©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Definition of cis-Regulatory Elements of the Mouse Interleukin-5 Gene Promoter
INVOLVEMENT OF NUCLEAR FACTOR OF ACTIVATED T CELL-RELATED FACTORS IN INTERLEUKIN-5 EXPRESSION (*)

(Received for publication, December 22, 1994; and in revised form, May 2, 1995)

Hyun Jun Lee , Esteban S. Masuda (1), Naoko Arai (1), Ken-ichi Arai , Takashi Yokota (§)

From the Department of Molecular and Developmental Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan and the Department of Cell Signaling, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 94304

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have previously reported that the promoter region of the mouse interleukin-5 (IL-5) gene, extending from a nucleotide position about -1,200 to +33 relative to the transcription initiation site, can mediate transcriptional stimulation by phorbol 12-myristate 13-acetate and dibutyryl cAMP (BtcAMP) in mouse thymoma EL-4 cells. Here, we describe identification of four cis-regulatory elements necessary for full activity of the IL-5 promoter, using deletion and mutation analyses. We designated these elements as IL-5A (-948 -933), IL-5P (-117 -92), IL-5C (-74 -56), and IL-5CLE0 (-55 -38). We found that IL-5P bears homology to the binding site for the nuclear factor of activated T cells (NF-AT) and interacted with protein factors in nuclear extracts prepared from EL-4 cells stimulated with phorbol 12-myristate 13-acetate and BtcAMP (designated NFIL-5P). NFIL-5P complex was inhibited in the presence of an excess NF-AT and AP1 oligonucleotides and supershifted by antisera raised against NF-ATp, c-Fos, and c-Jun. It thus seems likely that an NF-AT-related factor is involved in the regulation of IL-5 gene transcription.


INTRODUCTION

Interleukin-5 (IL()-5) is primarily a T cell-derived lymphokine with multiple regulatory functions, including stimulation of growth and differentiation, on eosinophils (1, 2) and, in the mouse, on B cells(3) . The IL-5 gene is located on mouse chromosome 11 and human chromosome 5, within a cluster of the IL-3, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4, and IL-13 genes (4, 5, 6) which are commonly expressed in T helper 2 (Th2) cells.

Mouse Th cells have been classified into two subsets on the basis of their distinct lymphokine production patterns(7) . Th1 cells produce IL-2, interferon , and lymphotoxin and promote cell-mediated immunity, whereas Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and promote humoral immunity(8) . Polarized Th1 and Th2 responses have been shown to be a feature of a number of disease states in both mice and humans, and a proper balance between the two subsets is crucial for the effective responses(9, 10, 11) . Despite their physiological significance, molecular mechanisms which account for the difference in lymphokine gene expression in Th1 and Th2 cells are not well understood. One mechanism may entail differences in transcription factors interacting with cis-acting elements in lymphokine genes. Another mechanism may involve differences in signal transduction pathways between Th1 and Th2 cells(12) . There are reports suggesting that Th1 and Th2 cells use different signal transduction pathways; several groups have demonstrated that compounds which elevate intracellular cAMP levels have differential effects on lymphokine production in Th1 and Th2 cells(13, 14, 15) .

We have previously reported that cAMP has differential effects on the production of Th1- and Th2-type lymphokines in EL-4 cells, a cell line which produces both types of lymphokines when stimulated with PMA(16) . Effects of cAMP on PMA-induced expression of IL-2 and IL-5 genes were opposite; i.e. cAMP activated the IL-5 gene synergistically with PMA, while it suppressed PMA-induced expression of the IL-2 gene, by modulating respective promoter activities. Mechanisms for differential regulation of the IL-2 and IL-5 genes by cAMP in EL-4 cells may be related to those in Th1/Th2 cells and should provide some insight at the molecular level.

Transcriptional control of the IL-2 gene has been extensively studied, and the major regulatory elements have been shown to reside within a region of approximately 300 bp upstream of the transcription initiation site(17, 18) . Of several transcription factors involved in regulation of the IL-2 gene, NF-AT is essential for transcription of the IL-2 gene upon T cell activation and plays a major role in various aspects of regulation of the IL-2 gene, including T cell-specific expression(19) . NF-AT has also been implicated in the transcriptional regulation of other lymphokine genes, such as GM-CSF, IL-3, IL-4, and tumor necrosis factor-, hence, a role in the coordinated expression of these genes was suggested(20, 21) . We also have data that NF-AT is a major target of the inhibitory action of cAMP on the IL-2 promoter(22) .

On the other hand, details on the transcriptional control of the IL-5 gene have not been explored at the molecular level. Within the 5`-flanking region, the IL-5 gene is associated with the conserved lymphokine elements (CLE)0, CLE1, and CLE2 which are also found in the promoters of IL-3, GM-CSF, and IL-4 genes, and may have a role in the regulation of coordinated lymphokine expression (23, 24) (Fig. 1).


