Stimulating Factor (GM-CSF) Gene Promoter: Nuclear Factors That. Interact with an Element ... those of the granulocyte-macrophage colony-stimulating fac-.
Vol. 11, No. 12
MOLECULAR AND CELLULAR BIOLOGY, Dec. 1991, p. 5894-5901 0270-7306/91/125894-08$02.00/0
Copyright © 1991, American Society for Microbiology
Characterization of the Mouse Granulocyte-Macrophage ColonyStimulating Factor (GM-CSF) Gene Promoter: Nuclear Factors That Interact with an Element Shared by Three Lymphokine GenesThose for GM-CSF, Interleukin-4 (IL-4), and IL-5 SHOICHIRO MIYATAKE,' JOSEPH SHLOMAI,12 KEN-ICHI ARAI,"3 AND NAOKO ARAI'* Department of Molecular Biology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, California 94304-1104'; Department of Cellular Biochemistry, The Hebrew University of Jerusalem, Hadassah Medical School, Jerusalem 91010 Israel2; and Department of Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108, Japan3
The region extending from -40 to -54 of the 5'-flanking region of the mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) gene shows homology to sequences found in the 5'-flanking regions of other cytokine genes, those encoding interleukin-4 (IL-4), IL-5, and granulocyte colony-stimulating factor (G-CSF). This sequence element is referred to as conserved lymphokine element 0 (CLEO). Saturation mutagenesis of the CLEO element indicates that in addition to the previously mapped region between -73 and -91 (CLE2+GC box), the CLEO element is necessary for induction of the mouse GM-CSF gene by phorbol myristate acetate/Ca ionophore (A23187) stimulation in T cells. The presence of the CLEO element is necessary to observe stimulation of the transcription activity of the mouse GM-CSF promoter in vitro. Mobility shift assays revealed that this region forms an inducible DNA-protein complex, NF-CLEO, which consists of two complexes of similar mobility, NF-CLEOa and NF-CLEOb. NF-CLEOa and NF-CLEOb recognize the 3' half and 5' half of the CLEO element, respectively, with an overlapping region recognized by both proteins. The recognition sequence of NF-CLEOa corresponds to the region required for induction by phorbol myristate acetate/A23187, while the recognition sequence of NF-CLEOb contains bases that have inhibitory activity. The CLEO elements of the IL-4 and IL-5 genes but not the G-CSF gene are also recognized by NF-CLEOa and -b, suggesting that the NF-CLEOa and -b proteins play an important role in the coordinate induction of these genes in activated T cells. The nuclear factor that recognizes the G-CSF CLEO element seems to be different from NF-CLEOa and NF-CLEOb; however, it weakly recognizes the DNA sequence for the NF-CLEOa.
Activation of T cells by antigen triggers the sequential activation of transcription of various sets of genes (1, 2, 16, 30). These include several oncogenes, lymphokine genes, and genes for cell surface molecules such as the interleukin-2 (IL-2) receptor a chain that lead to the proliferation and activation of the various biological functions of T cells (7). This process requires the activation of phosphoinositide turnover that leads to the production of inositol trisphosphate and diacylglycerol (DG) (4, 23). The increase of inositol trisphosphate induces the release of Ca2+ from internal stores and the influx of Ca2+ from outside to elevate the Ca2+ concentration in the cytoplasm. This event is mimicked by a Ca2+ ionophore such as A23187. DG is an activator of protein kinase C (PKC), and phorbol esters such as phorbol myristate acetate (PMA) are able to substitute for the effect of DG on PKC. The optimum transcriptional activation of lymphokine genes in T cells requires both an increase in the cytoplasmic Ca2+ concentration and activation of PKC; however, the biochemical events that take place downstream are not known. One approach to study these processes is to identify transcription factors required for the activation of these genes induced in T cells and to study the regulatory mechanism of the activity of these transcription factors. We have been characterizing regulatory elements of several lymphokine promoters, including those of the granulocyte-macrophage colony-stimulating fac*
Corresponding author. 5894
tor (GM-CSF) gene. We have previously reported that the region of the mouse GM-CSF gene extending from -95 to
-73 (CLE2+GC box element) is required for activation by PMA and A23187 in T cells (11, 17, 18). This region contains two DNA-binding elements. One sequence (-91 to -81) is recognized by the inducible factor NF-GM2, which resembles NF-KB, and the other is a GC box element (-84 to -73) recognized by a set of constitutive factors, A1, A2, and B (28). Mutations in these regions completely abolish the inducibility of the GM-CSF promoter in vivo as well as the binding of these recognition factors. In this report, another cis-acting DNA element, conserved lymphokine element 0 (CLEO), is described. The requirement of this region for induction of the GM-CSF gene was previously reported by Heike et al. (11) and Nimer et al. (20, 21). This element extends from -40 to -54 of the mouse GM-CSF promoter located downstream of the CLE2+GC box element. Highly homologous sequences are found in the 5'-flanking regions of the IL-4, IL-5, and granulocyte colonystimulating (G-CSF) genes. The CLEO element is recognized by two factors, NF-CLEOa and NF-CLEOb, the recognition sequences of which are partially overlapping. These two factors can interact with the CLEO element of the IL-4 and IL-5 genes but not with that of the G-CSF gene, whose expression is not induced in T cells. The nuclear factor that recognizes the G-CSF CLEO element seems to be different from NF-CLEOa and -b, but it recognized the region for NF-CLEOa weakly.
