 4 B shows that the interleukin responses of plasmid pR, which contains segments A-D of the IL-2R 5`-FR, are the same as those of the previously described equivalent chloramphenicol acetyltransferase construct (pmIL-2RPrCAT1, see Ref. 11). When plasmid pR is transfected into unprimed PC60 cells, subsequent stimulation with IL-1 alone induces, on average, a 5-fold increase in reporter gene expression. As pointed out above, under these conditions, stimulation with IL-1 results in transient IL-2 secretion. IL-2 stimulates a small but reproducible response, whereas both cytokines together stimulate a 12-fold increase in reporter gene mRNA. When the same plasmid is transfected into PC60 cells that have been primed by culture in IL-1 for 72 h, the pattern of interleukin responsiveness changes. IL-2 alone induces a response that is almost as strong as that to IL-1 + IL-2. Comparison of these results with the interleukin response of the genomic IL-2R gene in primed and unprimed PC60 cells shows a strikingly similar pattern, although the responses of the genomic IL-2R gene are stronger than that of the reporter gene constructs. Such differences are observed in most transient transfection studies of promoters. There is considerable variability in the magnitude of the IL-2 response of pwt in primed cells (see e.g.Fig. 7 ), which may be related to similar variation in the inducibility of the genomic IL-2R gene, but the response pattern is completely reproducible. The interleukin inducibility of plasmid pwt (see Fig. 4A) does not differ significantly from that of pR, indicating that the insertion of restriction sites in the latter construct had no effect on interleukin responsiveness. Comparing the time course of IL-2-induced accumulation of IL-2R transcripts (Fig. 3) with that of plasmid pwt mRNA in unprimed and IL-1 primed cells (Fig. 4 C), we found that both responded without a measurable lag and with similar kinetics.


Figure 7: IL-2 responsiveness does not require promoter-proximal elements. IL-1 primed PC60 cells were transfected with reference plasmid and plasmids containing an intact region C but deletions in segment D (pRD1 to pRD6, see Fig. 6 B). Aliquots of the transfected cells were cultured for 48 h in the presence of the indicated interleukins. Reporter gene expression is compared with that in cells cultured in normal medium after transfection (=1). Each construct was tested at least three times. The figure shows the mean of two different experiments in which the entire series of plasmids was transfected into the same batch of primed cells, with the range of values obtained below each column.



These results show that many if not all of the cis-acting elements that control the complex response of the IL-2R gene to IL-1 and IL-2 are contained in segments A-D.

IL-2 Response Elements

In previous experiments, we had found that segment C conferred IL-2 responsiveness on a heterologous viral (HSV-tk) promoter (11) . This is consistent with the results shown in Fig. 4, demonstrating that a plasmid in which segment C has been deleted (pRC) can still respond to IL-1 in unprimed PC60 cells but completely fails to respond to IL-2 in either primed or unprimed cells. Note that deletion of segment C (or B+C, Fig. 5) does not significantly alter constitutive promoter activity. The orientation of segment C in the 5`-FR can be inverted (in plasmid pRi) without affecting the response to IL-2 (Fig. 5 A).

We have mapped the elements in segment C required for IL-2 responsiveness in three stages (Fig. 5). In the first step, we introduced a series of contiguous deletions of 160-250 bp into segment C. Comparing the IL-2 responsiveness of these plasmids in IL-1-primed PC60 cells (Fig. 5 A) we found that deletion of the segment between bp -1389 and -1228 (C3) completely abolished IL-2 responsiveness. The plasmid with this deletion (pRC3) failed to respond in cells stimulated with IL-2 for times between 1 and 48 h (Fig. 4, C and D). Deletion of segment C2, immediately upstream of C3, reproducibly reduced but did not abolish the IL-2 response. Deletion of other parts of segment C did not significantly affect IL-2 responsiveness. Note that the changes in IL-2 responsiveness do not correlate with variations in constitutive promoter activity (see legend to Fig. 5 A).

