Key regulatory regions necessary for the expression of the gene encoding FcεRI α-chain, a component of the high-affinity IgE receptor primarily responsible for IgE-dependent allergic response, were investigated. Two regions, −74/−69 and −55/−47, which contained binding motifs for proteins belonging to the Ets family and the GATA family, respectively, were shown to be necessary for the activation of the α-chain promoter. Both the regulatory elements enhanced the promoter activity only in α-chain-producing cells PT18 and RBL-2H3 (mast cell lines), indicating that the elements required specific trans-acting proteins present in the α-chain-producing cells. EMSA using nuclear extracts and in vitro-translated proteins revealed that Elf-1 and GATA-1 bound to the enhancer elements. This is the first report describing the regulation in the expression of the FcεRI α-chain.

Binding of allergen-IgE Ab complexes to the high-affinity IgE receptor (FcεRI)2 triggers the release of a variety of chemical mediators from activated mast cells, which results in allergic responses.

FcεRI is a tetrameric receptor composed of one α-, one β-, and two γ-chains. The γ-chain is composed of two critical regions, a single membrane-spanning domain and a cytoplasmic domain that contains the immunoreceptor tyrosine-based activation motif (1) sequence for the association of Syk tyrosine kinase. The γ-chain is also served as a common component of other Ig Fc receptors, such as low-affinity receptor for IgG (FcγRIII), high-affinity receptor for IgG (FcγRI), and Fc receptor for IgA (FcαR), and is also associated with the TCR-CD3 complexes (2, 3, 4, 5, 6, 7). Thus, in contrast with the expression of FcεRI in the limited cells, the γ subunit is found in various kinds of cells, suggesting that the γ subunit is not directly involved in the regulation of FcεRI expression. The β-chain, which also has immunoreceptor tyrosine-based activation motif to associate with Lyn (8), is known to amplify the γ-chain-mediated cell-activation signals (9, 10). However, the β-chain is shown to be unnecessary for the expression of functional human FcεRI on the cell surface (11). On the other hand, the α-chain is a specific component of FcεRI and is known to bind to the Fc region of IgE Ab (12, 13). Recently, the expression of FcεRI on human mast cells (14, 15, 16) or on eosinophils (17) was found to be up-regulated by IL-4. Messenger RNA for the α-chain was specifically increased by IL-4, while the transcription levels of the β-chain and the γ-chain were not changed through the stimulation. All of these observations suggest a crucial role of the α-chain in the regulation of the FcεRI expression. Necessity of the α-chain for FcεRI-mediated allergic reaction was also observed in α-chain-deficient mice as well as human (18). In addition, when human α-chain gene was introduced in mouse cells, the human FcεRI α-chain transgenic mice have a cell expression pattern of human α-chain that is indistinguishable from that seen in humans (10, 19). This indicated the presence of a common mechanism for α-chain expression in mammalian cells. To reveal mechanisms of the FcεRI expression, which might contribute to manipulation of the IgE-mediated allergic response, we analyzed regulatory elements for human FcεRI α-chain gene and determined trans-acting proteins binding to the elements, which enhance the transcription of the α-chain specifically in mast cells.

PT18 (mouse mast cell line) and Jurkat (human T cell line) cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% FBS (Life Technologies, Rockville, MD), glutamine (300 mg/L), penicillin (100 U/ml), and streptomycin (100 μg/ml), and RBL-2H3 (rat mast cell line) cells were grown in minimum essential α medium (α-MEM) (Life Technologies) supplemented with 10% FBS and penicillin/streptomycin at 37°C in 5% CO2 incubator.

The human genomic DNA of the FcεRI α-chain was isolated from a human genomic library (Stratagene, La Jolla, CA) by plaque hybridization using human FcεRI α-chain cDNA as the probe. The 5′-flanking region of about 1.3 kb including an untranslated region of the first exon of the α-chain gene was prepared by PCR using 5′-GCaagcttGATCTTCATGTGGAATGACTGG-3′ and 5′-CAGGAGccATGGTCTTCATGGA-3′ (replaced nucleotides were shown as small letters to introduce the HindIII and NcoI site, which were shown as italic, and the initiation codon was represented as bold) as the primers and the human FcεRI α-chain gene as the template, and subcloned into the HindIII-NcoI site of pGV-B2 (Promega, Madison, WI). The resulting plasmid was named pGV-B-αHN1.3 (Fig. 1; nt −1256 to nt +29). The plasmid pGV-B-αKN2.4 containing a further upstream region, as shown at the bottom bar in Fig. 1, was constructed as follows. An ∼1.8-kb (nt −2437 to nt −602) fragment of the 5′-franking region was obtained from the human FcεRI α-chain gene using original the KpnI/NheI site, and was ligated to pGV-B-αHN1.3 digested with KpnI/NheI. Other plasmids containing a variety of 5′-truncation of the α-chain promoter region connected to the upstream region of the luciferase structural gene were constructed in a similar way using several restriction endonucleases and exonuclease III (Takara Shuzo, Kyoto, Japan) and/or by PCR.

FIGURE 1.

Determination of FcεRI α-chain promoter activated in α-chain-producing cells by transient transfection luciferase assay. The relative promoter activity is represented as the ratio to SV40 promoter activity in Figs 1, 2, and 3. Each experiment was conducted in duplicate for each sample, and the results are expressed as mean + SD for more than three independent experiments. Each solid bar represents relative length of the upstream region of the α-chain gene in the construct. Nucleotide numbers when the transcriptional start site is expressed as +1 were also attached. N.T., not tested.

FIGURE 1.

Determination of FcεRI α-chain promoter activated in α-chain-producing cells by transient transfection luciferase assay. The relative promoter activity is represented as the ratio to SV40 promoter activity in Figs 1, 2, and 3. Each experiment was conducted in duplicate for each sample, and the results are expressed as mean + SD for more than three independent experiments. Each solid bar represents relative length of the upstream region of the α-chain gene in the construct. Nucleotide numbers when the transcriptional start site is expressed as +1 were also attached. N.T., not tested.