VOL.
CLEO, CIS-ACTING ELEMENT OF THE CM-CSF GENE
11, 1991 poly A
1t1
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FIG. 1. Structures of plasmids used for transfection and in vitro 3-lactamase gene; E=I, CAT transcription assays. Notation: gene; poly A, simian virus 40 early mRNA polyadenylation site; SJ, small t antigen splice junction. Numbers show positions with respect to the transcription initiation site. Shown below are the substitution mutations introduced into the same plasmid. Lines represent unchanged bases. The CLEO element is indicated by the solid line below the sequence. -,
MATERIALS AND METHODS
Plasmids and oligonucleotides. Plasmid pmoGMCAT-96 was described previously (18). All of the plasmid constructs shown in Fig. 1 were synthesized by replacing the BstEIISacl fragment with synthetic fragments harboring various mutations. Oligonucleotide CLEO encompassing the region between positions -33 and -60 of the GM-CSF promoter was prepared by annealing the chemically synthesized oligonucleotides 5'-GATCGTCACCATTAATCATTTCCTCTAAC TGT-3' (upper strand) and 5'-GATCACAGTTAGAGGAAA TGATTAATGGTGAC-3' (lower strand), resulting in double-stranded DNA with Sau3A overhangs. All of the oligonucleotides harboring substitution mutations shown in Fig. 5A were similarly prepared. A nonspecific oligonucleotide, GM96-61, that encompasses the region between positions -61 and -% of the mouse GM-CSF promoter was prepared by annealing the chemically synthesized oligonucleotides 5'-ACTCAGGTAGTTCCCCCGCC CCCCTGGAGTTCTGTG-3' (upper strand) and 5'-CACA
5895
GAACTCCAGGGGGGCGGGGGAACTACCTGAGT-3' (lower strand). The other nonspecific oligonucleotide, CYC35, was prepared by annealing the chemically synthesized oligonucleotides 5'-CCAAACTTGCATGGTATCTT TGGCAGACACTC-3' (upper strand) and 5'-GAGTGTCT GCCAAAGATACCATGCAAGTTTGG-3' (lower strand). Oligonucleotide IL-4-CLEO, which encompasses the region between positions -40 and -69 of the 5'-flanking region of the mouse IL-4 gene (3), was prepared by annealing the chemically synthesized oligonucleotides 5'-GATCCAATG TAAACTCATITTTCCCTTGGlTTCAG-3' (upper strand) and 5'-GATCCTGAAACCAAGGGAAAATGAGlTTACA TTG-3' (lower strand), resulting in double-stranded DNA with Sau3A overhangs. Oligonucleotide IL-5-CLEO, which encompasses the region between positions -32 and -61 of the 5'-flanking region of the mouse IL-5 gene (6), was prepared by annealing the chemically synthesized oligonucleotides 5'-GATCTTAGCA ATFATTCATTT7GCTCAGAGAGAGA-3' (upper strand) and 5'-GATCTCTCTCTCTGAGGAAATGAATAATTGCT AA-3' (lower strand), resulting in double-stranded DNA with Sau3A overhungs. Oligonucleotide G-CSF-CLEO, which encompasses the region between positions -43 and -72 of the mouse G-CSF promoter (29), was prepared by annealing the chemically synthesized oligonucleotides 5'-GATCCAGCCCCAGGTA ATTTCCTCCCGGGGCCTT-3' (upper strand) and 5'-GAT CAAGGCCCCGGGAGGAAATTACCTGGGGCTG-3' (lower strand), resulting in double-stranded DNA with Sau3A overhangs. Preparation of Jurkat nuclear extract. Jurkat cells, a human T-cell leukemia line, were cultured in RPMI 1640 medium containing 10%o fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were harvested at a cell density of 3 x 106, to 5 x 106/ml. To obtain cell extracts from PMA (Calbiochemical) and A23187 (Calbiochemical), Jurkat cells were cultured in the presence of 2% heat-inactivated fetal calf serum and sodium selenite medium supplement (product 11184; Sigma Chemical Co.) and stimulated with PMA (50 ng/ml) and A23187 (0.5 F.M) for 2 to 3 h. Nuclear extract was prepared by the method of Dignam et al. (9). Preparation of partially purified fraction of NF-CLEO. The crude nuclear extract (fraction I) was loaded onto a 45-ml phosphocellulose column. The column was washed with 10 column volumes of buffer D (20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES]-NaOH [pH 7.9], 1 mM EDTA, 20%o glycerol, 1 mM dithiothreitol, 1.5 mM phenylmethylsulfonyl fluoride) containing 50 mM KCI. The binding proteins were eluted by 2 column volumes each of buffer D containing 0.3 and 1.0 M KCI, yielding fraction II. The 1.0 M fraction was dialyzed against buffer D containing 50 mM KCl and loaded onto a 45-ml hydroxylapatite column. The column was washed- with 10 column volumes of buffer H (20 mM imidazole-HCl [pH 6.8], 50 mM KCl) containing 10 mM potassium phosphate (pH 6.8). The binding protein was eluted by a linear (10 to 500 mM) gradient of potassium phosphate (pH 6.8). The peak of NF-CLEO activity was collected (fraction III) and dialyzed against buffer H until the potassium phosphate concentration was below 50 mM. To concentrate the fraction, this fraction was loaded onto a 2.5-ml hydroxylapatite column (1 ml of resin for 2 mg of protein) and eluted with 2 column volumes of buffer H containing 1 M potassium phosphate buffer (pH 6.8). The protein was precipitated by (NH4)2SO4 (67% saturation at 0°C) and resuspended in buffer D (fraction IV). This concen-
5896
MOL. CELL. BIOL.