Figure 1: Nucleotide sequence of the 5`-flanking region encompassing the SphI cleavage site and the translation initiation site of the IL-5 gene. Numbers are relative to the transcription initiation site. The nucleotide sequence from position -1174 to -545 was determined by sequencing both strands; the sequence further downstream was reported previously(58, 59) . Locations of sequences that correspond to CLE0, CLE1, CLE2, NF-1, and AP2 are indicated (bold character). The transcription initiation site (58) and TATA-like sequence are indicated. Restriction endonuclease cleavage sites for NsiI and BclI used for generating mutant constructs and base substitutions to generate a HindIII site are also indicated. The IL-5A, IL-5P, IL-5C, and IL-5CLE0 elements are boxed.



In this study, we identified four cis-regulatory elements which are necessary for full activity of the IL-5 promoter in response to PMA and BtcAMP stimulation in the mouse thymoma line EL-4. We showed that one element, the IL-5P element, shares homology with the binding site of NF-AT of the IL-2 gene and interacts with the inducible nuclear factor NFIL-5P which is closely related to the transcription factor NF-AT.


MATERIALS AND METHODS

Cells

The mouse thymoma cell line EL-4 TB6 was maintained in RPMI 1640 medium supplemented with 2 mM glutamine, 50 units/ml penicillin, 50 µg/ml streptomycin, 50 µM 2-mercaptoethanol, and 5% fetal bovine serum under 5% CO. Jurkat, Ba/F3, and COS-7 cells were cultured in the same medium containing 10% fetal bovine serum. Recombinant mIL-3 (1 ng/ml) was additionally supplemented for maintenance of Ba/F3 cells. NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and the antibiotics described above.

Antibodies

Anti-mouse NF-ATp (NF-ATp), a rabbit polyclonal antiserum raised against recombinant NF-ATp, was purchased from Upstate Biotechnology Incorporated. The Fos antibody (Fos), a rabbit polyclonal IgG reactive with c-Fos, Fos B, Fra-1, and Fra-2 and Jun antibody (Jun), a rabbit polyclonal IgG reactive with c-Jun, Jun B, and Jun D and their cognate peptides purchased from Santa Cruz Biotechnology.

DNA Sequencing

The DNA sequence of both strands of the region extending from position -1174 to -545 was determined using an Applied Biosystems 373A DNA sequencer.

Plasmid Constructs

Plasmids pUC00Luc and pmIL5Luc(1.2) were described previously(16) . pmIL5Luc(1.2) was linearized by SalI digestion and a series of nested 5` deletions was generated by BAL-31 exonuclease treatment. Then, 7-bp SalI linkers were attached and the SalI-HindIII fragments were inserted into pUC00Luc. The 5` border of each deletion mutant was determined by DNA sequencing and was named according to its 5`-end point. To generate pmIL5Luc(1.8), the 5`-EcoRI site of the pmIL5EHE (16) was replaced by the SalI site and the 1.8-kb SalI-HindIII fragment was inserted into pUC00Luc. The linker-scanning mutants were generated by replacing nucleotide sequences in various positions in the 1.2-kb IL-5 promoter with a 7-bp SalI or 6-bp XhoI linker sequences, using polymerase chain reaction (PCR). The resulting linker-scanning mutants contained 3-7-bp substitutions out of 6 or 7 bp at various positions in the context of the 1.2-kb IL-5 promoter (Fig. 3). These mutants were named according to the positions at which substitution linkers were introduced. To make the linker-scanning mutants LS971/965, PCR was performed with pmIL5Luc(1.2) as the template, M13 universal primer ((M13(-47)), and 1SA, an antisense strand oligonucleotide primer containing a SalI recognition site at nucleotide position -972 (Table 1). The resulting PCR product was digested with SacI (at the polylinker region) and SalI and was inserted into the SacI-SalI site of p964, a deletion mutant with a 5`-end point of -964 (Fig. 2). LS946/940, LS939/933, LS110/104, LS103/97, LS91/85, LS69/63, and LS57/51 were generated by essentially the same procedure. For the latter five mutants, p161, a deletion mutant whose 5`-end point is -161, was used as the template to minimize PCR-derived sequence and a sequence further upstream was attached using the SacI-NsiI (at nucleotide position -141) fragment of pmIL5Luc(1.2). To obtain LS954/949 and LS932/927, a pair of PCR amplifications was performed for each construct, as described(25) . Fragments upstream of the introduced mutations were generated using pmIL5Luc(1.2) as the template and M13(-47), and primers containing the XhoI recognition sites at the 5`-ends of the antisense strand oligonucleotides (Table 1). To generate downstream fragments, we used sense-strand oligonucleotide primers containing the XhoI linker sequence at the 5`-ends to overlap with the XhoI site of the upstream fragment, and antisense-strand oligonucleotide primer C1A (Table 1). The resulting upstream and downstream fragments were digested with SacI and XhoI, XhoI and BclI (at nucleotide position -800), respectively, and ligated between the SacI and BclI sites of pmIL5Luc(1.2). LS123/118, LS117/112, LS80/74, LS62/57, LS37/32 were generated essentially by the same procedure used for LS955/949 and LS933/927. For these constructions, we used p161 as the template and GLprimer2, a primer corresponding to the antisense strand of the luciferase structural gene (Promega Corp., Madison, WI) instead of C1A to generate downstream fragments. The resulting PCR fragments were cloned into the NsiI and HindIII (at nucleotide position +33) sites of pmIL5Luc(1.2). To generate pM1 to pM4 containing mutations in the IL-5P site, two steps of PCR were performed as described(25) . First, fragments upstream and downstream of point mutations were generated, using the same procedure as for LS124/118, except for use of sense and antisense oligonucleotides (Table 2, PM1 to PM4) used in the electrophoretic mobility shift assays (EMSAs) instead of primers containing the XhoI linker sequence. Second step PCR amplifications were done using one-tenth of the fragments generated by first step PCR and M13(-47) and GLprimer2. The resulting fragments were inserted into the NsiI and HindIII sites of pmIL5Luc(1.2). To increase fidelity, PCR amplifications were performed for 15 cycles (four cycles with annealing at 45 °C and 11 cycles at 65 °C) with a large amount (100 ng) of template. The PCR-derived sequences of each construct were confirmed by DNA sequencing. The expression plasmids of NF-ATc and NF-ATx were constructed by inserting corresponding cDNAs downstream of the SR promoter in pME18S(26) .