Close modal

All of the constructs in which 3–6 bp were replaced with others were produced by site-directed mutagenesis using Quickchange site-directed mutagenesis kit (Stratagene).

The cells were grown to ∼90% confluence (RBL-2H3), and 1 × 106 cells/ml (PT18 and Jurkat) were washed with ice-cold medium including 10% FBS and resuspended in the same medium. The cells (5 × 106 cells in 0.5 ml) were cotransfected with 5 μg of the test construct and 25 ng of pRL-CMV (Promega) by electroporation using a Bio-Rad Gene Pulsar II (Hercules, CA) set at 300 V and 950 μF. The pRL-CMV plasmid carrying Renilla luciferase gene was cotransfected and used for normalizing the transfection efficiency. The pGV-P2 (Toyo ink; Tokyo, Japan) was used as an internal control to compare each different experiment. The measurement of luciferase activity was conducted using a Dual-luciferase assay kit (Promega) essentially according to manufacturer’s instructions. Briefly, cells were harvested 20–24 h after transfection, washed twice with PBS, pH 7.4, and then lysed with 100 μl of lysis buffer (included in the Dual-luciferase assay kit) for 15 min. Ten microliters of the lysate was mixed with 100 μl of Luciferase Assay Reagent II, and the luminescence was immediately measured by a luminometer (Model LB9501; Berthold, Postfach, Germany). After the measurement of luciferase activity, 100 μl of Stop & Glo Reagent included in the Dual-luciferase assay kit was added to the reaction mixture, and the Renilla luciferase activity was determined as an internal control to normalize the transfection efficiency.

PT18 cells (2 × 107 cells) were washed with ice-cold PBS and resuspended in 1.2 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin) for 10 min at 4°C. The cells were incubated for an additional 15 min with 0.5% Nonidet P-40 for cell lysis. The lysates were centrifuged at 6000 × g for 1 min, and the pellets were resuspended in 100 μl of extract buffer (20 mM HEPES, pH 7.9, 400 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). After incubation for 1 h on ice, the lysates were centrifuged at 6000 × g for 5 min and the supernatant was collected. Glycerol was added to 15%, and the extract was stored at −70°C until use. Protein concentrations were determined by a commercially available kit (Bio-Rad).

The nuclear extract from RBL-2H3 was prepared by the method slightly different from that described above. RBL-2H3 cells (5 × 107) washed with ice-cold PBS were resuspended with 1.0 ml buffer A and were immediately centrifuged at 1000 × g for 5 min. The pellets were resuspended with 1.0 ml buffer A including 0.05% Nonidet P-40 and incubated for 1 min on ice. The lysates were centrifuged at 3000 × g for 1 min and the pellets were resuspended with 315 μl of modified extract buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 10% glycerol). After addition of 35 μl of 3 M (NH4)2SO4 to the nuclear extract, the suspension was incubated for 30 min on ice. After centrifugation at 6000 × g for 15 min, equal volumes of 3 M (NH4)2SO4 were added to the supernatants to precipitate the nuclear proteins. The pellets obtained by centrifugation at 10,000 × g for 15 min were resuspended with appropriate volume of modified extract buffer.

The double-stranded oligonucleotide probes for EMSA were prepared as follows. The probes 5′-GATACAGAAAACATTTCCTTCTGCTTTTTGGTTTTA-3′ and 5′-GGCTTAAAACCAAAAAGCAGAAGGAAATGTTTTCTG-3′ were annealed by incubation at 95°C for 5 min and successive gradual cooling to 37°C. The double-stranded DNA was 5′-end-labeled with [32P]ATP and T4 polynucleotide kinase (Promega). After Klenow fragment (Promega) treatment, labeled oligonucleotides were purified using a ProbeQuant G-50 micro column (Pharmacia Biotech, Uppsala, Sweden). The labeled double-stranded DNA containing the sequence motif for Ets (nt −68 to nt −29) was named probe 1. Another labeled double-stranded DNA named probe 2 containing the sequence motif for GATA (nt −77 to nt −58) was prepared by two oligo nucleotides, 5′-ACCAGATATGATACAG-3′ and 5′-TTTTCTGTATCATATC-3′, in a similar way. The reaction mixtures (15 μl), containing 4 μg of nuclear extract, 350 ng of poly(dI-dC), 1 mM MgCl2, 30 mM KCl, 10 mM HEPES, pH 7.9, 1 mM DTT, and 5% glycerol were preincubated at room temperature in the absence of the probe for 15 min and incubated for an additional 15 min at room temperature with the radiolabeled probe (5 × 104 cpm). The reaction mixtures were then subjected to electrophoresis with a native 4% polyacrylamide gel at 120 V for 2.5 h in 0.5× TBE buffer (45 mM Tris-borate, pH 8.0, 45 mM boric acid, 1 mM EDTA). For competition experiments, unlabeled blunt-ended competitor oligonucleotides were added to the binding reaction mixtures before the addition of the radiolabeled oligonucleotide probe. For the supershift or inhibition experiments by Abs, 1.0 μg of Abs was added to the reaction mixtures and incubated for 15 min. The labeled probe was then added to the reaction mixture and the mixture was incubated for an additional 15 min, then applied onto a native 4% polyacrylamide gel for electrophoresis. The anti-Elf-1, anti-Ets-1/2, anti-GATA-1, anti-GATA-2, and anti-GATA-3 Abs were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). All gels were dried and subjected to autoradiography. Modifications are described in the figure legends.

