Regulation of human Fc{epsilon}RI ß chain gene expression by Oct-1

Yushiro Akizawa1,2, Chiharu Nishiyama1, Masanari Hasegawa1, Keiko Maeda1, Tatsutoshi Nakahata3, Ko Okumura1, Chisei Ra1,4 and Hideoki Ogawa1

1 Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 2 Pharmacobioregulation Research Laboratory, Hanno Research Center, Taiho Pharmaceutical Co. Ltd, 1-27, Misugidai, Hanno-shi, Saitama 357-8527, Japan 3 Department of Pediatrics, Faculty of Medicine, Kyoto University, 54 Shogoin Sakyo, Kyoto 606-8507, Japan 4 Present address: Advanced Medical Research Center, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan

Corresponding to: C. Nishiyama; E-mail:chinishi{at}med.juntendo.ac.jp
Transmitting editor: S. Koyasu


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The ß chain, a component of the high-affinity receptor for IgE (Fc{epsilon}RI), plays an important role in IgE-mediated allergic reaction. The ß chain accelerates the function of Fc{epsilon}RI by amplification of its surface expression and of signal transduction in effector cells such as mast cells and basophils. Two regulatory regions, +60/+66 and +70/+76, for the human ß chain gene that are required for the cell-type-specific transcriptional activation were identified by transient reporter assay. Electrophoretic mobility shift assay demonstrated that Oct-1 binds both the regions, among which the +70/+76 Oct-1 motif was critical for the transactivation of the ß promoter responsive to Oct-1 overexpression. Regulation of ß chain gene expression is discussed.

Keywords: allergy, IgE receptor, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The high-affinity IgE receptor (Fc{epsilon}RI) composed of three subunits, {alpha}, ß and {gamma} chains, plays an important role in allergic reactions mediated by antigen-specific IgE antibodies. The ß chain is believed to be unnecessary for the expression of human Fc{epsilon}RI on the cell surface, because Fc{epsilon}RI is expressed as a trimer composed of one {alpha} and two {gamma} chains in monocytes (1), eosinophils (2), Langerhans cells (3,4) and dendritic cells (5), although the human Fc{epsilon}RI in mast cells and basophils is a tetramer of one {alpha}, one ß and two {gamma} chains. However, it was recently reported that the ß chain enhances the expression of Fc{epsilon}RI on the cell surface by association with the {alpha} chain (6). In addition, the ß chain also amplifies signal from the immunoreceptor tyrosine-based activation motif of the {gamma} chain (7,8). The ß chain is thus a key molecule in the allergic reaction. The ß chain is known to be one of the genes causing atopic diseases. Fc{epsilon}RIß is mapped on chromosome 11q13, the locus of which was assigned to be related to atopic diseases (9). Several polymorphisms of Fc{epsilon}RIß have been identified (1013) by a large number of studies on this linkage between the variations in Fc{epsilon}RIß and allergic diseases. However, at least, amino acid variations due to polymorphic missense mutations so-far identified do not show any detectable difference in its function in vivo (14,15). This may suggest that the polymorphism which affects the transcription and/or translation could be a candidate for the diseases. However, mechanisms for the expression of the ß chain have not yet been investigated.

In this study, we analyzed transcriptional regulatory elements for human Fc{epsilon}RI ß chain gene expression by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay, and demonstrated that Oct-1 transactivates the ß chain promoter by recognizing its binding site present in the 5'-untranslated region (5'-UT) of the gene.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines
The human mast cell line HMC-1 was established from a patient suffering from mast cell leukemia and was provided by Dr J. H. Butterfield (Mayo Clinic, Rochester, MN). HML/SE is a cytokine-dependent cell line established from childhood acute megakaryoblastic leukemia (16). KU-812 and HeLa cell lines were purchased from Riken Cell Bank (Tsukuba, Japan). HMC-1 cells were cultured in Iscove’s DMEM. HML/SE and KU-812 cells were cultured in RPMI 1640. HeLa cells were cultured in DMEM. All media were supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 10% FCS. For the cultivation of HML/SE cells, 5 ng/ml recombinant human granulocyte macrophage colony stimulating factor (Genzyme Techne, Minneapolis, MN) was added to the medium.