Figure 3: Linker-scanning analysis of the 5`-upstream region of the IL-5 promoter. Serial linker-scanning mutations were introduced into the 1.2-kb IL-5 promoter, and constructs obtained were transfected into EL-4 cells and analyzed, as described in Fig. 2. The positions in which substitutions were introduced are shown schematically on the left panel (SalI and XhoI linkers are indicated by solid boxes). The 1.2-kb promoter region and the positions of the IL-5A, IL-5P, IL-5C, and IL-5CLE0 elements are indicated. The promoter activity of each construct in response to PMA (solid bars) or PMA-BtcAMP stimulation (cross-hatched bars) was measured. Results are expressed as the percentage of activity of the wild type promoter in response to PMA-BtcAMP stimulation and represent the means ± S.D. of at least three independent experiments with at least two different DNA preparations.






Figure 2: Deletion analysis of the 5`-upstream region of the mouse IL-5 promoter. EL-4 cells were transfected with the serial deletion mutants shown schematically in the left panel. The cells were stimulated 24 h after transfection with either PMA (solid bars) or PMA and BtcAMP (cross-hatched bars), cell lysates were prepared 16 h later, and tested for luciferase activity, as described under ``Materials and Methods.'' Results represent the mean value of duplicate transfections. Two other independent experiments gave similar results. The regions responding to PMA and BtcAMP are indicated by shadowed boxes.





Transfections

Transient transfections were done using a DEAE-dextran procedure, as described previously(16) . At 24 h after transfection, cells were stimulated with 1 mM BtcAMP (Sigma) and 10 ng/ml PMA (Calbiochem). After another 16 h, the cells were harvested for luciferase assays. Luciferase activity was determined using the luciferase assay substrate (Promega) with a luminometer (Berthold, Postfach, Germany). Protein concentration in an aliquot of each sample was measured using BCA reagents (Pierce), and the results were calculated as (relative light unit-background)/µg protein.

Preparation of Extracts and EMSAs

Cells were stimulated for 3 h with various drugs, as described in figure legends, and nuclear extracts were prepared either by the method of Dignam et al.(27) or as described(28) . EMSAs were done using the double-stranded oligonucleotides given in Table 2. Each single-stranded oligonucleotide was purified on denaturing polyacrylamide gels before annealing. The annealed oligonucleotides were P-labeled with Klenow fragment and purified on a 12% polyacrylamide gels. The DNA-binding reactions were performed at room temperature for 30 min with 3-5 µg of nuclear extracts, 0.5 µg of poly(dI-dC), 10 mM HEPES, pH 7.9, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 100 mM KCl, and 50,000 counts/min of probe (0.5 ng) in a total volume of 10 µl. The samples were resolved on a 4% nondenaturing polyacrylamide gel at 120 V in 1 Tris-glycine-EDTA buffer, and the results were visualized by autoradiography. Extracts from transfected COS-7 cells were prepared as described(26) .


RESULTS

Functional Analysis of the 5`-Flanking Region of the IL-5 Gene

We have previously reported that the 5`-flanking region of the IL-5 promoter extending from the SphI site at a nucleotide position about -1,200 to +33 relative to the transcription initiation site mediated induction by PMA and BtcAMP in EL-4 cells(16) . Since only a partial nucleotide sequence has been reported, we first determined the complete nucleotide sequence of this region and found a number of potential regulatory elements (Fig. 1).