MAYATAKE ET AL.
trated fraction (10 mg/ml) was gel filtered on a 50-ml Superose 6 column (Pharmacia). Fractions containing NF-CLEO activity were pooled (fraction V) and used as the partially purified fraction. Transfection. A DEAE-dextran method was used for transfection (10, 27). Cells (5 x 106) washed with Trisbuffered saline were resuspended in 1 ml of Tris-buffered saline containing 0.5 mg of DEAE-dextran per ml and 10 ,ug of DNA, incubated for 25 min at room temperature, resuspended in 10 ml of tissue culture medium containing 0.1 mM chloroquine diphosphate (Sigma), and incubated for 1 h at 37°C. Chloroquine-containing medium was then replaced with normal medium, and the culture was incubated for an additional 40 h. For PMA/A23187 stimulation, cells were incubated with 50 ng of PMA (Sigma) per ml and 0.5 ,uM A23187 (Calbiochem-Behringer) in medium containing 1% fetal bovine serum for 8 h and harvested for the chloramphenical acetyltransferase (CAT) assay. Each construct was transfected at least three times, and average values are presented. In vitro transcription. A 5- to 10-,ug amount of protein of the crude nuclear extract was incubated with 0.2 ,ug of various template DNAs, linearized by EcoRI, in a mixture of 25 mM HEPES-NaOH (pH 7.9), 0.5 mM EDTA, 0.5 mM dithiothreitol, 4 mM MgCl2, 50 mM KCI, 2% (wt/vol) polyvinyl alcohol, 10% glycerol, 0.25 mM ATP, 0.25 mM CTP, 0.25 mM UTP, and 25 ,uM [oc-32P]GTP (40 Ci/mmol) for 30 min at 30°C. The reaction mixture (20 RIl) was phenolchloroform extracted, ethanol precipitated, and resuspended in 10 ILI of formamide dye. The radioactive RNA was separated by electrophoresis on a denaturing polyacrylamide gel containing 8 M urea and detected by autoradiography. Mobility shift assay. A modified mobility shift assay was performed as described previously (28). The binding reaction between the nuclear extract or the partially purified fraction and the radiolabeled probe was performed in a 12.5-,ul reaction mixture containing 8 mM Tris-HCI (pH 7.9), 14.4 mM HEPES-NaOH (pH 7.9), 98 mM KCI, 14.4% glycerol, 1.0 mM EDTA, 1.6 mM dithiothreitol, 1.76% (wt/vol) polyvinyl alcohol, 0.4 mg bovine serum albumin per ml, 0.1 mg of poly(dI-dC) per ml, and 105 cpm (3 ng) of various probes. Reactions were carried out at 30°C for 30 min and then chilled on ice. DNA-protein complexes were separated from free DNA by electrophoresis on 4% polyacrylamide gels (29:1 acrylamide/bisacrylamide), and dried gels were exposed at -70°C to X-ray films. RESULTS Precise determination of the region required for PMA and A23187 induction in T cells. We identified the region extending from -73 to -91 (CLE2+GC box) of the mouse GMCSF promoter as the PMA/A23187-inducible element in T cells (11, 17, 18). PMA/A23187-inducible factor (NF-GM2) and constitutive factors (A1, A2, and B) bind to the CLE2+GC box element (28). One motif located between the CLE2+GC box element and the TATA sequence (-25 to -29) is conserved in the 5'-flanking regions of other cytokine genes such as the genes for IL-4, IL-5, and G-CSF (see Fig. 7A). This element is located within 40 bp upstream of the TATA sequence in all of these cytokine genes and is referred to as the (CLEO element. The requirement for this region for PMA/A23187 induction was reported previously by Heike et al. (11) and Nimer et al. (20, 21). To determine the region more precisely, the sequence extending from positions -31 to -54 was divided into four regions, and each region was
TABLE 1. Effects of substitution mutations on induction by PMA/A23187 Relative CAT activity Plasmid
pmoGMCAT-96 pmoGMCAT-60 p31-36 p37-42 p43-48 p49-54
Fold
Unstimulated
PMA/A23187
induction
1.90 1.00 0.79 0.64 0.93 1.92
4.84 0.91 3.22 1.33 0.67 2.50
2.5 0.9 4.1 2.1 0.7 1.3
