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A Novel -66T/C Polymorphism in FcRI -Chain Promoter Affecting the Transcription Activity: Possible Relationship to Allergic Diseases¹

Masanari Hasegawa^*,, Chiharu Nishiyama^2,*, Makoto Nishiyama^¶, Yushiro Akizawa^*, Kouichi Mitsuishi^*,, Tomonobu Ito^*, Hiroshi Kawada^*,, Susumu Furukawa, Chisei Ra^*,||, Ko Okumura^*, and Hideoki Ogawa^*,

^* Atopy (Allergy) Research Center, and Departments of Dermatology and Immunology, Juntendo University School of Medicine, Tokyo, Japan; Department of Pediatrics, Yamaguchi University School of Medicine, Yamaguchi, Japan; ^¶ Biotechnology Research Center, University of Tokyo, Tokyo, Japan; and ^|| Department of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We found a novel polymorphism, -66T/C, in the promoter region of human FcRI, the specific component of the high affinity receptor for IgE (FcRI), which is essential for the cell surface expression of FcRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at -66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (-80/-59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous -66T/C genotype, while most of allergic individuals have homozygous -66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcRI polymorphism related to the allergic diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Allergic (atopic) diseases such as atopic dermatitis (AD)³ and asthma are a major concern as morbidity in developed countries because patients carrying the diseases have been increasing in population. The high affinity IgE receptor, FcRI, composed of three subunits (, , and ) is mainly expressed on the surface of effector cells such as mast cells and basophils. Cross-linking of IgE Abs bound to FcRI by multivalent Ags induces activation of these cells, resulting in secretion of allergic mediators and induction of cytokine gene transcription. Thus, FcRI plays a central role in the induction and maintenance of allergic responses. Until now, a large number of studies on this linkage between the variations in FcRIand allergic diseases has been reported by many research groups, because chromosome 11q13 on which FcRI is mapped was also assigned to relate to atopic diseases (1, 2). In contrast, there are no reports on the polymorphism of FcRI, despite that the -chain is the specific component of FcRI and directly binds to IgE. Involvement of the -chain in FcRI-mediated allergic reaction was definitively proved by the absence of the allergic reaction in -chain-deficient mice (3). Recently, the binding itself of monomeric IgE to FcRI was shown to promote the survival of mast cells without cross-linking of the receptor (4, 5), also suggesting that an increase in FcRI -chain on the cell surface accelerates the IgE-mediated allergic reaction. In addition, a genetic linkage to AD was recently assigned on human chromosome 1q21, which is very close to the chromosomal locus where FcRIis mapped (6). This prompted us to examine that possible polymorphism in FcRI may associate with allergic diseases.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell culture

PT18 (mouse mast cell line), RBL-2H3 (rat mast cell line), and KU812 (human mast cell line) were cultured, as described previously (7).

Plasmid construction

As a reporter plasmid carrying luciferase gene under the control of the -chain promoter (-66T allele), pGV-B2-NN0.6 (-605/+29) or pGV-B2-PN0.1 (-90/+29) was used (7, 8). To obtain -66C allele of promoter, site-directed mutagenesis was conducted on -66T allele of the promoter by using a Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA) and synthetic oligonucleotides: 5'-GTTAACCAGATATGACACAGAAAACATTTC-3' and 5'-GAAATGTTTTCTGTGTCATATCTGGTTAAC-3' (Invitrogen, San Diego, CA).

As an expression plasmid for wild-type GATA-1, pCR-GATA-1 (7, 8) was used.

Transfection and luciferase assay

Harvested cells were suspended in each culture medium supplemented with additional 10% FCS. The cells (5–10 x 10⁶ cells in 0.5 ml) were cotransfected with 5 µg of the test construct and 25 ng of pRL-CMV (Promega, Madison, WI) by electroporation by using Bio-Rad Gene Pulsar II (Hercules, CA) set at 300 V and 950 µF. On the coexpression analysis, 3 µg of pCR-GATA-1 or pCR3.1-self was added into the cell suspension. In this transient expression system, the amount of exogenous GATA-1 increased 10 times more than that of endogenous GATA-1 by Western blotting analysis (data not shown). The measurement of luciferase activity was performed, as described previously (9, 10).