Next, to identify the region responsible for inducible expression of the IL-5 promoter in response to PMA and BtcAMP, the luciferase reporter plasmids carrying sequential 5`-deletions of the mouse IL-5 promoter were transiently transfected into EL-4 cells. The plasmid pUC00Luc, which has no upstream sequence derived from the IL-5 gene(16) , showed an almost negligible response to PMA or a combination of PMA and BtcAMP (Fig. 2). Removal of the 5` sequence up to position 964 did not affect the promoter activity responding to PMA and BtcAMP, but deletions beyond this position through -932 did reduce the response by 50%. Further deletions up to position -140 led to no further reduction. Remarkably, the subsequent removal of nucleotides -140 through -80 diminished the promoter activity by 90%. Thus, the response to a combination of PMA and BtcAMP appears to decline in two stages, at the -964 to -933 (region I) and at the -140 to -80 (region II) (Fig. 2). On the other hand, the IL-5 promoter responded weakly to PMA alone, and removal of the 5` sequences up to position -140 did not affect promoter activity responding to PMA, while deletions beyond this position almost completely abolished the response. Thus, the sequences between -140 to -80 (region II) of the IL-5 promoter are required for a response, albeit a weak one, to the PMA signal.

Four Cis-regulatory Elements Are Required for Full Activity of the IL-5 Promoter

To further delineate regulatory elements in the IL-5 promoter, we dissected regions shown to be important for promoter activity. Using the linker-scanning method, we replaced the sequence in various positions between -972 and -932, and between -124 and -31 in the 1.2-kb IL-5 promoter with a 7-bp SalI or 6-bp XhoI linker sequence (see ``Materials and Methods''). We did not introduce mutations around -140, because we found that in further deletion analysis, the proximal regulatory region is located between -127 and -103 (data not shown). Consistent with results of the 5`-deletion analysis, mutations introduced between -948 and -933 (designated IL-5A) reduced promoter activity by 60% in response to PMA and BtcAMP but not to PMA alone (Fig. 3). Mutations introduced in the region extending from -117 to -92 (designated as IL-5P) reduced promoter activity by about 80%, in response to a combination of PMA and BtcAMP as well as to PMA alone. Mutation analyses allowed for definition of third and fourth elements, extending from -74 to -56 (designated IL-5C) and from -55 to -38 (designated IL-5CLE0), respectively. The IL-5C and IL-5CLE0 elements are located in close proximity, and mutation analysis has heretofore shown no clear boundary between them. However, we defined IL-5CLE0 in analogy with CLE0 of the GM-CSF gene, and the remaining sequence as IL-5C. These two elements are the most crucial for promoter activity, because mutations in these regions almost completely abolished promoter activity in response to PMA as well as to PMA and BtcAMP.

Characterization of IL-5P

Deletion and mutation analyses revealed that downstream sequences of -124 are critical for IL-5 promoter activity. Comparison of the mouse and human IL-5 promoters showed a high degree of homology within this region (Fig. 4A). Interestingly, closer inspection revealed that IL-5P is homologous to the binding site for NF-AT (Fig. 4B). NF-AT has also been implicated in transcriptional regulation of other lymphokine genes(20, 21) . We recently noted that the NF-AT site is a major target of the inhibitory action of cAMP on the IL-2 promoter(22) . Thus, we speculate that IL-5P may be involved in coordinated and differential regulation of the IL-5 gene and focused on further characterization of IL-5P.


Figure 4: The common sequence between the IL-5P and NF-AT sites. A, comparison of the mouse and human IL-5 promoter regions. Nucleotide sequence extending from -127 in the mouse promoter through the ATG is compared with human IL-5 genomic sequence (60) . The IL-5P, IL-5C, and IL-5CLE0 elements are boxed. Identical nucleotides are indicated by vertical bars. B, comparison of the nucleotide sequence of IL-5P with that of the distal NF-AT site of the IL-2 promoter. Gaps are introduced into the NF-AT site to maximize matches. Differences in the human sequence are shown below the murine sequence. The purine-rich sequence and an AP1-like site are indicated by closed circles and shadowed bars, respectively.



To identify nuclear factors that can mediate inducibility through IL-5P, EMSAs were done using a probe corresponding to the region extending from -119 to ;89 of the IL-5 promoter (Table 2). Nuclear extracts prepared from EL-4 cells stimulated with a combination of PMA and BtcAMP formed several complexes with the IL-5P probe (Fig. 5A). The major complex (designated NFIL-5P) appeared only when we used extracts prepared from stimulated cells (compare lanes 1 and 2), and this complex formation was specifically inhibited in a dose-dependent manner by excess unlabeled IL-5P oligonucleotide (lanes 2-5) but not by the Sp1 oligonucleotide (lanes 6-8). Other minor complexes with different mobilities could be detected, but appearance was variable, depending on different nuclear extract preparations, and they did not seem to be specific for IL-5P because binding was also inhibited by the Sp1 oligonucleotide (lanes 6-8).


Figure 5: Binding of nuclear factors to the IL-5P sequence. A, NFIL-5P is the inducible, sequence-specific factor which binds to the IL-5P sequence. EMSAs were done with unstimulated (lane 1) or PMA-BtcAMP stimulated (lanes 2-8) EL-4 nuclear extracts in the presence of increasing amounts of unlabeled IL-5P (lanes 3-5) or Sp1 (lanes 6-8) oligonucleotides, as indicated. B, regulation of NFIL-5P binding activity. Nuclear extracts from EL-4 cells untreated (lanes 1 and 5) or stimulated with either PMA (lanes 2 and 6), BtcAMP (lanes 3 and 7), or the combination of both (lanes 4 and 8) were incubated with the IL-5P (lanes 1-4) or Sp1 (lanes 5-8) probes. The positions of the NFIL-5P complexes induced by PMA (II), BtcAMP (I), and combination of both (III) and the Sp1 complex (arrowhead) are indicated.