substituted with a sequence of a restriction site (SpeI site for p31-36, p37-42, and p43-48; StuI site for p49-54) (Fig. 1). These mutations were introduced into plasmid pmoGMCAT96, which carries the 5'-flanking region of the mouse GMCSF gene extending from -96 to +27 in front of the reporter CAT gene. These plasmids were introduced into human T-cell leukemia line Jurkat by the DEAE-dextran method; 40 h after transfection, cells were either stimulated by PMA (50 ng/ml) and A23187 (0.5 puM) or left unstimulated for 8 h. Cell extracts prepared by freezing and thawing were assayed for CAT activity. The substitution mutations at -37 to -42, -43 to -48, and -49 to -54 completely abolished the induction by PMA and A23187 despite the presence of the CLE2+GC box element (Table 1). This finding suggests that the region including the CLE0 element is also necessary for the GM-CSF promoter to respond to PMA and A23187 in T cells. To identify the sequences required for induction, saturation mutagenesis of this region was carried out. As shown in Fig. 1, every base in the region extending from -37 to -54 was mutated in plasmid pmoGMCAT-96, and these plasmids were transfected into Jurkat cells. As a control, pmoGMCAT-60, which lacks the CLE2+GC box element, was also used for transfection. As reported previously, deletion of the CLE2+GC box element completely abolished the inducibility of the promoter (Fig. 2). Mutations at -39 and -40 showed a lower induced level following stimulation. The mutations introduced at the region extending from -41 to -49 completely abolished induction except for the mutation at -45. The mutation at -45 decreased both the basal and PMA/A23187-induced levels. Mutations at -50 and -52 upregulated both the basal and induced levels. Mutation at -51 upregulated the basal level but not the induced level, while mutation at -53 abolished induction. This experiment indicated that every base encompassing -39 and -49 and the base at -53 are required for induction by PMA/A23187. On the other hand, bases -50, -51, and -52 are involved in suppression of promoter activity. The CLEO element is required for transcription of the GM-CSF promoter in vitro. To study the role of this region for promoter activity, the same mutated templates were transcribed in vitro. Linearized DNA templates were incubated with crude nuclear extract prepared from PMA/ A23187-activated Jurkat cells in the presence of [a-32P]GTP. The synthesized RNA was separated by denaturing polyacrylamide gel electrophoresis. As controls, the wild-type template pmoGMCAT-96 was included, as well as pmoGM CAT-72, which lacks the CLE2+GC box element. As shown in Fig. 3, removal of the CLE2+GC box element severely (>80%) reduced transcription activity of the GM-CSF promoter in vitro. Every mutation introduced at positions -39 to -49 reduced transcription activity in vitro. The fact that
VOL . 1 l, 1991
CLEO, CIS-ACTING ELEMENT OF THE CM-CSF GENE
5897
B
A
Competitor Molar Excess of Competitor
Mof Excmetitr
CLE0 GM96-61 CYC35 e r-F l- [- -1 F 10° 1 10 50 1 10 50 1 10 50
PMAA23187 Stimulation Jurkat Cell Extract(ig)
0.7 1.4 0.7 1.4
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FIG. 2. Transfection experiment using plasmids with mutations in the CLEO motif and the flanking regions. CAT activity is normalized relative to the activity directed by plasmid pmoGM CAT-60 in unstimulated cells. Open bar, uninduced cells; hatched bar, induced cells. Each construct was transfected at least three times, and average values are shown.
this region almost completely overlaps the region required for PMA/A23187 activation in the transfection experiment suggests that this region functions as a positive cis-acting element. However, derepression observed as a result of the introduction of the mutations at -50, -51, and -52 in the
1.2-
0.0
E E
FIG. 3. Relative transcription activity in vitro of the GM-CSF promoters harboring single and double mutations. The transcription assay was carried out with Jurkat cell nuclear extract prepared from PMA/A23187-activated Jurkat cells and 0.2 ,ug of the various plasmid DNAs linearized by EcoRl in the presence of [a-32P]GTP. Synthesized RNA was fractionated on a 6% polyacrylamide gel containing 8 M urea, and the amount of the synthesized RNA was measured by densitometric autoradiography. Transcription activity is normalized relative to the activity directed by the wild-type template pmoGMCAT-96. Each assay was repeated at least three times, and average values are shown.