EMSA

GATA-1 proteins were synthesized by using pCR-GATA-1 as the template in vitro. In vitro transcription and translation were performed by using a TNT T7 Quick coupled transcription/translation system, according to the manufacturer’s instructions (Promega) (8).

As the probe for EMSA, double-stranded oligonucleotides were prepared by annealing FITC-labeled oligonucleotides: 5'- TTAACCAGATATGATACAGAAA-3' and 5'-TTTCTGTATCATATCTGGTTAA-3' (probe 1, see Fig. 3B); 5'-TTAACCAGATATGACACAGAAA-3' and 5'-TTTCTGTGTCATATCTGGTTAA-3' (probe 2, see Fig. 3C); 5'-TTAACCACCAATGATACAGAAA-3' and 5'-TTTCTGTATCATTGGTGGTTAA-3' (probe 5'T, see Fig. 3D); and 5'-TTAACCACCAATGACACAGAAA-3' and 5'-TTTCTGTGTCATTGGTGGTTAA-3' (probe 5'C, see Fig. 3D), respectively. EMSA was performed under the same conditions, as described previously (9, 10), by using 1 µl of reaction product prepared with in vitro transcription/translation system, as described above. Anti-GATA-1 Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All gels were subjected to a fluorescence detector, FluorImager 595 (Molecular Dynamics, Sunnyvale, CA).

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Binding profile of GATA-1 protein to -chain promoter with T or C at position -66. A, Nucleotide sequences of oligonucleotides used as competitors 1, 2, 3, and 4. B and C, Binding profile of GATA-1 to probe 1 (-66T allele promoter) and probe 2 (-66C allele promoter), respectively. Lane 1, Probe only; lanes 2–15, probe and in vitro translated GATA-1; lane 3, with anti-GATA-1 Ab; lanes 4-6, with competitor 1; lanes 7-9, with competitor 2; lanes 10-12, with competitor 3; lanes 13-15, with competitor 4. D, Binding profile of GATA-1 to probe 5'T and 5'C. The 5'T and 5'C lacking 5'-GATA motif contain T and C at -66, respectively. Free probes and specific bands corresponding to GATA-1/DNA complex were shown by arrows. Arrows with asterisks were the bands nonspecific to GATA-1 protein.

Flow cytometric analysis of transfectants

Luciferase genes in plasmids pGV-B2-NN0.6–66T and -66C were replaced with human FcRI -chain cDNA obtained by RT-PCR from KU812 mRNA to generate plasmids p-66T- and p-66C- carrying human -chain cDNA under the control of each allele of -chain promoter, respectively. Human FcRI -chain and -chain cDNAs were also cloned by RT-PCR and inserted into pCR-HA (11) (kindly provided from H. Nakano, Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan) to yield pCR-HA- and pCR-HA-, respectively. Nucleotide sequence of each cDNA was determined by an ABI PRISM377 DNA sequencer (Applied Biosystems, Foster City, CA). PT18 cells (2 x 10⁶) were cotransfected with 5 µg of pCR-HA-, 5 µg of pCR-HA-, and totally 10 µg of p-66T- and/or p-66C- by electroporation under the same conditions as described above. After 3-wk cultivation in the presence of 0.5 mg/ml of G418, cell surface expression of human FcRI -chain was analyzed by flow cytometry with FACSCalibur (BD Biosciences, Franklin Lakes, NJ) after staining with FITC-conjugated anti-human FcRI -chain Ab, which is not cross-reactive with mouse -chain (12) or FITC-conjugated mouse IgG2b (isotype control; BD PharMingen, San Diego, CA).