When EMSAs were done with nuclear extracts from EL-4 cells stimulated with different combinations of PMA and BtcAMP, the NFIL-5P complex was induced by either PMA or BtcAMP. The combination of both agents intensified the band (Fig. 5B, lanes 2-4). Interestingly, the NFIL-5P complex migrated with a different mobility, depending on the stimulation given. The control Sp1 binding was not altered by any of these stimuli (lanes 5-8).

To identify specific bases critical for NFIL-5P complex formation, segments of IL-5P sequence were mutated and tested for effects on complex formation (Fig. 6A). The PM1 mutant oligonucleotide with a mutation in the GGA sequence did not form the NFIL-5P complex, but did retain the ability to form other minor complexes (Fig. 6B, lane 4). NFIL-5P binding was gradually restored as mutations were introduced into the segments closer to the 3` portion of the IL-5P sequence (compare lanes 6, 9, 12, and 15). Competition analyses gave consistent results (data not shown).


Figure 6: Effects of mutations in the IL-5P sequence on the NFIL-5P binding and IL-5 promoter activity. A, location of the nucleotides substituted in PM1 to PM5. The sequences are aligned with the wild type IL-5P sequence, and identical bases are denoted by dashes. B, EMSAs using a panel of mutant IL-5P probes. Identical amounts of each probe depicted in panel A were incubated with nuclear extracts from EL-4 cells stimulated with PMA and BtcAMP. For each binding reaction, the competition was performed with 20 ng of unlabeled self or wild type oligonucleotides. The NFIL-5P complex is indicated by the arrow. C, functional analysis of site-specific mutations in the IL-5P site. Mutations corresponding to those analyzed by EMSAs in panel B were introduced into the 1.2-kb IL-5 promoter. The wild type pmIL5Luc(1.2) and mutant constructs (pM1 through pM4 and LS103/97) were transiently transfected into EL-4 cells, and luciferase activity was assayed at 16 h after stimulation with PMA and BtcAMP, as described under ``Materials and Methods.'' Results are expressed as a percentage of the activity of the wild type (shadowed bars), and represent the means ± S.D. of at least three independent experiments with at least two different DNA preparations. Relative intensity of NFIL-5P band elicited by each probe (B) was quantitated by PhosphorImage analyzer and plotted on the same graph (cross-hatched bars).



To search for a possible correlation between the in vitro formation of the NFIL-5P complex and in vivo activity of the IL-5 promoter, we introduced site-specific mutations corresponding to mutations introduced into the IL-5P probe for the EMSAs described above, in the context of the 1.2-kb IL-5 promoter. Constructs were transiently transfected into EL-4 cells and analyzed for luciferase activity (Fig. 6C). The results are shown along with the intensity of the NFIL-5P band elicited by each probe (Fig. 6B), as determined by PhosphorImage analyzer (Molecular Dynamics). Introduction of PM1, PM2, PM3, and PM5 mutations decreased IL-5 promoter activity by about 80%, in response to PMA and BtcAMP, whereas the PM4 mutation caused only a 50% decrease in promoter activity. Therefore, the in vivo activity of the IL-5 promoter correlated with the binding activity of NFIL-5P to the IL-5P site, i.e. the bases which were required for formation of the NFIL-5P complex in vitro were the same as those required for promoter activity. These results strongly suggest that NFIL-5P is the factor which regulates the IL-5 promoter through the IL-5P sequence.

The IL-5 gene is expressed mainly in activated T cells. To determine the cell-type specificity of the NFIL-5P complex, EMSAs were done using nuclear extracts from a variety of cell types either unstimulated or stimulated with PMA, A23187, and BtcAMP (Fig. 7). The NFIL-5P complex appeared only in the stimulated EL-4, Jurkat T cell line, and pro-B cell line Ba/F3 cells (left panel, lanes 2, 4, and 6, respectively), while the AP1 complex was detected in all cell types tested (right panel).


Figure 7: Cell type distribution of the NFIL-5P complex. Nuclear extracts were prepared from EL-4 (lanes 1 and 2), Jurkat (lanes 3 and 4), Ba/F3 (lanes 5 and 6), NIH3T3 (lanes 7 and 8), and COS-7 cells (lanes 9 and 10) untreated (lanes 1, 3, 5, 7, and 9) or treated for 3 h either with PMA and BtcAMP (lanes 2, 8, and 10) or with PMA, A23187, and BtcAMP (lanes 4 and 6). EMSAs were done using IL-5P probe, as described under ``Materials and Methods.''