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FIG. 4. Mobility shift assay with the Jurkat cell nuclear extract and oligonucleotide probe harboring the CLEO element. (A) Speecficity of the complex that interacts with the CLEO element. A 32P-labeled DNA fragment (10W cpm) containing the CLEO element was incubated with about 2 ,ug of Jurkat cell nuclear extract protein and the oligonucleotide competitors shown above the lanes in the indicated molar excess relative to the CLEO probe. The sequences of nonspecific competitors are given in Materials and Methods. (B) Induction of the NF-CLEO by PMA/A23187. 32P-labeled CLEO probe (105 cpm) was incubated with nuclear extracts prepared from stimulated (+) or nonstimulated (-) cells.
transfection experiment was not observed in transcription assays in vitro. In addition, the inhibitory effect of the mutation introduced at position -53 in the in vivo assay (Fig. 2) was not clear in vitro. Identification of the CLEO-binding proteins in the nuclear extract. Gel mobility shift assays using radiolabeled oligonucleotides extending from -33 to -60 were performed to detect proteins that specifically recognize this region. Three prominent DNA-protein complexes (I, II, and III) were detected (Fig. 4). Only complexes I and II were abolished by the addition of the same unlabeled oligonucleotides but not by a 50-fold excess of two different unrelated oligonucleotides of the same length (GM96-61 and CYC35). Complex III was abolished by both the same and unrelated oligon icleotides, suggesting that complexes I and II are formed by proteins with higher affinity to a specific sequence in this oligonucleotide. To determine whether this binding activity is inducible, mobility shift assays were performed with nuclear extracts prepared from both PMA/A23187-stimulated and nonstimulated Jurkat cells. As shown in Fig. 4B, complexes I and II were induced severalfold by PMA/ A23187 stimulation; however, the extract prepared from nonstimulated cells also contained a significant amount of binding activity to form complexes I and II. The amount of nonspecific complex III did not change upon stimulation. Further competition analysis showed that complex I is not detected by the probe that does not have Sau3A overhangs. Furthermore, after several fractionation steps, the proteins that form complex I were separated from the proteijus that form complex II; therefore, proteins that form complex II are referred to as NF-CLEO. Further analysis of NF-CLEO was performed with a partially purified fraction (fraction V) described in Materials and Methods. Sequence requirement of NF-CLEO. A series of oligonucleotides harboring double mutations covering the region extending from -40 to -55 were synthesized and used for competition assays with the partially purified fraction (frac-
MAYATAKE ET AL.
5898
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FIG. 5. Sequence requirement of the NF-CLEO protein. (A) Sequences of synthesized oligonucleotides of the wild type (CLEO) and of a series of double-base substitutions (GM40.41 to GM54.55). Dashes, unchanged bases; underline, CLEO element. (B) Competition assay with oligonucleotides harboring double-base substitutions. The oligonucleotides shown above the lanes were added to the reaction mixture as competitors. The amount of competitor DNA relative to that of CLEO probe is shown in fold molar excess above each lane.
tion V) (Fig. 5A). All of the mutations in the region extending from -42 to -53 reduced the affinity of the binding of NF-CLEO (Fig. SB). Analysis of the pattern of the competition assay revealed that the competitors harboring mutations extending from -42 to -45 reduced the upper part of the band representing the NF-CLEO complex, while the competitors harboring mutations extending from -50 to -53 reduced the lower part of this band, suggesting that NFCLEO consists of two complexes of different mobilities. The region extending from -46 to -49 seems to be required for the formation of both complexes, since competitors harboring mutations extending from -46 to -49 were not able to reduce either complex. The existence of two complexes became clearer when the mutated oligonucleotides shown in Fig. 5A were radiolabeled and gel mobility shift assays were performed (Fig. 6A). Oligonucleotides harboring mutations from -42 to -45 were able to form the low-mobility but not the high-mobility complex. The mutations at -46 and -47 abrogated both complexes. Oligonucleotides harboring mutations from -48 to -53 were able to form the high-mobility but not the low-mobility complex. Although the oligonucleotide harboring mutations at -48 and -49 is able to form the highmobility complex, the affinity is severely reduced. The complexes of high and low mobilities are referred to as NF-CLEOa and NF-CLEOb, respectively. This result indicates that every base encompassing -42 and -47 is absolutely required and that bases -48 and -49 are partially required for the binding of NF-CLEOa. This region is almost identical to the region mapped by the transfection experiments and in vitro transcription experiments. The region extending from -46 to -53 is required for the binding of NF-CLEOb. To confirm that NF-CLEOa and NF-CLEOb are proteins with different sequence specificities, a competition assay was performed. As shown in Fig. 6B, NF-CLEOa formed by the oligonucleotide with mutations at -52 and -53 was competed for by the same oligonucleotide but not by the oligonucleotide harboring mutations at -44 and -45 that are required for NF-CLEOb binding. On the other hand, the NF-CLEOb complex formed by the oligonucleotide with mutations at -44 and -45 was competed for by the same oligonucleotide but not by the oligonucleotide harboring mutations at -52 and -53 that are required for NF-CLEOa binding. This result supports the idea that the NF-CLEOa
and NF-CLEOb proteins have different sequence specificities. NF-CLEOa and NF-CLEOb can interact with the CLEO element in the 5'-flanking regions of the IL-5 and the IL-4 genes but not the G-CSF gene. As shown in Fig. 7A, the A M
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FIG. 6. (A) DNA mobility shift assay with oligonucleotides harboring substitution mutations. For this assay, 105 cpm of each oligonucleotide was mixed with 0.1 ,ug of partially purified NFCLEO protein. (B) DNA mobility shift assay that distinguishes NF-CLEOa and NF-CLEOb. Oligonucleotides GM44.45 and GM52.53 (105 cpm each) were incubated with no competitor or with competitors as indicated.