Cloning of the human ß chain promoter region
Plaque hybridization against a human genomic library (Stratagene, La Jolla, CA) was carried out to obtain the human ß chain genomic DNA by using a portion of the human ß chain cDNA as the probe. The probe DNA was labeled by the ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). Among five hybridization-positive clones, we chose a single clone and isolated a DNA fragment containing a transcription start site for the ß chain gene as an EcoRI–BamHI fragment of 1.6 kb which was inserted into EcoRI–BamHI-digested pBluescript II SK+ (Stratagene). The nucleotide sequence of the region containing the putative human ß chain gene promoter was determined by the dideoxy chain termination method and registered in GenBank/EMBL/DDBJ under accession no. AB080913.

RT-PCR analysis
Total RNA from each cell line was purified using TRIzol reagent (Invitrogen, Leek, The Netherlands). By using total RNA, Superscript II (Invitrogen) and oligo(dT) primer (Invitrogen), the reverse transcription reaction was performed according to the supplier’s instructions. One microliter from 40 µl of the reverse transcription reaction mixture was used as the template to detect the expression of Fc{epsilon}RI ß chain mRNA. For this purpose, the synthetic oligonucleotides shown below were used as PCR primers. Primer sets were: 5'-AGAGCAAATCTTGCTCTCCC-3' / 5'-GGTGAGAAACAGCATCATCA-3' for the human Fc{epsilon}RI ß chain and 5'-GCGCTCGTCGTCGACAACGG-3'/5'-CATCGGAACCGCTCATTGCC-3' for ß-actin. A thermal cycle of 94°C for 30 s, 62°C for 30 s and 72°C for 1 min was repeated 30 times.

Reporter gene constructs
Reporter plasmids carrying the luciferase gene under the control of various length of the human ß chain promoter were generated as follows.

Nucleotide replacement was performed to introduce a NcoI site at the translation start site of the ß chain genomic DNA fragment inserted in pBluescript II SK+ by using a Quick change site-directed mutagenesis kit (Stratagene) with oligonucleotide 5'-CGGTTAATGAAAccATGGACACAGA-3' (NcoI sites are shown as bold, replaced nucleotides are in lower case letters and the translational start site are underlined) and its complementary oligonucleotide. An NcoI fragment –1079/+102 was inserted into NcoI-digested pGL3-Basic (Promega, Madison, WI). The resulting plasmid with the insert in the correct orientation is named pGLß(–1079/+102).

To obtain plasmids, pGLß (–950/+102), pGLß (–750/+102), pGLß (–500/+102), pGLß (–227/+102) and pGLß (–95/+102), PCR was carried out using pGLß(–1079/+102) as the template. The following oligonucleotides (the added XhoI site is shown as bold), 5'- GAGCTCGAGCCACCAAAAAAACCACAC-3' (–950/–933), 5'-GAGCTCGAGTAGGCATACTACTCAATC-3' (–750/–733), 5'-GAGCTCGAGAAAAGACGGAAAG AGAGA-3' (–500/–483), 5'-GAGCTCGAGCTCCTACTAAAATGTCTC-3' (–227/–210) and 5'-GAGCTCGAGATCACAAGTAAAAGCCTG-3'(–95/–79), were used as the sense primers, and 5'-CTTTATGTTTTTGGCGTCTTCC-3' (GLprimer2; Promega) was used as antisense primer. The resultant PCR products were inserted into pGL3-Basic after XhoI–NcoI digestion. pGLß(–352/+102) was constructed from pGLß(–1079/+102) by the deletion of the HindIII fragment. To obtain pGLß(–95/+48), a NcoI site was introduced at +48/+53 of pGLß(–95/+102) by site-directed mutagenesis, and a fragment isolated from the resultant plasmid by XhoI (in MCS) and NcoI (newly introduced) digestion was inserted into XhoI/NcoI-digested pGL3-Basic. A series of the mutants, m1–m6, in which nucleotide substitutions of 2–6 bp were introduced, was constructed by site-directed mutagenesis using pGLß(–95/+102) as the template.

All constructs were subjected to sequencing analysis to verify the nucleotide sequence and orientation.