NFIL-5P Is Related to NF-AT

We next asked whether NFIL-5P and NF-AT were related. NF-AT is composed of a pre-existing cytoplasmic component, referred to as NF-ATp (29) or NF-ATc(30) , which is translocated into the nucleus in response to a calcium signal, and a newly synthesized nuclear component, which belongs to the AP1 family (30, 31) .

In EMSAs using nuclear extracts from EL-4 cells stimulated with PMA and BtcAMP, NFIL-5P and NF-AT complexes migrated with a similar mobility (Fig. 8A, lanes 1 and 7). NFIL-5P complex formation was completely inhibited by excess unlabeled NF-AT as well as by IL-5P oligonucleotides (lanes 2 and 5) but not by those mutated in GGA sequences (lanes 3 and 6). As expected, AP1 oligonucleotide also inhibited NFIL-5P complex formation (lane 4). Conversely, NF-AT binding was inhibited specifically by both excess unlabeled NF-AT and IL-5P oligonucleotides, indicating that NFIL-5P is an NF-AT-related complex (lanes 7-11).


Figure 8: Relationship between NFIL-5P and NF-AT. A, nuclear extracts from EL-4 cells stimulated with PMA and BtcAMP were incubated with the IL-5P (lanes 1-6) or the NF-AT (lanes 7-11) probes in the presence of 20 ng of the unlabeled competitor oligonucleotides. The left and right arrowheads indicate positions of NFIL-5P and NF-AT complexes, respectively. B, nuclear extracts were prepared from EL-4 cells stimulated with PMA and BtcAMP in the presence of different concentrations of CsA or CHX, as indicated. EMSAs were performed with IL-5P, NF-AT, and Sp1 probes. The positions of NFIL-5P, NF-AT, and Sp1 complexes are indicated by arrowheads. C, the NFIL-5P complex contains NF-ATp and AP1. Nuclear extracts from EL-4 cells stimulated with PMA and BtcAMP were incubated without antiserum (lanes 1, 5, and 9), with 0.2 µl of an antiserum to the NF-ATp (lanes 3, 7, and 11), or with non-immune serum (lanes 2, 6, and 10), followed by binding assays with IL-5P, NF-AT, and Sp1 probes. Nuclear extracts were also incubated without (lanes 13 and 18) or with 1 µl of antibodies raised against c-Fos (lanes 14 and 15) and c-Jun (lanes 16 and 17) in the presence (lanes 15 and 17) or absence (lanes 14 and 16) of their cognate peptides, followed by binding assays with IL-5P probe. D, nuclear extracts were prepared from Jurkat cells stimulated with PMA and A23187 (lane 7) and EL-4 cells stimulated with PMA and BtcAMP (lane 8). Cytosolic extracts were prepared from COS-7 cells transfected with vector alone (lanes 1 and 2) or expression vector containing cDNA encoding NF-ATx (lanes 3 and 4) or NF-ATc (lanes 5 and 6). EMSAs were done with IL-5P and NF-AT probes (left and right panels, respectively) using above extracts in the absence(-) or presence (+) of purified AP1.



NF-AT binding is known to be inhibited by either cyclosporin A (CsA) (30) or the protein synthesis inhibitor cycloheximide (CHX)(19, 32) . We then tested sensitivity of the NFIL-5P complex to CsA and CHX; treatment of CsA inhibited the induction of NFIL-5P complex in cells stimulated with PMA and BtcAMP, in a dose-dependent manner similar to that seen for NF-AT binding (Fig. 8B, top and central panels, lanes 2 and 3). Induction of both the NFIL-5P and NF-AT complexes was almost completely inhibited by treatment with 10 µg of CHX (lanes 4 and 5). This inhibition was not caused by nonspecific effects of these reagents on nuclear proteins, since there was no change in Sp1 binding, under the same conditions (bottom panel).

To determine whether an NF-AT component is involved in the NFIL-5P complex, supershift EMSAs were done (Fig. 8C). Both NFIL-5P and NF-AT complexes (lanes 1-3 and 5-7, respectively, arrow) from nuclear extracts stimulated with PMA and BtcAMP were supershifted by an antiserum raised against NF-ATp (lanes 3 and 7, respectively, arrowhead). This antiserum did not supershift the Sp1 complex (lanes 9-11), indicating these supershifts are specific. Antibodies raised against c-Fos and c-Jun which can recognize several members of the AP1 family also supershifted the NFIL-5P complex (lanes 14 and 16, respectively).

We then examined whether recombinant NF-AT would bind to the IL-5P site. It has been shown that the preexisting cytoplasmic component of NF-AT is heterogenous (33) and in fact is composed of a family of homologous proteins(34, 35) . We recently isolated cDNA clones encoding two of these, namely NF-ATc and NF-ATx(26) . EMSAs were done using extracts from recombinant NF-ATx- or NF-ATc-transfected COS-7 cells. As shown in Fig. 8C, both NF-ATx and NF-ATc bound to the IL-5P, in combination with AP1 purified from Jurkat cells (left panel, lanes 4 and 6, upper and lower bands, respectively). The mock transfected COS-7 cells showed no specific band (left panel, lanes 1 and 2). These results indicate that IL-5P can be recognized by different forms of the cytoplasmic component of NF-AT. The mobility of the complex formed with NF-ATx was slightly slower than that formed with NF-ATc (lanes 7 and 8), and the binding specificity of NF-ATx and NF-ATc to the IL-5P and NF-AT sites was somewhat different, i.e. NF-ATc but not NF-ATx can bind to the NF-AT site without AP1, while neither bound to the IL-5P without AP1 (compare left and right panels, lane 5). Taken together, these results strongly suggest that NFIL-5P is related to, if not identical to, NF-AT.