Accession number

Human FcRI, L14075.

Sequencing analysis of human FcRI

Genomic DNA was prepared from peripheral blood by a DNA quick (Dainippon Pharmaceutical, Osaka, Japan). PCR was conducted with the human genomic DNA by using Advantage 2 polymerase (Clontech, Palo Alto, CA) with following two oligonucleotides as primers: 5'-TTAGTTGCTGCTGTTTTATTCTGCTCTCCC-3' (-339/-310) and 5'-TCTAGCTTGGCTCCACTACAGAGTCCCTGG-3' (+237/+208). A thermal cycle of 95°C for 30 s, 65°C for 30 s, and 68°C for 30 s was repeated 30 times after 95°C incubation for 1 min and followed by 68°C for 1 min. After the purification of the PCR product by a MinElute Gel Extraction kit (Qiagen, Valencia, CA), the nucleotide sequences of the DNA fragments were determined by using ABI PRISM377 DNA sequencer (Applied Biosystems). An oligonucleotide 5'-GATAGGGAGTGGAGTAAGTG-3' (-182/-163) was used as the sequencing primer.

Subjects

AD individuals were patients of Juntendo University Hospital. The diagnosis was based on the morphological appearance of skin lesions and clinical course. A physician-administered questionnaire, which included the diagnostic criteria for AD of Hanifin and Rajka (13), was completed for each individual. In brief, the presence of at least three basic features from the following four features: 1) pruritus, 2) typical morphology and distribution, 3) chronic or chronically relapsing dermatitis, and 4) personal or family history of atopy, as listed by Hanifin and Rajka.

We selected as controls those who had no symptoms or history of allergic diseases, as written in Table I, based on physician-administered questionnaire.

View this table: [in this window] [in a new window]   Table I. -66T/C polymorphism in FcRI in Japanese populations

Measurement of FcRI on basophil surface by FACS

Mononuclear cells were enriched from 5 ml of peripheral blood treated with EDTA by using a Percoll-based density-gradient centrifugation technique, as described previously (14). According to the method in a previous report (15), cells were analyzed by flow cytometry with FACSCalibur (BD Biosciences) after staining with PE-conjugated anti-human CD3, CD9, CD14, and CD19 (BD PharMingen); with FITC-conjugated anti-human FcRI -chain Ab, which is noncompetitive with IgE (12); or FITC-conjugated mouse IgG2b (isotype control; BD PharMingen), and mean fluorescence intensity (MFI) is calculated. Total serum IgE was measured by enzyme immunoassay (SRL, Tokyo, Japan), and individuals showing low IgE (<250 IU/ml according to a previous report (16)) were used as control.

Statistical analysis

Statistical analysis of reporter assays (Figs. 1 and 2) and of MFI of FcRI on basophils (Fig. 6A) was performed using the Student t test. The association of the polymorphism at -66 with allergic diseases was analyzed by ² test for T/T vs T/C between the controls and allergy groups. Multiple linear regression analysis was used to test for independent predictors of MFI by entering genotypes, and total serum IgE into the model (SPSS version 11.0; SPSS, Chicago, IL). A p value less than 0.05 was considered statistically significant. The values of MFI and IgE were shown as mean ± SEM.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Effect of -66T/C polymorphism on the transcription activity of human FcRI -chain promoter. A, Nucleotide sequences of human FcRI -chain promoter (-80/-50). B, PT18 cells or RBL-2H3 cells were transiently transfected with pGV-B2-NN0.6–66T, pGV-B2-NN0.6–66C, or pGV-B2 (=pGL3-Basic). The relative luciferase activity is represented as the ratio of the activity to the luciferase activity in the cells transfected with the empty control vector pGV-B2. Each experiment was conducted in duplicate for each sample, and the results are expressed as mean +SE for three independent experiments in this figure and Fig. 2. *, p < 0.05; **, p < 0.01; ***, p < 0.005, as determined by a paired t test.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Effect of overexpression of GATA-1 on T allele or C allele promoter activity. A total of 5 µg of each of reporter plasmids, pGV-B2, pGV-B2PN0.1–66T, pGV-B2PN0.1–66C, or pGV-P2 (=pGL3-Promoter (SV40)), was transiently transfected with 3 µg of pCR3.1-self (mock) or pCR-GATA-1 to KU812 cells. The relative luciferase activity is represented as the ratio of the activity to the luciferase activity in the cells transfected with the empty control vector pGV-B2 and pCR3.1-self. *, p < 0.001.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Cell surface expression of FcRI on basophils from peripheral bloods. A, Basophils from peripheral bloods of T/C control (n = 6), T/T control (n = 8), and T/T patients (n = 5) were stained with FITC-conjugated anti-human FcRI -chain Ab, which is noncompetitive with IgE (see Materials and Methods). *, p < 0.05; **, p < 0.005, as determined by a paired t test. B, MFI vs concentration of serum IgE of each individual. Open circle, T/T control; closed circle, T/C control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