Transient expression and luciferase assay
Cells were transfected by electroporation as previously described (17,18). Briefly, HMC-1 (0.25–0.5 x 107cells), KU-812 (0.5–1 x 107cells), HML/SE (1.5–3 x 107cells) and HeLa (0.25–0.5 x 107cells) were electroporated at 950 µF and 300 V with 5 µg reporter plasmid in 20% FCS/RPMI 1640. pRL-CMV (5 ng; Promega) was also introduced into these cells as a control construct for normalizing the transfection efficiency. The pGL3-Promoter (Promega) and the promoter-less pGL3-Basic vectors were used as the positive and negative controls respectively. After electroporation, HML/SE cells were incubated for 6 h and the other cells were incubated for 24 h in culture medium. The cells from 1 ml of culture medium were lysed with 0.1 ml of reporter lysis buffer (Promega). Then the luminescence was measured by a microplate luminometer LB96V (Berthold, Postfach, Germany) using 20 µl from 0.1 ml of the cell lysate.

Co-expression study
The coding region of Oct-1 cDNA (accession no. X13403) was amplified by PCR using 5'-GAATTCATGAACAATCCGTCAGAAACCAGT-3' (the EcoRI site shown in bold was attached to +60/+83 of Oct-1 cDNA) as the sense primer and 5'-CTCGAGTCACTGTGCCTTGGAGGCGGTGGT-3' (the XhoI site shown in bold was attached to complementary sequence of +2291/+2268) as the antisense primer. The resultant PCR product was cloned into EcoRI–XhoI-digested pCR-2F expression plasmid [kindly provided from Dr Nakano (19)] to generate pCR-2F-Oct-1 which carries 2 x Flag-tagged Oct-1 cDNA. KU-812 cells were co-transfected with the pCR-2F-Oct-1 or pCR-2F and each reporter plasmid carrying wild-type or mutant ß chain promoter, and the luciferase activities were measured after 24 h incubation as described above.

EMSA
The following oligonucleotides labeled with FITC at the 5' site were used as the probes for EMSA (only the nucleotide sequence of the sense strand of each probe is shown): probe A (+54/+69, 5'-CTCAATATAATAATAT-3'), probe B (+68/+83, 5'-ATTCTTTATTCCTGGA-3') and probe C (+84/+102, 5'-CAGCTCGGTTAATGAAAAA-3'). The nucleotide sequences of non-labeled oligonucleotides used as the competitor are shown in Fig. 5(A and B). Nuclear extracts and double-stranded oligonucleotides probes were prepared according to the previously reported method (17,18). In vitro transcription and translation to prepare Oct-1 protein were performed with the TnT T7 Quick coupled transcription/translation system (Promega) according to the manufacturer’s instructions, using pCR-Oct-1, which was generated by insertion of full-length Oct-1 cDNA prepared from pCR-2F-Oct-1 by EcoRI–XhoI digestion into pCR3.1 as the template for the reaction. One microliter out of 50 µl of the in vitro reaction mixture was used for EMSA. The FITC-labeled probe was mixed with nuclear extracts (5 µg) in 10 mM HEPES (pH 7.9) containing 100 µg/ml poly(dG–dC), 1 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol and 5% glycerol, and incubated in the presence or absence of the competitor probes for 20 min at room temperature. For the supershift assay, the mixture was incubated with 1 µg of antibodies for the transcription factor. The reaction mixtures were then subjected to 4% polyacrylamide gel for electrophoresis. The gel was analyzed with a FluorImager 595 (Molecular Dynamics, Sunnyvale, CA).