DISCUSSION

We have previously reported that both PMA and cAMP are required for the activation of the IL-5 promoter(16) . In the present work, we analyzed regulatory elements in the promoter which were necessary to respond to these stimuli. By deletion and mutation analyses, we identified four cis-regulatory elements, designated IL-5A, IL-5P, IL-5C, and IL-5CLE0, which are important for the full activity of the IL-5 promoter in response to PMA and cAMP. We showed that the IL-5P, IL-5C, and IL-5CLE0 elements but not the IL-5A element were necessary to respond weakly to PMA alone (Fig. 3). The sequences within the former three elements are highly conserved between human and mouse IL-5 genes (Fig. 4A), and these elements are also crucial for the function of the human IL-5 promoter (data not shown). Therefore, the regulatory mechanism of the IL-5 gene is conserved between the two species.

Although IL-5A elicits activation of the IL-5 promoter in response to PMA and cAMP, it shares no homology with the cAMP response element (CRE). EMSA with an oligonucleotide probe containing the IL-5A element showed several sequence-specific complexes distinct from cAMP response element-binding protein (CREB).()The 3` portion of the IL-5A element, 5`-TGGGTCAAGGCCA-3`(-940 -928), has a close sequence similarity to the NF-1 consensus binding sequence, 5`-TGGNNNNNNGCCA-3` (36, 37) (Fig. 1). However, it is unlikely that NF-1 is involved in regulation through the IL-5A element, since LS932/927, in which the NF-1 site is mutated, showed activity similar to that of the wild type promoter.

The antisense strand sequence of the center of the IL-5C element, 5`-AGATAG-3` (-71 -66), matches the GATA consensus sequence, 5`-WGATAR-3`. Indeed, the mutant LS69/63 in which the GATA-binding site was destroyed lost promoter activity almost completely (Fig. 3). So far, four members of GATA family transcription factors, GATA-1 through GATA-4, have been reported, each GATA factor has a somewhat distinctive pattern of expression and is involved in cell-specific expression of certain genes(38, 39) . Among them, GATA-3 is the predominant GATA protein present in T cells (40, 41) and is involved in the regulation of a number of T cell-specific genes, including T cell receptor genes (42) . We are currently testing the role of the GATA family transcription factors in regulation of the IL-5 gene through this element.

The IL-5CLE0 element includes the CLE0 motif, which also occurs in the promoter proximal regions of the GM-CSF and IL-4 genes. The CLE0 motifs of these genes are also crucial for their promoter activities(24, 43, 44) . The sequence of these motifs is identical between humans and mice, although it is somewhat different among lymphokines(24) . Thus, the CLE0 motif may be involved in the coordinated and/or specific expression of these genes and related studies are ongoing. Interestingly, a recent report indicated that a similar region of the mouse IL-5 promoter is functionally important in D10.G4.1 (Th2 cell clone) and an inducible nuclear factor bound to this region is detected only in D10.G4.1 but not in HDK1 (Th1 cell clone)(45) .

The fourth cis-regulatory element, IL-5P, was investigated in detail because of its similarity to the NF-AT-binding site. We first identified an inducible nuclear factor (NFIL-5P) bound to the IL-5P element. Next, we demonstrated that NFIL-5P binding is required for in vivo functioning of this element by showing that mutations in IL-5P which prevent this interaction also inhibit promoter activity (Fig. 6). Finally, we showed that NFIL-5P is similar to the transcription factor NF-AT in several respects: the sequence specificity of NFIL-5P binding is closely correlated with that of the NF-AT (Fig. 6B),(46, 47) ; an excess NF-AT oligonucleotide inhibits NFIL-5P binding to IL-5P (Fig. 8A); the formation of both complexes is inhibited by CsA and CHX (Fig. 8B); the presence or absence of NFIL-5P in the several cell lines tested is correlated with that of NF-AT (Fig. 7); an antiserum raised against NF-ATp supershifts both complexes (Fig. 8C); and finally, recombinant NF-ATx and NF-ATc expressed in COS-7 cells in combination with AP1 can bind to the IL-5P site, as well as to the NF-AT site, with mobilities similar to those seen with stimulated EL-4 and Jurkat cell nuclear extracts (Fig. 8D).

