Characterization of the human Fc{gamma}RIIB gene promoter: human zinc-finger proteins (ZNF140 and ZNF91) that bind to different regions function as transcription repressors

Tadahiro Nishimura, Tadashi Narita, Emi Miyazaki, Tohru Ito, Norihiro Nishimoto1,, Kazuyuki Yoshizaki1,, Joseph A. Martial2,, Eric J. Bellfroid2,, Henrik Vissing3, and Tadayoshi Taniyama

Laboratory of Immunoregulation, Department of Immunology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
1 Department of Immunology, School of Health and Sport Science, Osaka University, Suita 565-0871, Japan
2 Laboratory of Molecular Biology and Genetics, University of Liege, Institute of Chemistry B6, B-4000 Sart-Tilman, Belgium
3 Molecular and Cellular Biology, Novo Nordisk, DK-2880, Bagsvaerd, Denmark

Correspondence to: T. Taniyama


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Expression of the human low-affinity Fc receptors for IgG (human Fc{gamma}RII) is differentially regulated. We report here the characterization of the promoter structure of the human Fc{gamma}RIIB gene and the isolation of the promoter region-binding proteins by a yeast one-hybrid assay. The minimal 154-bp region upstream from the transcription start site of the human Fc{gamma}RIIB gene was shown to possess promoter activity in a variety of cells. An electrophoretic mobility shift assay indicated that multiple nuclear factors in cell extracts bind to the two regions [F2-3 (–110 to –93) and F4-3 (–47 to –31)] of the human Fc{gamma}RIIB gene promoter. Mutation analysis indicated that GGGAGGAGC (–105 to –97) and AATTTGTTTGCC (–47 to –36) sequences are responsible for binding to nuclear factors respectively. By using GGGAGGAGC and AATTTGTTTGCC as bait sequences, we cloned two zinc-finger proteins (ZNF140 and ZNF91) that bind to the F2-3 and F4-3 regions within the promoter of the human Fc{gamma}RIIB gene respectively. When the ZNF140 and ZNF91 were transfected with reporter plasmid, both showed repressor activity with additive effects. Thus, these results indicate that these cloned ZNF140 and ZNF91 proteins function as repressors for the human Fc{gamma}RIIB transcription.

Keywords: Fc{gamma} receptor II, repressor, transcription, zinc-finger protein


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In humans, there are three classes of Fc receptor for IgG, i.e. Fc{gamma}RI, Fc{gamma}RII and Fc{gamma}RIII ( 13 ), and these have been shown to be involved in phagocytosis ( 4 ), clearance of immune complexes ( 5 ), antibody-dependent cellular cytotoxicity ( 6 , 7 ), release of inflammatory mediators ( 8 ), regulation of Ig synthesis ( 9 ) and superoxide production ( 10 ). Fc{gamma}RI and Fc{gamma}RIII are expressed in macrophages, and in natural killer cells, neutrophils, macrophages and a subset of T cells respectively. On the other hand, Fc{gamma}RII is found in various types of cells, including macrophages, neutrophils, platelets, B cells and epithelial cells.

Three human Fc{gamma}RII genes (Fc{gamma}RIIA, Fc{gamma}RIIB and Fc{gamma}RIIC) that contain eight exons and seven introns were previously cloned ( 11 , 12 ). Alternative splicing of at least two (Fc{gamma}RIIA and Fc{gamma}RIIB) of these three genes has been shown to result in the production of multiple transcripts ( 13 ). These transcripts are Fc{gamma}RIIa1, Fc{gamma}RIIa2, Fc{gamma}RIIb1, Fc{gamma}RIIb2 and Fc{gamma}RIIb3. Myelomonocytic cells contain all three Fc{gamma}RII transcripts, predominantly the Fc{gamma}RIIa1 transcript, the Fc{gamma}RIIb1 and Fc{gamma}RIIb2 transcripts, and the Fc{gamma}RIIC transcript. B cells do not express Fc{gamma}RIIA, but contain both the Fc{gamma}RIIb1 and Fc{gamma}RIIb2 transcripts and the Fc{gamma}RIIC transcript. Megakaryocytic cells contain predominantly Fc{gamma}RIIA transcripts. Further, human epithelial cells and trophoblasts also express all three Fc{gamma}RII transcripts, predominantly the Fc{gamma}IIC transcript ( 14 , 15 ). Thus, these findings indicate that different regulatory mechanisms exist among these three Fc{gamma}RII gene expressions in a variety of cell types.

McKenzie and his group ( 16 ) characterized the 5' region of the human Fc{gamma}RIIA gene. They reported that the Fc{gamma}RIIA gene, which consists of nine exons, has two discrete transcription start sites. One start site was mapped to a 5'-untranslated (5'-UT) exon ~1 kb 5' to the ATG translation initiation codon and the second start site was mapped near the ATG codon. However, these authors did not report the precise functional characterization of the human Fc{gamma}RIIA promoter and the molecular mechanism underlying the regulation of the human Fc{gamma}RII gene expression is still obscure.