To delimit more precisely the sequences within segment C3 that were required for IL-2 responsiveness, we introduced six contiguous deletions within this segment into plasmid pwt that contains the entire IL-2R 5`-FR. Two of these deletions, C3.1 and C3.3, abolished IL-2 responsiveness, and removal of the intervening segment, C3.2, strongly reduced IL-2 responsiveness (Fig. 5 B), whereas deletions in the remaining part of segment C3 (C3.4, C3.5 and C3.6) had no significant effect on the capacity of the corresponding plasmids to be stimulated by IL-2. Again, there was no correlation between constitutive promoter activity and IL-2 responsiveness among the plasmids of this series (see the legend to Fig. 5 B).

To test whether the segment defined by deletions C3.1, C3.2, and C3.3 was sufficient to confer IL-2-inducible enhancer activity on a heterologous promoter, we inserted the fragment between nucleotides -1406 and -1301 upstream of the minimal -globin promoter of pG1 (37) that does not respond to stimulation by IL-2. The resulting plasmid, pG1.C3, responds to IL-2 as well as pG1.BC, which contains the entire segment B+C (Fig. 5 C). Addition of segment C2, in plasmid pG1.C2+C3, does not significantly improve IL-2 inducibility.

Finally, we mapped the elements required for IL-2 responsiveness of the IL-2R gene by scanning the segments C3.1, C3.2, and C.3.3 with a series of mutations involving 4-9 nucleotides each. The results (Fig. 5 D) show that IL-2 responsiveness of pwt depends on three separate stretches that are between 6 and 12 nucleotides long. These sites are defined by mutations M4, M5, and M6 (site I), mutation M9 (site II) and mutations M12 and M13 (site III). Each of them presumably corresponds to the binding site of a transcription factor required for IL-2 responsiveness (see ``Discussion'').

IL-1 Response Elements

The results shown in Fig. 4B demonstrate that removal of segment C does not abolish IL-1 responsiveness of the IL-2R 5`-FR. The data presented in Fig. 2indicate that the early protein synthesis-independent response of the genomic IL-2R gene to IL-1 requires activation of NF-B, and studies on the human promoter proximal region have shown that the IL-1 response mediated by this segment depended on a NF-B site (18) , which is conserved in the mouse gene (Fig. 6 A, B1 in Fig. 6 B). This led us to investigate the effect of deletions in the conserved promoter proximal region on the IL-1 responsiveness of the mouse gene. Since stimulation of PC60 with IL-1 induces secretion of IL-2, we used for these experiments recombinants that had been made IL-2 unresponsive by the removal of region C.

Fig. 6B compares the constitutive expression and IL-1 responsiveness of plasmids missing various segments of the region between position -585 and -54. Note that the ratio between reference plasmid and IL-2R recombinant is not the same in all the transfections (see legend to Fig. 6C). It should also be pointed out that the normalization of signals derived from IL-2R recombinant transcripts ( i) to the signals derived from reference plasmid mRNA ( r) is very precise and allows reliable determination of 2-fold inductions. The expression of the plasmid containing an intact D region (pRC) is increased about 5-fold by stimulation with IL-1. Deletion D3 completely abolishes this response, indicating that all IL-1-responsive elements in pRC are contained in the segment between positions -585 and -54. Note that the constitutive expression of plasmid pRCD3 is not significantly different from that of pRC. Deletion D4, which removes 23 bp, containing the NF-B site (B1) conserved in the mouse and the human genes, reduces IL-1 responsiveness to 2-3-fold. Removal of the segment 5` of this stretch (D1) reduces responsiveness to the same extent as D4, indicating that elements upstream of the conserved NF-B site are required for a complete IL-1 response. Deletion D6 does not reduce IL-1 responsiveness, confirming that elements between position -585 and -246 contribute most of the response. However, the plasmid with the complementary deletion, D2, still responds reproducibly, albeit weakly, to IL-1, indicating that elements downstream of B1 may also contribute to the IL-1 response of the IL-2R gene. The increased base-line expression of pRCD1 suggests a role for the conserved negative regulatory element-2 (see Fig. 6 A) that reduces expression of human IL-2R constructs in phorbol 12-myristate 13-acetate-stimulated Jurkat cells (17) , but in this case, pRCD2 would be expected to show a similarly increased constitutive activity.