Though NF-AT binding is induced by PMA alone in EL-4 cells, it is inhibited by CsA which is known to block Ca-dependent pathway. Two possible explanations for this discrepancy were raised and but are yet to be proven: either calcineurin is activated by other mechanisms in EL-4 cells or the resting Ca concentration is sufficient to activate calcineurin in EL-4 cells(48) . Our in vitro result indicating that NFIL-5P is inhibited by CsA appears to disagree with a report showing that the level of IL-5 mRNA induced by PMA was not inhibited CsA(49) . However, in the subline of EL-4 cells of our use only low level of IL-5 mRNA is induced with PMA alone, and the combination of PMA and cAMP synergistically augments the level of expression(16) . This maximal level expression of IL-5 mRNA was inhibited by addition of CsA. In addition, other reports (50, 51, 52) and our unpublished results()also indicate that CsA inhibits the production of IL-5 from several T cell lines both in human and mouse systems. Thus, the disagreement may be caused from the difference of cell line and stimuli adopted.

Recent cloning of the cytoplasmic components of NF-AT has revealed that they are encoded by at least three members of related clones containing the Rel-homology domain(26, 34, 35) . Moreover, mRNA of each NF-AT clone has distinct tissue distribution and induction characteristics. Considering the diversity of the AP1 family, it is tempting to speculate that distinct NF-AT complexes composed of different combinations of members of the cytoplasmic component of NF-AT and AP1 families are formed depending on signals and cell types. Indeed, the NFIL-5P complexes with different mobilities were induced depending on the stimulation given (Fig. 5B). We demonstrated that IL-5P can be bound by multiple NF-AT complexes and the mobilities of complexes containing NF-ATx and NF-ATc were slightly different (Fig. 8D). Whether NF-ATp, NF-ATx, NF-ATc, and other members of the NF-AT family are induced differently by PMA and cAMP remains to be clarified.

We found that the NF-AT site is the target of the inhibitory action of cAMP on the IL-2 promoter in EL-4 cells(22) . However, NF-AT binding to this site increased with the addition of cAMP. Thus, it appears that IL-5P and the IL-2 promoter NF-AT site, though they are closely related, are regulated oppositely by cAMP. PMA and cAMP may induce distinct NF-AT-related complexes which have different transcriptional activation functions on the IL-5P and NF-AT sites. Furthermore, PMA and cAMP may differentially affect the components of the NF-AT-related complexes by protein modifications to either promote or to inhibit their transcriptional activation functions. For instance, certain members of the AP1 family have been shown to be regulated not only at transcriptional but at post-translational levels in response to PMA and cAMP(53) . At present, we cannot distinguish between these possibilities. Nevertheless, the identification of IL-5P and NFIL-5P provides a system for exploring the mechanism for differential regulation through NF-AT-related sites.

It is important to note that the cAMP effect on the IL-5 promoter was not limited to the IL-5P site. The regulation of many genes by cAMP is mediated by either the AP2 or CREB/activating transcription factor family of nuclear proteins(54, 55, 56) . However, we did not find CRE or the AP2 binding sequence within the elements which we identified in this study. Since all four regulatory elements were required for maximal activity of the IL-5 promoter responding to PMA and cAMP, they may function cooperatively, as was suggested for another system(57) . Thus, the effect of cAMP may be exerted at the level of assembly of a functional transcription machinery including factors bound to these elements.

In summary, we have delineated the cis-regulatory elements essential for expression of the IL-5 gene and obtained the first evidence for an NF-AT related factor regulating expression of the gene. The results presented here narrow down the elements involved in the mechanisms underlying the differential effect by cAMP on expression of the IL-5 and IL-2 genes in EL-4 cells. It will be interesting to check whether the same elements are involved in the differential lymphokine expression by Th1 and Th2 cells.


FOOTNOTES

*
This work was supported in part by a grant from the Ministry of Education, Science and Culture of Japan. DNAX Research Institute of Molecular and Cellular Biology is supported by the Schering-Plough Corporation. 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(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) D14461[GenBank® Link].

§
To whom correspondence should be addressed: Dept. of Molecular and Developmental Biology, The Institute of Medical Science, The University of Tokyo. Tel.: 81-3-5449-5661; Fax: 81-3-5449-5424; tyokota{at}ims.u-tokyo.ac.jp

The abbreviations used are: IL, interleukin; GM-CSF, granulocyte-macrophage colony stimulating factor; PMA, phorbol 12-myristate 13-acetate; BtcAMP, dibutyryl cAMP; NF-AT, nuclear factor of activated T cells; EMSA, electrophoretic mobility shift assay; CsA, cyclosporin A; CHX, cycloheximide; Th, helper T; bp, base pair(s); kb, kilobase; CLE, conserved lymphokine element; PCR, polymerase chain reaction; CRE, cAMP response element.

H. J. Lee, unpublished observation.

Y. Naito, and N. Arai, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank N. Koyano-Nakagawa, H. Yssel, D. Wylie, and M. Ohara for critical reading of the manuscript and for helpful discussions, L. Tsuruta and J. Nishida for helpful discussions, and D. Ligget at DNAX Research Institute and the staff at the Laboratory of Molecular Genetics, The Institute of Medical Science, The University of Tokyo (IMSUT) for synthesizing oligonucleotides. Computational time was provided by the Human Genome Center, IMSUT.


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