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Fig. 5. Nuclear proteins binding to the critical elements in the 5'-UT of the human ß chain gene. (A) Identification of nuclear protein binding motif A using competitors and antibodies. EMSA was performed with FITC-labeled probe A and nuclear extract from HMC-1. Lane 1, probe only; lanes 2–25, probe with nuclear extracts; lanes 3–5, 6–8, 9–11, 12–14, 15–17 and 18–20, with competitor WT1, m54, m57, m60, m63 and m66 respectively; lanes 21–25, with anti-Oct-1, -Oct-2, -Oct-4, -PU.1 and -Elf-1 antibodies respectively. (B) Identification of nuclear protein binding motif B using competitors and antibodies. EMSA was performed with a FITC-labeled probe B and nuclear extract from HMC-1. Lane 1, probe only; lanes 2–25, probe with nuclear extracts; lanes 3–5, 6–8, 9–11, 12–14, 15–17 and 18–20, with competitor WT2, m69, m72, m75, m78 and m81 respectively; lanes 21–25, with anti-Oct-1, -Oct-2, -Oct-4, -PU.1 and -Elf-1 antibodies respectively. (C) Mobility profile of in vitro translated Oct-1. EMSA with probe A. Lanes 2 and 3, with nuclear extract; lanes 4 and 5 with in vitro translated Oct-1. Lanes 1, 2 and 4, without antibody; lanes 3 and 5, with anti-Oct-1 antibody.

 

    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Analysis of the transcriptional regulatory region of the human ß chain gene
By plaque hybridization against a human genomic library, we cloned a DNA fragment containing the Fc{epsilon}RI ß chain gene. Determination of the nucleotide sequence of the 5'-non-coding region (1.2 kb) of the ß chain gene and its analysis using a MOTIF search service (http://motif.genome.ad.jp) revealed the presence of possible DNA-binding sites for several transcription factors, Oct-1, GATA-family, AP-1, AP-4 and NF-AT, in the region (Fig. 1).



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Fig. 1. Nucleotide sequence of the 5'-flanking region of the human ß chain gene. The transcription initiation site is indicated as +1 (33). Possible transcription factor binding sites (found by publicly available MOTIF search service) and TATA box (33) are shown.

 
The human ß chain is known to be expressed in limited cells such as mast cells, basophils and megakaryocytes. In order to confirm the expression of the ß chain gene in human cell lines, HMC-1 and KU-812 (mast cell lines) and HML/SE (megakaryocyte cell line), which were cultivated in our laboratory, we performed RT-PCR analysis, and found that the transcript was detected in HMC-1, KU-812 and HML/SE cells, but not in HeLa cells (Fig. 2). To identify the regulatory elements required for the cell-type-specific expression of the human ß chain gene, we generated a series of deletion constructs in which 5'-upstream region of ß chain gene was connected in various lengths at upstream of the luciferase gene and measured the luciferase activity in HMC-1, KU-812, HML/SE or HeLa cells transfected with these reporter plasmids. As shown in Fig. 3, the longest region (–1079/+102) showed promoter activity slightly higher than that of the basic control. Deletion of the –1079/–951 region had no effect on the promoter activity. On the other hand, the promoter activity was gradually increased as the 5' deletion increased. These results suggest that suppressive elements are present between –950 and –96 of the ß chain gene. The pGLß(–95/+102) gave distinct luciferase activity in every ß chain+ cell line, but not in HeLa cells. This indicates that –95/+102 region contains the promoter of the Fc{epsilon}RI ß chain and elements required for the cell-type-specific expression. Unexpectedly, further deletion of +49/+102 region reduced the reporter activity nearly equal to that of the promoter-less construct, suggesting that positive cis-acting element(s) is present in the 5'-UT region of the ß chain gene.



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Fig. 2. RT-PCR analysis for human ß chain expression in each cell line. Ten microliters from each 25 µl of PCR reaction mixture was applied onto 1% agarose gel and stained with ethidium bromide.

 


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Fig. 3. Determination of the cell-type-specific cis-enhancing region in the human ß chain gene. The relative promoter activity is represented as the ratio to the promoter activity of pGL3-Basic, and the results expressed as the mean ± SD for more than three independent experiments conducted in duplicate for each sample in Figs 3, 4 and 6.

 
In the region from +49 to +102, three putative binding sites of Oct-1 were found [motif A (+57/+69), motif B (+67/+79) and motif C (+89/+101)] (Fig. 1). To examine the role of these putative Oct-1 binding sites for the regulation of the promoter, we generated a series of plasmids carrying the ß chain gene in which nucleotide substitutions of 2–6 bp were introduced to pGLß(–95/+102) around its putative Oct-1 binding sites (Fig. 4). The luciferase activities directed by pGLß(–95/+102)m2, m3 and m4 were markedly decreased to a level nearly equal to that of the promoter-less construct. pGLß(–95/+102)m5 conferred moderately decreased luciferase activity. However, the nucleotide substitutions at +57/+59 and +92/+96 had slight or negligible effects in each cell line. These results suggest that the region from +61 to +79 containing motif A and motif B is required for initiating human ß chain transcription, but the region containing motif C was not essential (Fig. 4).