The -66T/C polymorphism affecting the transcription activity of -chain promoter

We previously determined the nucleotide sequence of a full-length genomic DNA encoding human FcRI -chain (17, 18) and analyzed the -chain promoter (7, 8) with T at position -66. By sequencing analysis of several genomic DNA clones in the present study, we found T/C polymorphism at -66. Because the polymorphism was found at the promoter region, we examined the role of the single nucleotide at -66 for the promoter function. We then constructed two reporter plasmids, each of which carried either of the both types of the promoter (-605/+29) in front of the luciferase gene, and analyzed their transcription activity by transient reporter assay in the -chain-positive cells, PT18 and RBL-2H3. As shown in Fig. 1, luciferase activity derived from the -66T promoter was significantly higher than that from the -66C promoter in both of the cell lines. Similar results were obtained, when human cell line KU812 was used for the transient reporter assay, although the promoter activity detected in KU812 was lower than those in other two cell lines (data not shown).

The above-mentioned results suggested that nucleotide T at -66 was critical for showing the strong transcription activity of the FcRI -chain promoter. Interestingly, in the promoter with -66T, an additional GATA-1-like motif (3'-GATA) is present at -68/-65, although the sequence, TGATAC, is not completely identical with the optimum sequence (A/T)GATA(A/G) for GATA-1 binding (19). To confirm the involvement of GATA-1 in -chain promoter activity, we performed coexpression experiments. The luciferase activity derived from -66T promoter was 4-fold higher in the presence of overproduced GATA-1, and that from -66C promoter was increased about only 2-fold (Fig. 2). Thus, -66T promoter responded to GATA-1 better than -66C promoter.

Binding profile of GATA-1 to DNA around -66

We next performed EMSA using several labeled and nonlabeled oligonucleotides. Specific binding between GATA-1 protein and probe (-80/-59) was observed with both probe 1 (-66T allele, lane 2 in Fig. 3B) and probe 2 (-66C allele, lane 2 in Fig. 3C). When nonlabeled wild-type (-66T) double-stranded oligonucleotide, competitor 1, was added as the competitor, 50-fold excess of the competitor was sufficient for disappearing the specific bands (lane 6 in Fig. 3, B and C). In contrast, both the competitors, competitor 2 lacking 5'-GATA motif and competitor 3 lacking 3'-GATA motif, inhibited the binding between GATA-1 and the wild-type probe, but the inhibitory activity was obviously lower than that of competitor 1. It should be noted that the band intensity in the presence of competitor 2 or 3 was always weaker for probe 2 (Fig. 3, B and C). This suggests that GATA-1 has higher affinity to probe 1 than to probe 2. To confirm the possibility that 3'-GATA can be also recognized by GATA-1, we performed EMSA by using probes 5'T and 5'C, which carried GATA and GACA at 3'-GATA site, -68/-65, respectively, but lacked 5'-GATA motif. As shown in Fig. 3D, shift band was observed with probe 5'T (lane 3), but not with probe 5'C (lane 4). From these observations, it was concluded that 3'-GATA-1 motif of -66T allele had a potential to be recognized by GATA-1, although the affinity was lower, compared with that of 5'-GATA. Actually, Scatchard plot analysis (20, 21) using the data of EMSA indicated that the binding affinity of GATA-1 for T allele probe was about 2 times higher than that for C allele probe, and the affinity for T allele probe lacking 5'-GATA was significantly lower than that for T allele probe, but still retained in the same order of magnitude (1/4 for T allele probe). These results indicate that 3'-GATA itself possesses the binding potential to GATA-1 and increases the binding affinity of GATA-1 for the FcRI -chain promoter.