Both of the rat GATA-1 cDNA and rat Elf-1 cDNA were obtained with mRNA from RBL-2H3 cells by using Trizol reagent (Life Technologies) and a RT-PCR kit (Takara Shuzo). Two synthetic oligonucleotides, 5′-GTTAAACCCCAGTGTCCACATGGATTTTCCTGGTCTA-3′ and 5′-CCTTCAAGAACTGAGTGGAGACACTACGCTAG-3′, were used as primers to amplify rat GATA-1 cDNA according to reported nucleotide sequence (20). As primers for amplification of rat Elf-1 cDNA, we used the following oligonucleotides derived from mouse Elf-1 cDNA (21); 5′-ATGGCTGCTGTTGTCCAACAGAACGACCTA-3′ and 5′-TTAAAAAGAGTTGGGCTCTAGCAGTTCATT-3′, because the nucleotide sequence of rat Elf-1 cDNA has not been published until now. Rat Elf-1 cDNA was obtained using a rat intestine cDNA library (Clontech Laboratories, Palo Alto, CA) using the amplified PCR products as the probe, and its nucleotide sequence was determined (C. Nishiyama et al., manuscript in preparation). These cDNA fragments, both of rat GATA-1 and rat Elf-1, were inserted to the expression plasmid pCR3.1 (Invitrogen, Leek, The Netherlands), and the resulting plasmids were used for in vitro transcription and translation using TnT; T7 Quick-coupled transcription/translation system (Promega).

Previous studies by Fung-Leung et al. with transgenic mice suggested that 1.3 kb upstream of the human FcεRI α-chain-coding region contained all the elements sufficient for cell type-specific expression of the FcεRI α-chain in mouse cells (19). For elucidation of the transcriptional control enhancer of human α-chain gene, we used two rodent cell lines, RBL-2H3 and PT18, which were known to express FcεRI constitutively. In this study, a DNA fragment containing 2.4 kb upstream of the translational initiation codon for the α-chain was cloned into the KpnI-NcoI site of pGV-B2 carrying the luciferase gene from Photinus pyralis as reporter. For identification of cis-activating elements of the promoter, several deletion constructs carrying the upstream sequences in various length ranging from 2.4 kb to 100 bp upstream of the translational start site were generated and introduced into FcεRI-positive cells, RBL-2H3 and PT18, and negative cells, Jurkat. As shown in Fig. 1, no constructs including the translational start site showed detectable luciferase activity in Jurkat. This indicated that Jurkat cells did not contain components promoting the transcription of FcεRI α-chain gene. Even the shortest region containing 100 bp upstream of the translational start site demonstrated promoter activity higher than that of SV40 promoter in RBL-2H3, suggesting the region contained the promoter of FcεRI α-chain gene that gave the transcription at a sufficient level. In PT18, each construct gave the luciferase activity at almost the same level, which was contrast with the case of RBL-2H3, where longer DNA fragment gave higher luciferase activity. This may suggest that the amount of transcription factors recognizing the promoter region and/or proteins affecting the transcription efficiency was different. In any cases, these results indicated that the α-chain promoter was functional only in the FcεRI-positive cells, although regulation was somewhat different between RBL-2H3 and PT18 cells.

The above-mentioned results suggested that the cloned upstream region of the α-chain gene included its own promoter and possible enhancer elements responsible for initiating the transcription of the α-chain gene in α-chain-producing cell lines. For determining the location of the elements, we used several constructs in which a portion of the promoter region of the α-chain gene was replaced by a heterologous promoter, SV40, or thymidine kinase promoter. All the constructs lacked the transcription initiation site of α-chain to decrease the transcription from α-chain promoter.

As displayed in Fig. 2,A, when the luciferase gene was connected upstream of SV40 or thymidine kinase promoter, the luciferase activity raised up to above four or five times compared with the basal activity by the construct containing only either of SV40 or thymidine kinase promoter in RBL-2H3. In addition, this effect was not observed in Jurkat as expected. This suggested that the fragment from nt −605 to nt −8 contained the element that increased the promoter activity in the specific cells. Consistent with this, when the fragment from nt −1256 to nt −8 was placed in the opposite orientation to the SV40 promoter, increased luciferase activity was again observed (Fig. 2 B). This indicated that the fragment contained cis elements up-regulating the promoter present in its neighborhood. As will be described below, two transcription factors actually bound the upstream region. Hereafter we therefore called the elements as the enhancers for α-chain gene. We next performed precise mapping of the putative enhancer elements through generating various deletion constructs. The α-chain gene fragment from nt −605 to nt −8 had transcription-enhancing activity equivalent to that demonstrated with the fragment from nt −1256 to nt −8 even on the heterologous promoter as well as its own promoter. This suggested that the region nt −605 to nt −8 contained major elements up-regulating the transcription of the α-chain gene. Further deletion from nt −605 to nt −368 had no effect on the promoter activity. However, the deletion from nt −85 to nt −8 drastically decreased the activity, suggesting that the region from nt −85 to nt −8 contained a enhancer element. Principally the same results were also obtained with PT18 as the host (data not shown).

FIGURE 2.

Mapping of enhancer elements in the FcεRI α-chain 5′-flanking region. A, Enhancer activity on heterologous promoters. B, Deletion mapping of enhancer elements in FcεRI α-chain promoter using RBL-2H3. The upstream region of FcεRI α-chain gene contained in the constructs are shown as solid bars. The third construct from the top shown in B possesses the same upstream region as that of the second one but their orientations to SV40 promoter were opposite.

FIGURE 2.

Mapping of enhancer elements in the FcεRI α-chain 5′-flanking region. A, Enhancer activity on heterologous promoters. B, Deletion mapping of enhancer elements in FcεRI α-chain promoter using RBL-2H3. The upstream region of FcεRI α-chain gene contained in the constructs are shown as solid bars. The third construct from the top shown in B possesses the same upstream region as that of the second one but their orientations to SV40 promoter were opposite.

Close modal

The aforementioned results indicated the presence of enhancer element in the region between nt −85 and nt −8. Therefore, we performed fine mapping of the enhancer element by introducing the mutations into the region from nt −91 to nt −8 of the −605/−8 fragment. For this purpose, nine mutant genes in which three to six nucleotides were replaced by others were generated by site-directed mutagenesis. The analysis revealed that the mutations around nt −75 and nt −50 drastically decreased the enhancing activity in both RBL-2H3 and PT18 cells (Fig. 3). This suggested the presence of two enhancer segments in this region.

FIGURE 3.

Determination of functional enhancer elements by site-directed mutagenesis. Mutations were introduced into −91 to −28 of FcεRI α enhancer region (−605/−8) linked to SV40 promoter. Only the nucleotides that differ from the original were shown. Lines represent unchanged nucleotides.