CLEO, CIS-ACTING ELEMENT OF THE CM-CSF GENE
VOL . 1 l, 1991
A
IL5-CLEO
Probe
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FIG. 8. Competition assay with oligonucleotides GM44.45 and GM52.53. The indicated probes (101 cpm each) were mixed with partially purified NF-CLEO protein in the absence of competitor or in the presence of competitor as indicated.
A
_
1 2 3 4 5 6 7 8 9 1011 1213 FIG. 7. Competition assay with the CLEO elements of the IL-5, IL-4, and G-CSF genes. (A) Comparison of the CLEO element of various mouse (m) and human (h) lymphokine genes. Underlines indicate identical bases with the CLEO element of the GM-CSF gene. (B) Competition assay with oligonucleotides CLEO, IL-5CLEO, IL-4-CLEO, and G-CSF-CLEO. CLEO (10 cpm) was mixed with partially purified NF-CLEO protein in the absence of competitor and in the presence of the indicated competitors.
CLEO element was identified in the 5'-flanking regions of other cytokine genes, the IL-4, IL-5, and G-CSF genes. To determine whether NF-CLEOa and -b interact with the CLEO elements in other cytokine promoters, a competition assay was performed. As shown in Fig. 7B, NF-CLEO formed by the CLEO oligonucleotide was competed for by the IL-4 and IL-5 CLEO elements but not by the G-CSF CLEO element. The G-CSF CLEO element seems to compete very weakly. To determine whether both NF-CLEOa and NF-CLEOb are able to interact with the CLEO element in the IL-4 and IL-5 genes, the complexes formed by the IL-4 CLEO element and the IL-5 CLEO element were challenged by oligonucleotides GM44.45 and GM52.53 (Fig. 8). For both elements, GM44.45 was displaced from the complex of higher mobility and GM52.53 was displaced from the complex of lower mobility, suggesting that CLEO elements of both the IL-4 and IL-5 genes are recognized by both NFCLEOa and NF-CLEOb. However, the affinity of NF-CLEOb for the CLEO element of the IL-5 gene was relatively weak. As a control, complex formation with the CLEO element of the G-CSF gene was examined by using oligonucleotides GM44.45 and GM52.53 as competitors. As predicted from the results of the previous experiment, neither competitor was able to compete even at an 80-fold molar excess. The complex formed with the CLEO element of the G-CSF gene was not competed for with oligonucleotide GM44.45 but was weakly competed for with oligonucleotide GM52.53. This finding suggests that the nuclear factor that binds to the G-CSF CLEO motif is different from NF-CLEOa and -b; however, this factor is able to recognize the sequence for NF-CLEOa weakly.
DISCUSSION The importance of the region located between the CLE2+GC box element and the TATA sequence of the GM-CSF promoter for induction has been reported previously (11, 18, 20, 21), and this study describes both the precise mapping of the region and the identification of factors that interact with this region. A battery of lymphokine genes are activated upon antigen stimulation of T cells; it has been proposed that there are common transcription factors involved in this process (1, 2, 16, 30). One candidate for a common factor is NF-KB, shown to be required for T-cell-inducible genes such as those for IL-2 (12), the human immunodeficiency virus long terminal repeat (19, 24), IL-2 receptor a chain (5, 8, 15), and possibly GM-CSF (26, 28). The CLEO element is identified as a highly homologous element for the GM-CSF, IL-4, IL-5, and G-CSF genes. The transfection experiments reveal that every mutation extending from -39 to -49 abolishes the ability of the promoter to respond to PMA/A23187 despite the presence of the CLE2+GC box element. This region is located within the CLEO element, implying that the CLEO element may be crucial for the coordinate regulation of these genes in T cells. As previously reported, deletion of the CLE2+GC box element also completely impairs promoter activity (18, 28); therefore, cooperation between the CLEO element and CLE2+GC box element is essential for the GM-CSF promoter to respond to PMA/A23187 stimulation. Mutation in position -53 also abolishes induction by PMA/A23187, while mutations in the 5' half of the CLEO element (-50, -51, and -52) upregulate the basal level; in the case of mutations at -50 and -52, the PMA/A23187-induced level is also elevated. The upregulation by these mutations may imply the existence of a suppressor molecule interacting with this region. The plasmid carrying mutations at -50, -51, -52, and -53 (p49-54; Fig. 1) did not respond to PMA/A23187 (Table 1), indicating that the effect of the mutation at -53 may overcome the derepression caused by mutations at -50, -51, and -52. In addition to the CLE2+GC box element, the region extending from -39 to -49, which is almost identical to the region mapped for induction in vivo, is required for the transcriptional enhancement in transcription assays in vitro utilizing the nuclear extract prepared from the PMA/A23187-