Promoter Proximal Elements Are Not Essential for IL-2 Responsiveness

Testing the effect of deletions D1-D6 on the IL-2 response of plasmids containing an intact C region in IL-1-primed cells we found that none of these deletions abolish the response (Fig. 7), although some markedly reduce it. Thus, the function of the IL-2 response elements in segment C does not depend on sequences between position -585 and -54. This is in agreement with the finding that segment C3 can confer IL-2 responsiveness on a heterologous promoter (Fig. 5 C). The observation that D2 does not significantly alter IL-2 responsiveness argues against any role for NF-B in this response since computer searches have not revealed any NF-B consensus site in the mouse IL-2R 5`-FR apart from B1 and B2 (see legend to Fig. 6 A for definition of NF-B consensus site).


DISCUSSION

PC60 Is a Model for Normal CD4CD8 Thymocytes-We have recently shown that IL-1 and IL-2 synergize to induce IL-2R mRNA levels and surface expression in normal CD4CD8 thymocytes. Analysis of transgenic mice lines (65) demonstrated that this regulation depends on the 5`-FR of the IL-2R gene. In this paper we have used a CD4CD8 cell line whose response to IL-1 and IL-2 is very similar to that of normal cells with the same phenotype to analyze more closely the mechanisms that mediate interleukin responsiveness of the IL-2R gene and to localize the cis-acting elements controlling it. Since these cells do not depend on interleukins for growth or survival, the responses analyzed are not the indirect consequences of the growth factor activity of these interleukins.

Stimulation of PC60 cells with IL-1 + IL-2 induces a biphasic increase in IL-2R mRNA levels. The first phase is a protein synthesis-independent response to IL-1 that can be blocked by inhibitors of NF-B activation. The second phase is due mainly to IL-2 and starts at about 10 h. This delay is due to the fact that unstimulated cells respond very slowly to IL-2 alone, whereas stimulation with IL-1 induces a strong increase in IL-2 responsiveness. This priming effect reflects, most likely, a rise in IL-2R and IL-2R expression that results in an increased number of high affinity IL-2R.

Previous work has shown that the interleukin induced rise in IL-2R mRNA is due mainly to an increase in IL-2R gene transcription. The experiments described in this paper have allowed us to map the cis-acting elements that control both phases of mRNA increase.

IL-1-responsive Elements Map to the Conserved Promoter Proximal Region

The elements that account for the direct response to IL-1 are contained within the promoter proximal segment (position -585 to -54) of the IL-2R gene. Between bp -335 and the transcription start site the mouse gene is 78% homologous to the human gene (Fig. 6 A). Within this segment, a conserved motif that was recognized as a NF-B consensus binding site (B1) appears to play a major role as an IL-1 response element. This is consistent with the finding that inhibitors of NF-B activation prevent the early response of the endogenous IL-2R gene in PC60. It is also in agreement with a previous study in which the human 5`-FR was analyzed in murine EL4 cells (18) . However, sequences upstream of the conserved NF-B site are required to obtain a full response. The contribution of this segment (D1) may be due to a second NF-B site (B2). But this motif is not conserved in the human gene, and it seems more likely that an AP-1 site present in both species around position -335 contributes to the IL-1 response (Fig. 6 A). We have previously shown that AP-1 elements can mediate IL-1 responsiveness in PC60. AP-1-dependent mRNA increases are slower than those mediated by NF-B and are not yet detectable after 4 h of IL-1 treatment (23) . Thus, NF-B may mediate the very early response of the endogenous IL-2R gene to IL-1, which is completely blocked by PDTC, while AP-1 may act in a later response phase.

In spite of the strong homology between the promoter proximal regions of the mouse and the human IL-2R genes the binding sites for SRF and Sp1 (GC-box) that are immediately downstream of the NF-B site in the human gene and that have been implicated in its phorbol 12-myristate 13-acetate response (19, 43, 44) , are not conserved in the mouse gene. The sequence called NF IL-2RA or UE-1 (19, 44) is partially conserved, but the specificity of the protein(s) binding to this element has not been sufficiently well characterized to determine whether their binding site is present in the mouse gene.