FIGURE 3.

Determination of functional enhancer elements by site-directed mutagenesis. Mutations were introduced into −91 to −28 of FcεRI α enhancer region (−605/−8) linked to SV40 promoter. Only the nucleotides that differ from the original were shown. Lines represent unchanged nucleotides.

Close modal

We next analyzed, by EMSA, the nuclear factors from the α-chain-producing cells that specifically bound to the upstream region around nt −50. As shown in Fig. 4, nuclear extract prepared from PT18 contained protein(s) that could bind to this fragment (−68/−29). Electrophoretic mobility shift, showed by an arrow in Fig. 4, was inhibited when excess amounts of the same but unlabeled fragment was used as a self-competitor (Fig. 4, lanes 3, 4, and 5). Furthermore, the competitive effect was not observed when the double-stranded DNA with the following sequence, GATACAGAAAACATaTgcgTCTGCTTTTTGGTTTTAAGCC, which contained the base substitutions −54/−50, decreasing enhancer activity, was applied as a competitor (see Fig. 3 and Fig. 4, lanes 6, 7, and 8). This result suggested that the shifted band was the complex of the oligonucleotide with the nuclear protein responsible for the promoter-enhancing activity. The region around −54/−50 contained the nucleotide sequence TTTCCTT, which was similar to that of the Ets motif, TTCC, especially the Elf-1 motif, T/CTTCC T/G (22). Then, we synthesized double-stranded oligonucleotide containing the Ets motif of human GM-CSF promoter (23) and performed similar EMSA using the oligonucleotide as the competitor. The competitor inhibited the binding of the nuclear protein(s) to the probe DNA (Fig. 4, lanes 9, 10, and 11). Therefore, this observation suggests that the nuclear protein binding to the Ets motif of GM-CSF promoter also bound to the enhancer element of the α-chain gene in mast cell line PT18.

FIGURE 4.

Competition assay on Ets motif. EMSAs were performed with a radiolabeled probe 1 (see Materials and Methods and Fig. 7) and nuclear extracts from PT18. Competitive binding assays were performed with unlabeled competitor 1 (probe 1), competitor 2 (mutant probe 1), and competitor 3 (CLE0 motif in human GM-CSF promoter) probes. Lane 1, probe 1 only; lanes 2–11, probe 1 with nuclear extracts; lane 2, without competitor; lanes 3, 4, and 5, with competitor 1; lanes 6, 7, and 8, with competitor 2; lanes 9, 10, and 11, with competitor 3. The specific binding of nuclear protein(s) to the boxed sequence in the probe is shown with an arrow.

FIGURE 4.

Competition assay on Ets motif. EMSAs were performed with a radiolabeled probe 1 (see Materials and Methods and Fig. 7) and nuclear extracts from PT18. Competitive binding assays were performed with unlabeled competitor 1 (probe 1), competitor 2 (mutant probe 1), and competitor 3 (CLE0 motif in human GM-CSF promoter) probes. Lane 1, probe 1 only; lanes 2–11, probe 1 with nuclear extracts; lane 2, without competitor; lanes 3, 4, and 5, with competitor 1; lanes 6, 7, and 8, with competitor 2; lanes 9, 10, and 11, with competitor 3. The specific binding of nuclear protein(s) to the boxed sequence in the probe is shown with an arrow.

Close modal

The promoter of GM-CSF contains AP-1 motif just upstream of the Ets motif, forming conserved lymphokine element 0 (CLE0) motif. Elf-1 or Ets-1 was identified as the transcription factor binding to CLE0 of GM-CSF promoter and IL-5 promoter in T cells (23, 24, 25, 26, 27). To identify nuclear protein(s) binding to the α-chain enhancer, we examined the effects of anti-transcription factor Abs on the EMSA profile. As demonstrated in Fig. 5 A, when anti-Elf-1 mAb was added to the reaction mixture, the shifted band drastically decreased and faint but obvious super shift was observed. On the other hand, anti-Ets-1/2, GATA-1, GATA-2, and GATA-3 Abs did not show any effect on the EMSA profile.

FIGURE 5.

Identification of the nuclear protein binding to the Ets motif. A, Identification using anti-transcription factor Abs. The Abs used were anti-Ets-1/2, anti-Elf-1, and anti-GATA-1, anti-GATA-2, and anti-GATA-3. Additives are: lane 1, none; lanes 2, 3, and 4, anti-Elf-1; lanes 5, 6, and 7, anti-Ets-1/2; lane 8, anti-GATA-1; lane 9, anti-GATA-2; lane 10, anti-GATA-3. The specific shifted and super shifted bands were shown by lower and upper arrows, respectively. Reactions were separated by native electrophoresis in a 6% acrylamide gel (instead of 4% acrylamide gel as described in Materials and Methods) with 0.5 × TBE at 120 V for 2.5 h. B, Mobility profile of in vitro-translated Elf-1. In vitro translation reactions were conducted without template (lanes 1 and 2), with empty vector, pCR3.1 (lanes 3 and 4), and with pCR3.1-Elf-1 (lanes 5 and 6). Lanes 1, 3, and 5, without Ab; lanes 2, 4, and 6, with anti-Elf-1 Ab.

FIGURE 5.

Identification of the nuclear protein binding to the Ets motif. A, Identification using anti-transcription factor Abs. The Abs used were anti-Ets-1/2, anti-Elf-1, and anti-GATA-1, anti-GATA-2, and anti-GATA-3. Additives are: lane 1, none; lanes 2, 3, and 4, anti-Elf-1; lanes 5, 6, and 7, anti-Ets-1/2; lane 8, anti-GATA-1; lane 9, anti-GATA-2; lane 10, anti-GATA-3. The specific shifted and super shifted bands were shown by lower and upper arrows, respectively. Reactions were separated by native electrophoresis in a 6% acrylamide gel (instead of 4% acrylamide gel as described in Materials and Methods) with 0.5 × TBE at 120 V for 2.5 h. B, Mobility profile of in vitro-translated Elf-1. In vitro translation reactions were conducted without template (lanes 1 and 2), with empty vector, pCR3.1 (lanes 3 and 4), and with pCR3.1-Elf-1 (lanes 5 and 6). Lanes 1, 3, and 5, without Ab; lanes 2, 4, and 6, with anti-Elf-1 Ab.