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Fig. 4. Determination of the cis-enhancing elements in the 5'-UT of the human ß chain gene by site-directed mutagenesis. Mutations were introduced into +54/+102 of the ß chain cis-enhancing region of reporter plasmid pGLß(–95/+102) carrying the luciferase gene under the control of the wild-type minimum promoter. The nucleotides that differ from the original are shown and lines represent unchanged nucleotides in mutant plasmids. Motifs A, B and C are shown underlined.

 
EMSA for Oct-1 binding
The above-mentioned results suggest that Oct-1 binds to putative Oct-1 motifs (motif A and motif B) to activate the ß chain promoter. To confirm the binding between Oct-1 and these Oct-1 motifs, we performed EMSA using nuclear extract from HMC-1. EMSA with the 16-bp double-stranded oligonucleotides containing motif A showed several shift bands (Fig. 5A, lane 2). Among these bands, the most slowly migrating one was shifted by binding of Oct-1, because addition of anti-Oct-1 antibody caused a supershift of this band. We briefly discuss the other bands later (see Fig. 7 and Discussion). For further identification of the recognition sequence of Oct-1, we performed competition assays by using 5-, 10- or 20-fold excess amounts of the unlabeled wild-type or mutant oligonucleotide. The most slowly migrating band disappeared when the mixture was incubated in the presence of WT1, m54, m57 or m66, in a dose-dependent manner (Fig. 5A, lanes 3–11 and 18–20). On the other hand, addition of m60 or m63 was less effective to compete with the labeled probe (Fig. 5A, lanes 12–17). These results indicate that ATAATA (+60/+65) served as a core sequence for the binding of the nuclear protein. Since Oct-1 is known to recognize 5'-(A/T/G)TAAT(A/GA)-3' or 5'-ATGCAAAT-3' as a core sequence (20,21), the results suggest that Oct-1 actually binds to motif A via ATAATAA (+60/+66) in vitro.



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Fig. 7. EMSA profile using a probe A and nuclear proteins derived from ß chain gene positive and negative cells. Lane 1, HMC-1; lane 2, KU-812; lane 3, HML/SE; and lane 4, HeLa. Two specific bands observed by addition of ß chain+ cell nuclear extracts are shown with arrows.

 
Similar EMSA using probe B showed that Oct-1 also bound to the probe by recognizing 5'-TTTATT-3' (+72/+77), although the variation in the shifted band was less than that of EMSA using probe A (Fig. 5B). This observation suggests that Oct-1 binds to motif B with the complementary sequence of 5'-TCTTTAT-3' (+70/+76). To confirm that Oct-1 bound to these Oct-1 motifs, we performed EMSA with Oct-1 prepared by an in vitro transcription/translation system. As shown in Fig. 5(C), the specific shift band showing mobility identical to that seen with the nuclear protein was generated when probe A was used (Fig. 5C, lanes 2 and 4). Furthermore, disappearance of this specific band as well as appearance of a supershift band were observed at the same time in the presence of anti-Oct-1 antibody as seen in the case where nuclear proteins were added (Fig. 5C, lanes 3 and 5). Oct-1 also bound to probe C by recognizing +92/+97 in vitro (data not shown), although the binding was not essential for the promoter activation in vivo (Fig. 4). Shifted bands shown with an asterisk in Fig. 5(A and B) disappeared by addition of competitive oligonucleotides with a similar profile to that of Oct-1, suggesting that the binding of Oct-1 to its consensus sequences could be critical for recruiting uncharacterized binding proteins causing specific bands, alternatively uncharacterized binding proteins recognize closely located regions of Oct-1 binding sites.