IL-2 Responsiveness of the Mouse IL-2R Gene Depends on Three Closely Spaced Elements 1.3 kb Upstream of the Transcription Start Site

None of the deletions in segment D abolishes IL-2 responsiveness. Removal of the NF-B consensus sites B1 and B2 in deletion D2 does not significantly affect IL-2 inducibility. Since a computer search revealed no additional NF-B sites in segments A-D (see legend to Fig. 6 A for search conditions), it is unlikely that NF-B plays a direct role in the transcriptional regulation of the IL-2R gene by IL-2. Even pRD3 in which all IL-1 response elements have been removed is still significantly stimulated by IL-2. This result implies that IL-1-mediated transcriptional activation of the IL-2R gene is not a prerequisite for the activity of the IL-2-responsive elements in segment C. It is consistent with the observation that segment C3 acts as an IL-2-responsive enhancer on a heterologous promoter (Fig. 5 C) that cannot be stimulated with IL-1 (45) . Nevertheless, the reduced IL-2 responsiveness of plasmids with deletions in the promoter proximal region (particularly pD5, pD3, and pD6) suggests that maximal activity of the IL-2-responsive elements in segment C3 does depend on elements in the promoter region itself.

Using IL-1 primed cells, we have shown that the elements in segment C that are strictly required for IL-2 responsiveness of the IL-2R gene map to a 48-bp segment 1.3 kb upstream of the transcription start site. Elsewhere (65) we show that in normal T lymphocytes, a DNase I-hypersensitive site appears at this position when cells are stimulated to express the IL-2R gene. This provides completely independent evidence for an important regulatory role of this segment. Our results show that three separate elements within the 48-bp segment are required for full IL-2 responsiveness. Site I (defined by mutations M4-M6) contains a nucleotide sequence that is identical to the core of the GAS-1 element in the tryptophanyl-tRNA synthetase gene. This sequence binds several cytokine-inducible members of the STAT family of transcription factors (46, 47, 48) . Since it has recently been shown that IL-2 can activate STAT proteins that recognize similar motifs (49, 50) , it is likely that site I is the target of such a factor. However, band shift and footprinting experiments with PC60 nuclear extracts have not yet revealed any IL-2-inducible DNA-binding proteins specific for sequences in C3. The sequence of site II (defined by mutation M9) is very similar to that of site I and may also be a target of an IL-2-induced STAT protein. But it includes a potential binding site for factors of the GATA family, a member of which has been implicated in the regulation of T cell-specific gene expression (51, 52, 53) . Site III (defined by mutations M12 and M13) forms part of a consensus binding site for transcription factor Ets-1 (54) . The consensus site extends into the nucleotides altered in M14, but this mutation is compatible with Ets-1 binding. Site III overlaps with a DNase I footprint over position -1331 to -1314 that is obtained with extracts from induced as well as nonstimulated PC60 cells.() Further work is required to definitively identify the transcription factors that confer IL-2 responsiveness to the C3 segment, and to define their role in this response. We also plan to explore whether the finding that significant IL-2 responsiveness requires three distinct but closely spaced elements is a reflection of some form of direct cooperation between the proteins binding to these sites.

Stimulation of IL-2R transcription by IL-2 has been observed in human cells, and it seems likely that the proteins and sequence elements that mediate this regulation are the same in rodents and in man. The sequence of the 5`-FR of the human gene until position -1241 has been published. The two genes share highly homologous promoter-proximal regions, but apart from homology between repetitive Alu and B1 elements (see Fig. 4 A for repetitive elements in the mouse gene) there is no significant similarity upstream of position -335 in the mouse gene. Recombinants between the -1241/+93 segment of the human IL-2R and a chloramphenicol acetyltransferase reporter gene do not respond to stimulation with IL-2 in PC60 cells.() Thus, the human IL-2 response elements do not seem to be present in the part of the gene for which sequence data have been published.

IL-2 has the characteristics of a progression factor, in that it stimulates the proliferation of ``competent'', IL-2-responsive lymphocytes (55, 56, 57) . In PC60 cells, the effect on IL-2R expression is uncoupled from the growth factor activity of IL-2. This suggests that the signal transduction pathway leading from the IL-2 receptor to the IL-2R gene is different from the one that drives cells into S-phase. It may overlap with the pathways that mediate IL-2 stimulation of granzyme and perforin expression (28, 58) in T cells. In B cells, IL-2 stimulates IgJ-chain expression (59) . IL-2 responsiveness of the J-chain gene depends on a 28-bp stretch between position -74 and -41 (60) . This sequence has no significant similarity with segment C, suggesting that IL-2 stimulates IgJ expression via different transcription factors.