Close modal

To confirm that Elf-1 bound to the Ets motif, rat Elf-1 cDNA was cloned from RBL-2H3 cells and expressed by in vitro transcription/translation system. The rat Elf-1 produced in vitro actually bound to the oligonucleotide containing the Ets motif (Fig. 5 B), and the DNA-Elf-1 complex showed mobility identical with that seen with the nuclear protein (data not shown). When anti-Elf-1 Ab was added, faint but apparent super shift and disappearance of the shifted band were also observed. These results indicate that the nuclear protein binding to the Ets motif of FcεRI α-chain gene is a transcription factor, Elf-1.

To identify the transcription factor that recognizes the sequence around −75 of FcεRI α-chain gene, EMSA was conducted. For this purpose, a double-stranded DNA, probe 2 (nt −77 to nt −58), containing the critical region for the enhancer activity was used for EMSA. Specific binding to probe 2 was observed when nuclear proteins extracted from PT18 were applied (Fig. 6,A), which suggested specific nuclear factors actually bound to the DNA region. The promoter-enhancing region around −75 contained a motif of GATA, suggesting a member of the GATA protein family is responsible for the enhancing activity of FcεRI α-chain gene expression (see Fig. 3). A shifted band showing the probe-protein complex disappeared only by addition of anti-GATA-1 Ab among the three anti-GATA-1, -2, and –3 Abs. This indicated that the GATA motif was recognized by GATA-1. To confirm this possibility, similar EMSA was conducted with in vitro-translated GATA-1 (see Materials and Methods). The in vitro-translated GATA-1 caused a mobility shift similar to that shown by RBL-2H3 and PT18 nuclear extracts (Fig. 6 B). Furthermore, the addition of anti-GATA-1 Ab again inhibited the binding of the GATA-1 and the oligonucleotide. Therefore, we concluded that the transcription factor that bound to the GATA motif in the α-chain gene promoter was GATA-1.

FIGURE 6.

Identification of the nuclear protein binding to the GATA motif. A, Identification using anti-transcription factor Abs. EMSAs were performed with a radiolabeled probe 2 (see Materials and Methods and Fig. 7) and nuclear extracts from PT18. Reactions were separated by native electrophoresis in a 6% acrylamide gel (instead of 4% acrylamide gel as described in Materials and Methods) with 0.5 × TBE at 120 V for 2.5 h. Additives are: lane 1, none; lane 2, anti-GATA-1; lane 3, anti-GATA-2; lane 4, anti-GATA-3. The specific band shown with and arrow was disappeared with an anti-GATA-1 Abs in lane 2. B, Mobility profile of in vitro-translated GATA-1. Lane 1, nuclear extract from RBL-2H3 without anti-GATA-1 Ab; lane 2, nuclear extract from RBL-2H3 with anti GATA-1 Ab; lane 3, nuclear extract from PT18 without Ab; lane 4, nuclear extract form PT18 with Ab; lane 5, in vitro-translated GATA-1 without Ab; lane 6, in vitro-translated GATA-1 with Ab.

FIGURE 6.

Identification of the nuclear protein binding to the GATA motif. A, Identification using anti-transcription factor Abs. EMSAs were performed with a radiolabeled probe 2 (see Materials and Methods and Fig. 7) and nuclear extracts from PT18. Reactions were separated by native electrophoresis in a 6% acrylamide gel (instead of 4% acrylamide gel as described in Materials and Methods) with 0.5 × TBE at 120 V for 2.5 h. Additives are: lane 1, none; lane 2, anti-GATA-1; lane 3, anti-GATA-2; lane 4, anti-GATA-3. The specific band shown with and arrow was disappeared with an anti-GATA-1 Abs in lane 2. B, Mobility profile of in vitro-translated GATA-1. Lane 1, nuclear extract from RBL-2H3 without anti-GATA-1 Ab; lane 2, nuclear extract from RBL-2H3 with anti GATA-1 Ab; lane 3, nuclear extract from PT18 without Ab; lane 4, nuclear extract form PT18 with Ab; lane 5, in vitro-translated GATA-1 without Ab; lane 6, in vitro-translated GATA-1 with Ab.

Close modal

We here addressed that two transcription factors, GATA-1 and Elf-1, were produced in the specific cells and bound to the regions of the promoter for human FcεRI α-chain gene to up-regulate its expression. When the upstream sequence was aligned with the sequences of corresponding regions of murine and rat α-chain genes, the sequences of the two enhancer elements were found to be highly conserved (Fig. 7) (28, 29, 30). This suggests that the expression of the α-chain is regulated by the two transcription factors in rodents or other mammals as well as human.

FIGURE 7.

The sequences of 5′-flanking regions of FcεRI α-chain gene. The sequences critical for enhancer activity (Fig. 3) are shown in boxes. The sequences of the 5′-flanking regions of human and rodent genes are also shown. The regions used as probes for EMSAs are marked with arrows (probe 1 for Figs 4 and 5; probe 2 for Fig. 6).

FIGURE 7.

The sequences of 5′-flanking regions of FcεRI α-chain gene. The sequences critical for enhancer activity (Fig. 3) are shown in boxes. The sequences of the 5′-flanking regions of human and rodent genes are also shown. The regions used as probes for EMSAs are marked with arrows (probe 1 for Figs 4 and 5; probe 2 for Fig. 6).