Effect of T/C polymorphism for cell surface expression of FcRI -chain

To confirm whether -66T/C polymorphism affected cell surface expression of FcRI, we introduced the plasmids p-66T- and p-66C-, which carry -66T and -66C promoters followed by human -chain cDNA, respectively, along with expression vectors for human - and -chains into PT18 cells, and analyzed cell surface expression of FcRI by an anti-human FcRI -chain Ab, which recognizes human -chain specifically, but not rodent -chain. By FACS analysis, slight, but apparent expression of human -chain protein was detected on surface of PT18 cells transfected with these plasmids (Fig. 4). The highest expression of human -chain was observed when only a plasmid carrying -66T promoter was transfected, whereas introduction of a plasmid carrying -66C promoter resulted in faint expression of the -chain. Cotransfection with the plasmids carrying -66T and -66C promoters gave intermediate expression in comparison with the cases of -66T promoter alone and -66C promoter alone. These results indicated that T/C polymorphism actually affected the cell surface expression of FcRI -chain.

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Effect of the -66T/C polymorphism on cell surface expression of human FcRI -chain. Each plasmid carrying -66T or -66C promoter followed by human FcRI -chain cDNA (p-66T-, or p-66C-) was cotransfected with both pCR-HA- and pCR-HA-. After 3-wk cultivation in the presence of 0.5 mg/ml of G418, cell surface expression of human FcRI -chain was analyzed with anti-human -chain Ab (18 ) by FACS. No human -chain; cells transfected with 10 µg each of pCR-HA- and -, but not with plasmids encoding -chain, C/C; cells cotransfected with 10 µg each of p-66C-, pCR-HA-, and -, C/T; cells cotransfected with 5 µg each of p-66C- and p-66T-, and 10 µg each of pCR-HA- and -, T/T; cells cotransfected with 10 µg each of p-66T-, pCR-HA-, and -. Similar result was observed in other independent experiments.

Polymorphism in FcRI -chain gene regulatory region

As described above, the transcription activity of FcRI -chain promoter was influenced by a single nucleotide substitution at -66. To elucidate possible relationship between -66T/C polymorphism and atopic diseases, we next preliminarily analyzed the population with -66T/C polymorphism in Japanese individuals (Fig. 5). We sequenced 350 bp of FcRI, in which we found most of all the elements responsible for FcRI -chain gene expression in our previous study (7, 8, 22), and found polymorphism only at -66 (Fig. 5A). To elucidate the relationship between the polymorphism and allergic diseases, statistic analysis was performed. As shown in Table I, T/T was major (46 of 54, 85.2%), while T/C was minor (8 of 54, 14.8%) in population. Most (96.2%) of the patients with AD carried T/T at -66. Surprisingly, a significant portion (25.0%) of control individuals without allergic diseases, such as AD, atopic asthma, and allergic rhinitis, had T/C at this position. Frequency of heterozygous -66T/C was further increased (33.3%), when individuals without any symptoms or history of allergic diseases for allergic conjunctivitis, pollinosis, contact dermatitis, and family history in addition to above three allergic diseases were chosen. In both calculations, significantly small p values (<0.05) were obtained. These observations suggest that this polymorphism is possibly related to allergic diseases, and that the individuals (in Japanese population) carrying the -chain promoter with C allele at -66 are apt to be resistant to allergy.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Sequencing analysis of human FcRI. A, Nucleotide sequencing analysis was performed on 5'-flanking region, exon 1, and a part of intron 1. B, DNA sequence electrophoregrams of the polymorphism at -66 of FcRI with T/T and T/C. Arrows indicate the polymorphic sites.