A General Model for the Regulation of IL-2R Gene Transcription

The results presented in this paper are summarized in the model shown in Fig. 8. They show that the complex transcriptional response of the IL-2R gene in CD4CD8 thymocytes to IL-1 + IL-2 can be explained by the existence of two sets of cis-acting elements. Promoter proximal elements, among which a conserved NF-B site plays a dominant role, mediate an early response that is stimulated by IL-1 alone. IL-1 also increases IL-2 responsiveness of the cells by augmenting the number of functional IL-2R. IL-2 induces the second phase of the IL-2R gene, which is mediated by a complex enhancer 1.3 kb upstream of the transcription start site. Thus, the biphasic response correlates with a bipartite structure of 5`-FR.


Figure 8: Model for the transcriptional regulation of the mouse IL-2R gene by IL-1 (or antigen) and IL-2. The model postulates that the biphasic response of the IL-2R gene to stimulation by IL-1 and IL-2 in CD4CD8 thymocytes or to antigen and IL-2 in mature T cells is controlled by two independently functioning sets of regulatory elements. IL-1 or antigen activate the first phase of transcription via promoter proximal elements, among them a critical NF-B site. Simultaneously, these signals induce cells to become IL-2 responsive, and thus prime them for the second phase. This is mediated by IL-2-responsive elements 1.3 kb upstream of the promoter.



We believe that the model presented here also applies to the antigen-triggered induction of IL-2R expression in mature lymphocytes and that antigen plays a similar role to IL-1. Stimulation of T cells with antigen or anti-CD3 induces activation of NF-B, and we have found that the early phase of antigen-induced increase in IL-2R mRNA in a CD8 T cell clone shows the same sensitivity to inhibition by PDTC of NF-B activation.() In the same clone, IL-2R mRNA levels return to base line 24 h after exposure to antigen unless IL-2 is added, and we have shown that maximal and prolonged expression of both the normal IL-2R gene and a IL-2R 5`-FR/chloramphenicol acetyltransferase transgene in concanavalin A-stimulated peripheral T cells depends on IL-2 (65) .


FOOTNOTES

*
This work has been supported in part by grants from the Swiss National Science Foundation and the Swiss Cancer League (to M. N.) and by a fellowship from ICSC-World Laboratory (to S.-M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence reported in this paper has been entered into the GenBank/EMBL Data Bank under accession number M16398.

§
Contributed equally to the work described in this paper.

Present address: Div. d'Immunologie et d'Allergie, CHUV, CH-1011 Lausanne, Switzerland.

**
Present address: Dept. of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2135 N. Campus Dr., Evanston, IL 60208.

§§
Present address: Dept. Biologia Cellular, Universitat de Girona, E-17071 Girona, Spain.

¶¶
To whom correspondence should be addressed: ISREC, CH-1066 Epalinges, Switzerland. Tel.: 21 692 58 58; Fax: 21 652 69 33; E-mail: Markus.Nabholz@isrec.unil.ch.

The abbreviations used are: IL, interleukin; IL-2R, IL-2 receptor; 5`-FR, 5`-flanking region; bp, base pair(s); kb, kilobase; PCR, polymerase chain reaction; STAT, signal transducer and activator of transcription; PDTC, pyrrolidinedithiocarbamate.

S. Barangé, P. Sperisen, and G. Plaetinck, unpublished results.

M. Pla, P. Sperisen, and M. Nabholz, unpublished results.

P. Reichenbach and M. Nabholz, unpublished results.

S. Barangé and M. Nabholz, unpublished results.


ACKNOWLEDGEMENTS

We thank GLAXO for the generous gifts of interleukin 1 and 2. We thank Tasuku Honjo for the IL-2R genomic and cDNA clones and Guido Miescher for the mouse -actin construct. We also thank Charles Weissman and Pierre Chambon for plasmids Z-pCRI/pRChrG-1 and pG1; Claudine Ravussin, Pierre Dubied, and Marcel Allegrini for preparation of the manuscript and figures; and Bernhard Hirt for useful suggestions and criticisms concerning the manuscript.

Note Added in Proof Recently it has been shown (66) that an Elf-1 binding site around bp 92 in the human IL-2R gene is important for its response to phorbol 12-myristate 13-acetate. This site is conserved in the mouse gene (see Fig. 6 A).


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