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stimulated Jurkat cells. The effects of the two elements are rather additive, and the cooperation of these two elements seen in vivo is not observed in vitro. The transcription activity of the GM-CSF promoter with extracts prepared from nonstimulated cells is lower than that observed with extracts prepared from stimulated cells; however, both the combined CLE2+GC box element and the CLEO element alone show stimulatory effects (data not shown). Since the effects of mutations in these elements on the basal-level expression in unstimulated cells are not observed in the transfection experiments, these result may imply either that the nuclear extract is partially activated during the preparation or that some components required to coordinate the two elements are inactivated. Furthermore, neither the upregulation of the promoter activity by mutations at positions -50, -51, and -52 nor the downregulation by the mutation at -53 is observed in the transcription assay in vitro. The DNA-protein complex specific to the CLEO element was detected by mobility shift assays utilizing nuclear extract prepared from the PMA/A23187-stimulated cells. NFCLEO is induced severalfold by PMA/A23187 stimulation, suggesting that the induction of NF-CLEO may play a role in activation of the GM-CSF promoter. However, extracts obtained from uninduced cells also display significant levels of NF-CLEO. Mobility shift assays using oligonucleotides harboring substitution mutations indicate that the NF-CLEO complex consists of two complexes of slightly different mobilities, NF-CLEOa (higher mobility) and NF-CLEOb (lower mobility). The region extending from -42 to -47 is absolutely required for the binding of NF-CLEOa. In addition, the bases at -48 and -49 are partially required. This region is almost identical to the region that is required for the induction of promoter activity in vivo and that has stimulatory activity on transcription in vitro. Hence, the NF-CLEOa protein is likely a transcription factor that plays an important role in the induction of the GM-CSF gene in T cells. The recognition sequence of NF-CLEOb extends from -46 to -53, containing the region which is required for suppression in vivo. This may imply that NF-CLEOb could serve as a suppressor molecule, while NF-CLEOa might function as a positive regulator. Furthermore, suppression by NF-CLEOb is observed only when NF-CLEOa is functioning, although suppression is not clear in transcription assays in vitro. NF-CLEO is induced severalfold by PMA/ A23187 stimulation, suggesting that the induction of the NF-CLEO may play a role in activation of the GM-CSF promoter. However, extracts obtained from uninduced cells also display significant levels of NF-CLEO. The mobility shift assay with oligonucleotides GM44.45 and GM52.53 indicated that NF-CLEOb was strongly induced by PMAI A23187 stimulation, whereas NF-CLEOa was not detected in the crude nuclear extract (data not shown). The crude nuclear extract may contain inhibitory activity for NFCLEOa; therefore, the question of whether NF-CLEOa is induced by PMA/A23187 stimulation requires further investigation. The DNA-protein complex that is formed by the binding of NF-CLEOa and -b to the same oligonucleotide does not seem to be detected in the mobility shift assay, suggesting that the binding of NF-CLEOa and -b might be mutually exclusive. Another possible interpretation of this discrepancy is that under the conditions of the mobility shift assay, these two factors are not able to interact with the two elements on the same DNA molecule. The mutation at -53 abolishes induction of PMA/A23187 stimulation in the transfection experiment, indicating that there might be an additional positive regulator interacting
MOL. CELL. BIOL.
with this region. However, no additional proteins were detected by mobility shift assays. A homology search revealed that there are sequences homologous to the CLEO element of the GM-CSF gene in other cytokine genes, those for IL-5 (6), IL-4 (3), and G-CSF (29). All of these elements are 100% conserved between human and mouse genes. The extents of the homologies of the CLEO element of IL-5, IL-4, and G-CSF with that of GM-CSF are 14 of 15, 9 of 15, and 9 of 15 bases. The 5' end of the IL-4 CLEO element is not homologous but is similar to the CLEO element of GM-CSF, since this region is AT rich. The 5' end of the G-CSF CLEO element is not homologous at all. Another common feature of these CLEO elements is their proximity to the TATA sequence. All of them are located within 40 bp upstream from the TATA sequence. Competition assays showed that IL-5 CLEO and IL-4 CLEO elements compete with the protein-DNA complex formed with the GM-CSF CLEO element, indicating that both NF-CLEOa and -b can bind to these elements, whereas the G-CSF CLEO element does not seem to be recognized by the NF-CLEO proteins. As expected from the homology data, the affinity of the IL-5 CLEO element is higher than that of the IL-4 CLEO element. The competition assay with oligonucleotides GM44.45 and GM52.53 confirmed that both the IL-4 and IL-5 CLEO elements are recognized by two factors, NFCLEOa and -b, but the affinity of NF-CLEOb for IL-5 CLEO element is relatively weak despite the higher homology. These data may imply that NF-CLEOa and -b are common factors for the promoters of GM-CSF, IL-4, and IL-5. On the other hand, despite the high homology, the CLEO element in the G-CSF gene, which is not