Close modal

In the α-chain gene, the GATA motif (−74/−69) was recognized by GATA-1 and the Ets motif (−55/−49) was recognized by Elf-1 (Fig. 7). GATA-1 is known to be indispensable for erythropoiesis (31, 32) and is expressed in cells of myeloid lineage (erythroid cells, mast cells, megakaryocytes, eosinophils, and basophils) (33, 34, 35, 36, 37), with the exception of a testis-specific form transcribed from an alternative promoter (38). Elf-1 was initially found in T cells as the transcription factor that was required for the TCR-mediated trans-activation of HIV-2 gene expression (39). Recently, another role of Elf-1 as a trans-activation factor for cell-specific gene expression was found in megakaryocytes, B cells, and macrophages. In addition, there is evidence showing involvement of Elf-1 in the regulation of a number of lymphocyte-specific genes, IL-2 (40), IL-2 receptor α-chain (41), GM-CSF (23), CD4 (42), IL-3 (43), and IgH (44) genes. This study revealed an additional role of Elf-1 in the expression of FcεRI α-chain. Though either Elf-1 or GATA-1 is expressed in several cells, all the cells expressing FcεRI α-chain contain both of Elf-1 and GATA-1. This indicates both factors are essential for the expression of FcεRI α-chain gene. Because both the GATA-1 and Elf-1 transcription factors are thus obligatory required for the expression of FcεRI α-chain (Table I), other FcεRI α-chain-producing cells, such as Langerhans cells, could express these transcription factors.

Table I.

Cell-type specificity in the expression of FcεRI α-chain, Elf-1, and GATA-1

Cell TypeFcεRI α-ChainElf-1GATA-1
Mast cells 
(PT18; mouse mastocytoma)    
(RBL-2H3; rat basophil leukemia)    
Mast cells – ND − 
(P815; mouse mastocytoma)    
T cells − − 
B cells − − 
Erythroid − − 
Megakaryocytes 
Cell TypeFcεRI α-ChainElf-1GATA-1
Mast cells 
(PT18; mouse mastocytoma)    
(RBL-2H3; rat basophil leukemia)    
Mast cells – ND − 
(P815; mouse mastocytoma)    
T cells − − 
B cells − − 
Erythroid − − 
Megakaryocytes 

The CLE0 element is often found in the promoters of several cytokines, GM-CSF, IL-3, IL-4, and IL-5, and is known to play a crucial role in regulation of the expression of these cytokines in T cells and megakaryocytes (23, 24, 25, 26, 27, 45, 46, 47, 48). By activation of mast cells through the cross-linking of FcεRI, production and release of the Th2-type cytokines were also induced in mast cells (49, 50, 51). In T cells, several transcription factors, Elf-1, NF-AT, and HMG-1, were present and promote the expression of the genes under the control. Interestingly, although the IL-5 promoter has a GATA motif just upstream of a CLE0 site, GATA-3 but not GATA-1 binds to the GATA motif and regulates the expression of IL-5 in the combination with other factors, AP-1 and Elf-1 (24).

In mast cells, IL-4 gene expression is regulated by two GATA family proteins, GATA-1 and GATA-2, and PU.1, which belongs to Ets family (52). The expression of mast cell carboxypeptidase A is known to be regulated predominantly by GATA-1 in mast cells (53). However, in a mast cell line, P815, which is negative in both FcεRI (2) and mast cell carboxypeptidase A, the expression of GATA-1 was not observed (Table I). This also suggests that GATA-1 is essential for the expression of FcεRI α-chain and plays a crucial role in the expression of certain genes in mast cells.

The Ets motif was found in the FcεRI α-chain genes of mouse, rat, and human (Fig. 7). Furthermore, the sequence is also present in other Fc receptor genes, suggesting an indispensable role of the sequence in the regulation of the expression of Fc receptors in myeloid cells (54, 55). The fact that the motif is recognized by Elf-1 in this study indicates that all the Ets sequences in other Fc receptor genes might be also recognized by Elf-1 in each myeloid cell. The further involvement of Elf-1 in other Fc receptor genes remains to be analyzed.

Here, we have revealed a part of the cell-specific transcription mechanisms of the FcεRI α-chain, mediated by two transcription factors, Elf-1 and GATA-1. Recently, the expression of FcεRI were reported to be up-regulated by IL-4 (14, 15, 16, 17), which was known to transfer the signal to the STAT6 motif. For better understanding of the expression mechanisms of FcεRI α-chain, it would be necessary to examine whether the functional STAT6 motifs exist in the FcεRI α-chain gene. The present study has become the first step to elucidate the expression mechanism of FcεRI.

We thank Dr. Makoto Nishiyama (Biotechnology Research Center, University of Tokyo, Tokyo, Japan) for his advice in completing the manuscript.

1

Address correspondence and reprint requests to Dr. Chisei Ra, Department of Immunology, Allergy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail address: cra@med.juntendo.ac.jp

2

Abbreviations used in this paper: FcεRI, high-affinity IgE receptor; CLE0, conserved lymphokine element 0.