Cell surface expression of FcRI on basophils from peripheral bloods of T/T and T/C individuals

To evaluate the polymorphism in cell surface expression of FcRI, we measured the amount of FcRI on cell surface of basophils from 14 volunteers (6 T/C and 8 T/T) of nonatopic individuals and 5 patients of AD, all of which are T/T genotype. No basophils from AD patients with T/C genotype were subjected to this analysis, due to extremely low allelic frequency of T/C in AD patients (see Table I). As shown in Fig. 6A, the average of MFI of T/C (399 ± 89) is lower than that of T/T containing both T/T patients and T/T control (762 ± 77) (p < 0.01). When MFI is compared between T/T patients and T/T control, the average of T/T patients (991 ± 100) is higher than that of T/T control (620 ± 52) (p < 0.005). The average of T/T control (620) is significantly higher than that of T/C (399) (p < 0.05). We also measured concentration of serum IgE, because serum IgE affects cell surface expression of FcRI. A significant difference was not observed in the average of IgE value between T/C control (62 ± 27) and T/T control (72 ± 19) (p = 0.73), whereas T/T patients showed a markedly increased concentration of serum IgE (5568 ± 1410). We plotted MFI against serum IgE concentration for each individual of T/T control and T/C control (Fig. 6B), and analyzed these data using a multiple linear regression model, to evaluate the effect of -66T/C polymorphism and serum IgE level on MFI. According to this analysis, a multiple linear regression equation was obtained (R² = 0.687, adjusted R² = 0.630, and p = 0.0017), and -66T/C polymorphism (p = 0.030) and concentration of serum IgE (p = 0.0036) were significant independent predictors for MFI. From these observations, we conclude that basophils from AD patients with T/T allele express FcRI on the cell surface at the highest level probably due to high concentration of serum IgE, and that those of T/T allele control express higher amount of cell surface FcRI than those of T/C allele control, independently of serum IgE level.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Among three subunits, , , and , the gene encoding is mapped on chromosome 11q13. Because this region was also assigned to be related to atopic diseases (1), a large number of studies on this linkage between the variations in -chain gene and allergic diseases has been performed by many research groups (2). In contrast, there are no reports on the polymorphism of FcRI. In this study, however, we found a T/C polymorphism in the promoter at the nucleotide position of -66, and demonstrated that the promoter with -66T had stronger activity than that with -66C and the allelic frequency was associated with allergic diseases in the statistic analysis. Considering that -chain is the FcRI-specific molecule and essential for both functional expression of FcRI on the cell surface and binding to IgE Ab, it is probable that the polymorphism found in the -chain gene is related to allergic diseases. Recently, a genetic locus related to AD was mapped on chromosome 1q21, which is very close to 1q23 on which FcRI -chain gene is mapped (6). In that study, Cookson et al. also mapped determinants for the amount of total serum IgE on chromosome 16q-tel and weakly on chromosome 5 as well. These observations suggest that the increase in the amount of total serum IgE is not always a direct cause of the AD, but that possible increase in the FcRI -chain expressed on the cell surface may enhance the response to IgE Abs.