expressed in T cells, could be recognized by a different factor. This putative factor might be closely related to NF-CLEOa or -b, since the proteins are coeluted after several fractionation steps (data not shown), and it recognizes the NF-CLEOa binding site weakly. The CLEO element of the G-CSF gene was not found to be one of the regulatory regions of the G-CSF gene in a human carcinoma line. However, one construct which carries substitution mutations in the G-CSF CLEO element showed impaired promotor activity (22). The CLEO elements of the GM-CSF, IL-4, IL-5, and G-CSF genes are all located within 40 bp from the TATA sequence. This common feature of the location of this element and its absolute requirement for induction of the GM-CSF promoter may suggest that the factors interacting with the CLEO element function as components that link various transcription factors for regulatory elements located upstream and the basic transcriptional machinery. To analyze this possibility, the effect of shuffling of the CLE2+GC box element and the CLEO element on activation of the GM-CSF promoter needs to be studied. The recognition sequence of NF-CLEOa of the CLEO element contains the core motif (GGAA) of the PU box, the recognition sequence of proteins containing the ets domain, suggesting that NF-CLEOa may be a member of the ets gene family (13, 14, 25). Nimer et al. reported on the importance of the CATT repeat (20, 21). We found that the recognition sequence of NF-CLEOa is larger than the CATT-1 motif and that NFCLEOb, which recognizes a part of the region encompassing the CATT-2 motif, may be inhibitory rather than stimulatory. Purification of NF-CLEOa and -b is in progress. Complete purification and cloning of the genes for the NF-CLEOa and -b proteins are essential to further studies on the activation
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mechanism of transcription of the lymphokine genes in antigen-activated T cells. ACKNOWLEDGMENTS We are grateful to Felix Vega for synthesizing various oligonucleotides. We thank A. Tsuboi, Y. Yamaguchi-Iwai, K. Sugimoto, I. Matsuda, and E. S. Masuda for helpful discussions. DNAX Research Institute of Molecular and Cellular Biology is supported by Schering-Plough Co. REFERENCES 1. Arai, K., F. Lee, A. Miyajima, S. Miyatake, N. Arai, and T. Yokota. 1990. Cytokines: coordinators of immune and inflammatory responses. Annu. Rev. Biochem. 59:783-836. 2. Arai, K., T. Yokota, A. Miyajima, N. Arai, and F. Lee. 1986. Molecular biology of T-cell-derived lymphokines: a model system for proliferation and differentiation of hemopoietic cells. Bioessays 5:166-171. 3. Arai, N., D. Nomura, D. Villaret, R. De Waal Malefijt, M. Seiki, S. Yoshida, S. Minoshima, R. Fukuyama, M. Maekawa, J. Kudoh, N. Shimhizu, K. Yokota, E. Abe, T. Yokota, Y. Takebe, and K. Arai. 1989. Complete nucleotide sequence of the chromosomal gene for human IL-4 and its expression. J. Immunol. 142:274-282. 4. Berridge, M. J., and R. F. Irvine. 1984. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312:315-321. 5. Bohnlein, E., J. W. Lowenthal, M. Siekevitz, D. W. Ballard, B. R. Franz, and W. Green. 1988. The same inducible nuclear protein regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type I HIV. Cell 53:827-836. 6. Campbell, H. D., G. J. Sanderson, Y. Wang, Y. Hort, M. E. Martinson, W. Q. J. Tacker, A. SteUlwagen, M. Strath, and I. G. Young. 1988. Isolation, structure and expression of cDNA and genomic clones for murine eosinophil differentiation factor. Comparison with other eosinophilopoietic lymphokines and identity with interleukin-5. Eur. J. Biochem. 174:345-352. 7. Crabtree, G. R. 1989. Contingent genetic regulatory events in T lymphocyte activation. Science 243:355-361. 8. Cross, S. L., N. F. Halden, M. J. Lenardo, and W. J. Lenard. 1989. Functionally distinct NF-KB binding sites in the immunoglobulin K and IL-2 receptor a chain genes. Science 244:466469. 9. Dignam, J. D., R. M. Revovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase 11 in a soluable extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489. 10. Grosschedl, R., and D. Baltimore. 1985. Cell-type specificity of immunoglobulin gene expression is regulated by at least three DNA sequence element. Cell 41:885-897. 11. Heike, T., S. Miyatake, M. Yoshida, K. Arai, and N. Arai. 1989. Bovine papillomavirus encoded E2 protein activates lymphokine gene through DNA elements, distinct from the consensus motif, in the long control region of its own genome. EMBO J. 8:1411-1417. 12. Hoyos, B., D. W. Ballard, E. Boehnlein, M. Seikevitz, and W. C. Green. 1989. Kappa B-specific DNA binding proteins: role in the regulation of human interleukin-2 gene expression. Science 244:457-460. 13. Karim, F. D., L. D. Urness, C. S. Thummel, M. J. Klemsz, S. R. McKercher, A. Celada, C. Van Beveren, R. A. Maki, C. V. Gunther, J. A. Nye, and B. J. Graves. 1990. The ETS-domain: a new DNA-binding motif that recognizes a purine-rich core DNA sequence. Genes Dev. 4:1451-1453. (Letter.) 14. Klemsz, M. J., S. R. McKercher, A. Celada, C. V. Beveren, and R. A. Maki. 1990. The macrophage and B cell-specific transcrip-
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