1
Cambier, J. C..
1995
. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL).
Immunol. Today
16
:
110
2
Ra, C., M.-H. Jouvin, U. Blank, J.-P. Kinet.
1989
. A macrophage Fc receptor and the mast cell receptor for IgE share an identical subunit.
Nature
341
:
752
3
Kurosaki, T., J. V. Ravetch.
1989
. A single amino acid in the glycosylphosphatidylinositol attachment domain determines the memvrane topology of FcγRIII.
Nature
342
:
805
4
Ernst, L. K., A. M. Duchemin, C. L. Anderson.
1993
. Association of the high-affinity receptor for IgG (FcγRI) with the γ subunit of the IgE receptor.
Proc. Natl. Acad. Sci. USA
90
:
6023
5
Pfferkorn, L. C., G. R. Yeaman.
1994
. Association of IgA-Fc receptors (FcαR) with FcεRIγ2 subunits in U937 cells.
J. Immunol.
153
:
3228
6
Orloff, D. G., C. Ra, S. J. Frank, R. D. Klausner, J.-P. Kinet.
1990
. Family of disulfide-linked dimers containing the ζ and η chains of the T-cell receptor and γ chain of Fc receptors.
Nature
347
:
189
7
Letourneur, F., R. D. Klausner.
1991
. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor ζ family proteins.
Proc. Natl. Acad. Sci. USA
88
:
8905
8
Alber, G., L. Miller, C. L. Jelserna, N. Varin-Blank, H. Metzger.
1991
. Structure-function relationships in the mast cell high affinity receptor for IgE: role of the cytoplasmic domains and of the β subunit.
J. Biol. Chem.
266
:
22613
9
Lin, S., C. Cicala, A. M. Scharenberg, J.-P. Kinet.
1996
. The FcεRI β subunit functions as an amplifier of FcεRI γ-mediated cell activation signals.
Cell.
85
:
985
10
Dombrowicz, D., S. Lin, V. Flamand, A. T. Brini, B. H. Koller, J.-P. Kinet.
1998
. Allergy-associated FcRβ is a molecular amplifier of IgE- and IgG-mediated in vivo responses.
Immunity
8
:
517
11
Miller, L., U. Blank, H. Metzger, J.-P. Kinet.
1989
. Expression of high-affinity binding of human immunoglobulin E by transfected cells.
Science
244
:
334
12
Hakimi, J., C. Seals, J. A. Kondas, L. Pettine, W. Danko, J. Kochan.
1990
. The α subunit of the human IgE receptor (FcεRI) is sufficient for high affinity IgE binding.
J. Biol. Chem.
265
:
22079
13
Blank, U., C. Ra, J.-P. Kinet.
1991
. Characterization of truncated a chain products from human, rat and mouse high affinity receptor for immunoglobin E.
J. Biol. Chem.
266
:
2639
14
Toru, H., C. Ra, S. Nonoyama, K. Suzuki, J.-I. Yata, T. Nakahata.
1996
. Induction of the high-affinity IgE receptor (FcεRI) on human mast cells by IL-4.
Int. Immunol.
8
:
1367
15
Pawankar, R., M. Okuda, H. Yssel, K. Okumura, C. Ra.
1997
. Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the FcεRI, CD40L, IL-4, and IL-13, and can induce IgE synthesis in B cells.
J. Clin. Invest.
99
:
1492
16
Xia, H.-Z., Z. Du, S. Craig, G. Klisch, N. Noben-Trauth, J. P. Kochan, T. H. Huff, A.-M. A. Irani, L. B. Schwartz.
1997
. Effect of recombinant human IL-4 on Tryptase, Chymase, and Fcε receptor type I expression in recombinant human stem cell factor-dependent fetal liver-derived human mast cells.
J. Immunol.
159
:
2911
17
Terada, N., A. Konno, Y. Terada, S. Fukuda, T. Yamashita, T. Abe, H. Shimada, K. Ishida, K. Yoshimura, Y. Tanaka, C. Ra, K. Ishikawa, K. Togawa.
1995
. IL-4 upregulates FcεRI α-chain messenger RNA in eosinophils.
J. Allergy Clin. Immunol.
96
:
1161
18
Dombrowicz, D., V. Flamand, K. K. Brigman, B. H. Koller, J.-P. Kinet.
1993
. Abolish of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor α chain gene.
Cell
75
:
969
19
Fung-Leung, W.-P., J. D. Sousa-Hitzler, A. Ishaque, L. Zhou, J. Pang, K. Ngo, J. A. Panakos, E. Chourmouzis, F.-T. Liu, C. Y. Lau.
1996
. Transgenic mice expressing the human high-affinity immunoglobulin (Ig) E receptor α chain respond to human IgE in mast cell degranulation and in allergic reactions.
J. Exp. Med.
183
:
49
20
Matsuda, K., Y. Kobune, C. Noda, A. Ichihara.
1994
. Expression of GATA-binding transcription factors in rat hematocytes.
FEBS Lett.
353
:
269
21
Davis, J. N., M. F. Roussel.
1996
. Cloning and expression of the murine Elf-1 cDNA.
Gene
171
:
265
22
Wang, C.-Y., B. Petryniak, I-C. Ho, C. B. Thompson, J. M. Leiden.
1992
. Evolutionarily conserved Ets family members display distinct DNA binding specificity.
J. Exp. Med.
175
:
1391
23
Wang, C.-Y., A. G. Bassuk, L. H. Boise, C. B. Thompson, R. Bravo, J. M. Leiden.
1994
. Activation of granulocyte-macrophage colony-stimulating factor promoter in T cells requires cooperative binding of Elf-1 and AP-1 transcription facotrs.
Mol. Cell. Biol.
14
:
1153
24
Siegel, M. D., D.-H. Zhang, P. Ray, A. Ray.
1995
. Activation of the interleukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and CLE0 elements.
J. Biol. Chem.
270
:
24548
25
Jenkins, F., P. N. Cockerill, P. Bohmann, M. F. Shannon.
1995
. Multiple signals are required for function of the human granulocyte-macrophage colony-stimulating factor gene promoter in T cells.
J. Immunol.
155
:
1240
26
Thomas, R. S., M. J. Tymms, A. Seth, M. F. Shannon, I. Kola.
1995
. Ets1 transactivates the human GM-CSF promoter in Jurkat T cells stimulated with PMA and ionomycin.
Oncogene.
11
:
2135
27
Karlen, S., M. D’Ercole, C. J. Sanderson.
1996
. Two pathways can activate the interleukin-5 gene and induce binding to the conserved lymphokine element 0.
Blood
88
:
211
28
Tepler, I., A. Shimizu, P. Leder.
1989
. The gene for the rat mast cell high affinity IgE receptor α chain: structure and alternative mRNA splicing patterns.
J. Biol. Chem.
264
:
5912
29