Transactivation of human ß chain promoter by Oct-1 via the +72/+77 Oct-1 site
The above results suggest that Oct-1 positively regulates the expression of the ß chain gene. To confirm the transactivation of the ß chain promoter by Oct-1, we examined the effect of overexpression of Oct-1 on the ß chain promoter by co-expression study. As shown in Fig. 6(A), the luciferase activity was increased by co-transfection of the Oct-1 expression plasmid in a dose-dependent manner. By Western blotting analysis, we confirmed that the amount of Oct-1 protein actually increased as the amount of the expression plasmid introduced was increased and that the amount of exogenously produced Oct-1 reached at least 10 times more than that of endogenous one (data not shown). To elucidate the Oct-1 binding site critical for the up-regulation by Oct-1, we examined the effects of the mutation at the Oct-1 sites on the luciferase expression in the cells which overexpressed Oct-1 (Fig. 6B). The wild-type (–95/+102) promoter was up-regulated ~7-fold by exogenously expressed Oct-1. Similar up-regulation was observed when plasmid pGLß(–95/+102)m6 lacking motif C was used. The mutant promoter lacking motif A in pGLß(–95/+102)m2 was also up-regulated ~7-fold when Oct-1 was overproduced, although detected activity was low. On the other hand, extremely low activity was detected in the cells carrying pGLß(–95/+102)m3 or pGLß (–95/+48) even in the presence of overexpressed Oct-1. These results suggest that Oct-1 transactivates the human ß chain promoter through the Oct-1 site of motif B. The above results also suggest that motif A, which has a potential to be recognized by Oct-1, does not function as an Oct-1 binding site in vivo.



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Fig. 6. Transcription activation of the ß chain promoter by Oct-1 via the second Oct-1 site in the 5'-UT. (A) Promoter activity of the ß chain promoter (–95/+102) with or without Oct-1 expression plasmid. Five micrograms of reporter plasmid pGLß(–95/+102) and various amounts of pCR3-2F-Oct-1 (closed circles; flag tagged Oct-1 expression plasmid) or pCR3-2F (open circles; mock) were introduced into KU-812 cells. (B) Promoter activity of wild-type or mutant promoter with or without Oct-1 expression plasmid. Five micrograms of various reporter plasmids was introduced into KU-812 cells with (closed bars) or without (open bars) 20 µg of pCR3-2F-Oct-1.

 
Analysis of protein binding to motif A by EMSA
We next performed EMSA analysis using nuclear extracts prepared from various cells (Fig. 7). When the extract from ß chain HeLa cells was used, only two shifted bands containing the band caused by Oct-1 were found. On the other hand, when the extracts from ß chain+ cells were used, several shifted bands were observed other than that shifted by Oct-1. It should be noted that two shifted bands (shown by arrows) were found in all three ß chain+ cells. We also performed a similar analysis using probe B. However, no shifted bands, which were found only when the extracts from ß chain+ cells were used, were present (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we analyzed the regulatory mechanism for the expression of the human Fc{epsilon}RI ß chain gene. Our analysis with a series of truncated ß chain promoter/luciferase constructs demonstrated that the –95/+102 region of the human ß chain gene showed promoter activity in ß chain+ cells (HMC-1, KU-812 and HML/SE), but not in ß chain cells (HeLa) (Fig. 3). We further found that the 5'-UT contains the elements required for the expression of the ß chain gene in ß chain+ cells. Although a regulatory element for the gene expression is often found around its own promoter or upstream, the ß chain gene has the elements at the 5'-UT. This is not only found in the ß chain: similar involvement of the 5'-UT in transcriptional control through transcription factor binding has recently been reported by several groups (2225). It is interesting to reveal how transcriptional regulation via 5'-UT is different from other usual regulation mechanisms as the machinery controlling gene expression.

A motif analysis for the nucleotide sequence of the region suggested that the region contained three potential Oct-1 motifs at +60/+66 (motif A), +76/+70 (motif B) and +92/+98 (motif C). Although Oct-1 is known to bind the canonical octamer consensus sequence ATGCAAAT, the protein is also able to bind to A/T/GTAATA/GA with high affinity (20,21). Three potential motifs have sequences similar to the latter. EMSA suggested that Oct-1 had the ability to bind all the three motifs in vitro. Among the three motifs, only motif B was found to respond to Oct-1 overexpression in vivo. These results suggest that motif B has the ability to transactivate ß chain expression in an Oct-1-dependent manner. On the other hand, motifs A and C were not required for the transactivation by overproduced Oct-1. It may be contradicting our result that the region containing motif A is involved in up-regulating ß chain gene expression. This can be explained by assuming that other transcription factors activate the ß chain promoter by binding to a region overlapping with motif A or its close region. Actually, EMSA analysis suggests the presence of several proteins binding to the probe containing motif A (Fig. 5A). On the other hand, we could not find any contribution of motif C to the promoter activity in this study, although Oct-1 could bind this motif in vitro.