Recently, a single nucleotide polymorphism was found in the proximal CD14 promoter that is related to the expression level of total serum IgE. In that case, the promoter activity was modified by affecting the binding affinity for the promoter of Sp3, which regulated CD14 promoter repressively (23). The polymorphism in the promoter region of the -chain gene may become a similar example found in allergy-related genes. For further study of the relationship between T/C polymorphism and allergic diseases, it is necessary to analyze the polymorphism in a large number of individuals with allergic diseases in various countries, because the allele frequency of the -chain promoter may vary in other countries, as is the case for -590C/T polymorphism of IL-4 promoter (24).

The promoter with T at -66 displayed increased activity to initiate transcription, compared with the promoter with C at -66. Because GATA-1 binds to T allele promoter with the affinity higher than that to C allele promoter, it is likely that the difference in GATA-1-binding affinity is attributed to the difference in the promoter activity. Palindromic or repetitive GATA sites were found in several promoters and were shown to have higher GATA-1-binding affinity than a promoter with a single GATA site (20). Even in such cases, however, only a single GATA-1 protein could bind to the promoter, probably because of steric hindrance of the GATA-1 to its neighboring GATA motif. Then, a question is evoked; that is, how two GATA-1 binding sites serve to increase the affinity and up-regulate the FcRI -chain promoter activity. Recently, it has been shown that N-finger that has been thought to facilitate the formation of specific and stable DNA/C-finger complex has the DNA-binding ability (25). Based on the available information on structure and function of GATA-1, we speculate that specific binding of C-finger to 5'-GATA site could spatially dispose N-finger near 3'-GATA site to bind, and the possible weak binding between N-finger and 3'-GATA could be sufficient to enhance the affinity of GATA-1 to the promoter DNA. For elucidation of the mechanism for GATA-1-mediated up-regulation of the T allele promoter, three-dimensional structure of whole GATA-1/DNA (-66T) complex will be obviously required.

Comparison of cell surface expression of FcRI on basophils using MFI as the index revealed that AD patients possessed higher degree of cell surface FcRI than those of control individuals, which is consistent with other studies showing the elevated cell surface expression of FcRI in allergic patients (26). To eliminate the possible effect of IgE for increasing expression of FcRI, we compared cell surface expression of FcRI between T/C and T/T allele in nonatopic individuals with low IgE (<250 IU/ml) and found apparently higher surface expression in T/T control (620 ± 52) than that in T/C control (399 ± 89). Furthermore, the multiple linear regression analysis revealed that -66T/C polymorphism is actually related to cell surface expression of FcRI, irrespective of serum IgE level. All these data suggest that -66T/C polymorphism at FcRI, which affects the expression of FcRI -chain, could be one of the determinants for allergic diseases. To clarify the relationship between -66 polymorphism and atopic diseases, further detailed analysis with a numerous number of individuals including atopic patients of T/C genotype will be required. We also think that it is necessary to find polymorphism in other loci of the genome and analyze its association with -66T/C polymorphism for further elucidation of genomic background for allergic diseases.

	Acknowledgments

 
We are grateful to members of Biotechnology Research Center (University of Tokyo), and Atopy Research Center, Department of Dermatology, and Department of Immunology (Juntendo University School of Medicine) for providing peripheral blood. We are also grateful to Drs. Hiroyasu Nakano (Department of Immunology, Juntendo University School of Medicine), Atsuhito Nakao (Atopy Research Center, Juntendo University School of Medicine), and Takaomi Yasuhara (Asahi Breweries, Ibaraki, Japan) for helpful discussions. We thank Dr. Yutaka Kanamaru for technical support, Tomoko Tokura and Haruka Nishiyama for technical assistance, and Michiyo Matsumoto and Emiko Kawasaki for secretarial assistance.

	Footnotes

 
¹ This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to C.N.).

² Address correspondence and reprint requests to Dr. Chiharu Nishiyama, Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. E-mail address: chinishi{at}med.juntendo.ac.jp

³ Abbreviations used in this paper: AD, atopic dermatitis; MFI, mean fluorescence intensity.

Received for publication April 26, 2002. Accepted for publication June 9, 2003.

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