Ye, Z.-S., J.-P. Kinet, W. E. Paul.
1992
. Structure of the gene for the α-chain of the mouse high affinity receptor for IgE (FcεRI).
J. Immunol.
149
:
897
30
Pang, J., G. R. Taylor, D. G. Munroe, A. Ishaque, W.-P. Fung-Leung, C. Y. Lau, F.-T. Liu, L. Zhou.
1993
. Characterization of the gene for the human high affinity IgE receptor (FcεRI) α-chain.
J. Immunol.
151
:
6166
31
Pevny, L., M. C. Simon, E. Robertson, W. H. Klein, S.-F. Tsai, V. D’Agati, S. H. Orkin, F. Costantini.
1991
. Erythroid differentiation in chimeric mice blicked by a targeted mutation in the gene for transcription factor GATA-1.
Nature
349
:
257
32
Simon, M. C., L. Pevny, M. V. Wiles, G. Keller, F. Costantini, S. H. Orkin.
1992
. Rescue of erythroid development in gene targeted GATA-1-mouse embryonic stem cells.
Nat. Gen.
1
:
92
33
Evans, T., G. Felsenfeld.
1989
. The erythroid-specific transcription factor Eryf 1: a new finger protein.
Cell
58
:
877
34
Tsai, S.-F., D. I. K. Martin, L. I. Zon, A. D. D’Andrea, G. G. Wong, S. H. Orkin.
1989
. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mamalian cells.
Nature
339
:
446
35
Martin, D. I. K., L. I. Zon, G. Mutter, S. H. Orkin.
1990
. Expression of an erythroid transcription factor in megakaryotic and mast cell lineages.
Nature
344
:
444
36
Romeo, P.-H., M.-H. Prandini, V. Joulin, V. Migrotte, M. Prenant, W. Vainchenker, G. Marguerie, G. Uzan.
1990
. Megakaryocytic and erythrocytic lineages share specific transcription factor.
Nature
344
:
447
37
Yamamoto, M., L. J. Ko, M. W. Leonard, H. Beug, S. H. Orkin, J. D. Engel.
1990
. Activating and tissue-specific expression of the transcription factor NF-E1 multigene family.
Gene Dev.
4
:
1650
38
Ito, E., T. Toki, H. Ishihara, H. Ohtani, L. Gu, M. Yokoyama, J. P. Engel, M. Yamamoto.
1993
. Erythroid transcription factor GATA-1 is abundantly tranxcribed in mouse testis.
Nature
362
:
466
39
Leiden, J. M., C.-Y. Wang, B. Petryniak, D. M. Markovitz, G. J. Nabel, C. B. Thompson.
1992
. A novel Ets-related transcription factor, Elf-1, binds to human immunodeficiency virus type 2 regulatory elements that are required for inducible trans activation in T cells.
J. Virol.
66
:
5890
40
Thompson, C. B., C.-Y. Wang, I. Ho, P. R. Bohjanen, B. Petryniak, C. H. June, S. Miesfeldt, L. Zhang, G. J. Nabel, B. Karpinsky, J. M. Leiden.
1992
. Cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1.
Mol. Cell. Biol.
12
:
1043
41
Serdobova, I., M. Pla, P. Reichenbach, P. Sperisen, J. Ghysdael, A. Wilson, J. Freeman, M. Nabholz.
1997
. Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor α gene.
J. Exp. Med.
185
:
1211
42
Wurster, A. C., G. Siu, J. M. Leiden, S. M. Hedrick.
1994
. Elf-1 binds to a critical element in a second CD4 enhancer.
Mol. Cell. Biol.
14
:
6452
43
Gottschealk, L. R., D. M. Giannola, S. G. Emerson.
1993
. Moleculat regulation of the human IL-3 gene: inducible T cell-restricted expression requires intact AP-1 and Elf-1 nuclear protein binding sites.
J. Exp. Med.
178
:
1981
44
Grant, P. A., C. B. Thompson, S. Pettersson.
1995
. IgM receptor-mediated trans activation of the IgH 3′ enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.
EMBO J.
14
:
4501
45
Arai, K.-I., F. Lee, A. Miyajima, S. Miyatake, N. Arai, T. Yokota.
1990
. Cytokines: coordinators of immune and inflammatory responses.
Annu. Rev. Biochem.
59
:
783
46
Nimer, S., J. Fraser, J. Richards, M. Lynch, J. Gasson.
1990
. The repeat sequence CATT(A/T) is required for granulocyte-macrophage colony-stimulating factor promoter activity.
Mol. Cell. Biol.
10
:
6084
47
Miyatake, S., J. Shlomai, K.-I. Arai, N. Arai.
1991
. Characterization of the mouse granulocyte-macrophage colony stimulation factor (GM-CSF) gene promoter: nuclear factors that interact with an element shared by three lymphokine gene—those for GM-CSF, interleukin-4 (IL-4) and IL-5.
Mol. Cell. Biol.
11
:
5894
48
Nimer, S., J. Shang, H. Arraham, Y. Miyazaki.
1996
. Transcriptional regulation of interleukin-3 expression in megakaryocytes.
Blood
88
:
66
49
Brown, M. A., J. H. Pierce, C. J. Watson, J. Falco, J. N. Ihle, W. E. Paul.
1987
. B cell stimulatory factor-1/interleukin-4 mRNA is expressed by normal and transformed mast cells.
Cell
50
:
809
50
Plant, M., J. H. Pierce, C. J. Watson, J. Hanley-Hyde, R. P. Nordan, W. E. Paul.
1989
. Mast cell lines produce lymphokines in response to cross-linking of FcεRI or to calcium ionophores.
Nature
339
:
64
51
Paul, W. E., R. A. Seder, M. Plant.
1993
. Lymphokine and cytokine production by FcεRI+ cells.
Adv. Immunol.
53
:
1
52
Henkel, G., M. A. Brown.
1994
. PU. 1 and GATA: components of a mast cell-specific inerleukin 4 intronic enhancer.
Proc. Natl. Acad. Sci. USA
91
:
7737
53
Zon, L. I., M. F. Gurish, R. L. Stevens, C. Mather, D. S. Reynolds, K. F. Austen, S. H. Orkin.
1991
. GATA-binding transcription factors in mast cells regulate the promoter of the mast cell carboxypeptidase A gene.
J. Biol. Chem.
266
:
22948
54
Perez, C., J. Wietzerbin, P. D. Benech.
1993
. Two cis-DNA elements involved in myeloid-cell-specific expression and γ interferon (IFN-γ) activation of the human high-affinity Fcγ receptor gene: a novel IFN regulatory mechanism.
Mol. Cell. Biol.
13
:
2182
55
Gessner, J. E., T. Grussenmeyer, W. Kolanus, R. E. Schmidt.
1995
. The human low affinity immunoglobulin G Fc receptor III-A and III-B genes: molecular characterization of the promoter regions.
J. Biol. Chem.
270
:
1350