In several cases, Oct-1 served as a transcription factor for the genes that are expressed only in specific cells, although this protein is ubiquitously expressed in various cells (26). Then, why is the regulation of ß chain expression found only in specific cells? In this respect, it is possible to postulate that nuclear proteins expressed in ß chain+ cells regulate the cell-type-specific expression of the ß chain gene. EMSA using probe A with motif A suggests the presence of proteins capable of binding to the probe in a cell-type-specific manner (Fig. 7). These proteins may be candidates to activate the ß chain promoter in concert with Oct-1 bound to motif B. Such cell-type-specific and synergic transactivation of Oct-1 was reported with OBF-1 (27,28), VP-16 (29) and Pit-1 (30). However, we cannot exclude the possibility that other transcription factor(s) might determine cell-type-specific ß chain expression independent of Oct-1 which elicits the ß chain promoter activity. Thus, to elucidate the mechanism for ß chain gene expression, further analysis to identify cell-type-specific co-activators or transcription factors that might function cooperatively with Oct-1 is required.

When we compared the expression level of mRNA for the ß chain by RT-PCR analysis, a high level of expression was observed in KU-812 and HMC-1, while lower expression was found in HML/SE (Fig. 2). We obtained similar results in quantitative RT-PCR using ABI7700 (data not shown). We recently found a polymorphism in the ß chain promoter which might cause different transcription levels in each cell line—one is C/C in the genomic DNA prepared from HML/SE, whereas it is T/T in HMC-1 and KU-812 (details will be reported elsewhere). Since the relative luciferase activities derived from HML/SE were similar to those of KU-812 and HMC-1 in transient reporter assays, we could conclude that these cell lines possess similar sets of transcription factors or co-activators.

Recently, the ß chain gene was found to be expressed in eosinophils in human (31). It contrasts with the case for mice where expression of the ß chain was not observed in eosinophils (32), suggesting a difference in the mechanism for cell-type specificity of ß chain expression between human and mouse. When the genomic sequences of the transcriptional control region of the ß chain gene of human and mouse were compared, we found that putative TATA sequence present in human ß chain genomic DNA is not conserved in mouse ß chain genomic DNA. Furthermore, in mouse 5'-UT, 2- and 3-base substitutions are found in the regions corresponding to motif A and motif B, although motif C is completely conserved. These differences in the nucleotide sequence between human and mouse ß chain genomic DNAs may be possible causes of no or negligible expression of mouse ß chain in eosinophils. These observations suggest the presence of a human-specific mechanism for the regulation of ß chain gene expression.


    Acknowledgements
 
We thank Drs H. Nakano (Department of Immunology, Juntendo University School of Medicine) and J. H. Butterfield (Mayo Clinic, Rochester) for providing pCR-2F and HMC-1 respectively. We are grateful to Dr M. Nishiyama (Biotechnology Research Center, The University of Tokyo) for his advice in completing this manuscript. We thank members of the Atopy (Allergy) Research Center and Department of Immunology for helpful discussion. We thank Ms T. Tokura for technical assistance, and Ms M. Matsumoto and Ms E. Kawasaki for excellent secretarial assistance. 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.).


    Abbreviations
 
EMSA—electrophoretic mobility shift assay

Fc{epsilon}RI—high-affinity receptor for IgE

5'-UT—5'-untranslated region


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Maurer, D., Fiebiger, E., Reininger, B., Wolff-Winiski, B., Jouvin, M. H., Kilgus, O., Kinet, J. P. and Stingl, G. 1994. Expression of functional high affinity immunoglobulin E receptors (Fc epsilon RI) on monocytes of atopic individuals. J. Exp. Med. 179:745.[Abstract]
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