To unravel the molecular mechanism of the regulation of the human Fc{gamma}RII gene transcription, we isolated the human Fc{gamma}RIIB gene promoter and identified two different region-binding proteins (F2-3 and F4-3 binding proteins) within the promoter region of the human Fc{gamma}RIIB gene by electrophoretic mobility shift assay (EMSA). We also cloned two different zinc-finger proteins (ZNF140 and ZNF91) that can bind to the F2-3 and F4-3 regions within the human Fc{gamma}RIIB promoter respectively. These human zinc-finger proteins (ZNF140 and ZNF91) function as repressors for the human Fc{gamma}RIIB transcription. When they were simultaneously expressed, the two proteins demonstrated an additive suppressive effect.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Plasmids and phage library
The EMBL3 genomic phage library (kindly provided by the Japanese Cancer Research Resources Bank, Tokyo, Japan) was used for the screening of human Fc{gamma}RII genomic genes. The pSVOOCAT was obtained from Wako Pure Chemical Industries (Osaka, Japan). The pBluescript II KS+ (pBSIIKS+), and pCAT-enhancer (pCAT-EN), pGL3-Enhancer (pGL3EN) and pSV-ß-galactosidase control vector (pSVßgal) were purchased from Stratagene (La Jolla, CA) and Promega (Madison, WI) respectively. The pCAGGS was kindly supplied by Dr Miyazaki ( 17 ) and pcDNA3.1/Hygro(+) (pcDNA3.1) was obtained from Invitrogen (Carlsbad, CA).

Cloning of the human Fc {gamma} RIIB gene promoter region
Four clones were isolated from the EMBL3 phage library using the 32 P-labeled 700-bp Pst I fragment of the PC23 cDNA as a probe ( 18 ) under high-stringent conditions. Construction of deletion mutants using pBSIIKS+ plasmid was performed using exonuclease III (Stratagene) according to the manufacturer's instructions.

Construction of plasmids
The 1.5-kb Eco RI– Nar I fragment of the cloned human Fc{gamma}RIIB gene was filled in and ligated to Sal I linker, and then ligated to the Sal I site of pSVOOCAT to give pFcRCAT. The same 1.5-kb fragment was inserted into the Sal I sites of pCAT-EN to yield pFcRCAT-EN. The various deletion mutants were prepared using exonuclease III and sequenced, and the resulting inserts were ligated into the Sal I site of pCAT-EN. The pFcR (del-326) CAT-EN was prepared by deleting the –326 to +21 region and inserting into the Sal I site of pCAT-EN. The pFcR-CAT-EN/R was prepared by inserting the 1.5-kb Eco RI– Nar I fragment into the Sal I site of the pCAT-EN in a reversed orientation. The pFcR2-154/pGL3EN was prepared by inserting the –154-bp fragment into the Sma I site of the pGL3EN. The full-length ZNF140 ( 19 ) and ZNF91 ( 20 ) fragments were inserted into Eco RI site of the pcDNA3.1 and pCAGGS respectively.

Primer extension
Primer extension analysis was performed using a single-stranded synthetic oligonucleotide of sequence 5'-GGTAAGAATGACAGGATTCCCAT-3', which is complementary to nucleotides 1–23 of human Fc{gamma}RIIB cDNA. The primer was labeled with T4 polynucleotide kinase (Takara Shuzo, Tokyo, Japan) and annealed to 5 µg of mRNA in 25 µl of 50 mM Tris–HCl (pH 8.3), 100 mM KCl and 10 mM MgCl 2 , incubated at 95°C for 5 min, then at 55°C for 60 min and cooled slowly to room temperature. The sample was mixed with 25 µl of 50 mM Tris–HCl (pH 8.3), 100 mM KCl, 10 mM MgCl 2 , 2 mM dNTP, 20 mM DTT and 200 U of M-MLV reverse transcriptase (Life Technologies, Rockville, MD), and incubated for 60 min at 37°C. The reaction mixture was recovered by ethanol precipitation and loaded onto a polyacrylamide urea gel, followed by autoradiography.

Nuclear extracts and EMSA
Nuclear extracts were prepared by the method of Dignam et al . ( 21 ). EMSA was performed as follows. A fragment (–154 to +21 bp region) and oligonucleotides were end-labeled using [ 32 P]dCTP and Klenow fragment. Nuclear extracts (20 µg) were incubated at 16°C for 20 min with 32 P-labeled fragment (~1 ng, 25,000 c.p.m./ng) in 20 µl of binding buffer containing 40 mM Tris–HCl (pH 7.5), 200 mM NaCl, 2 mM DTT, 10% Glycerol, 0.05% NP-40, 5 mM MgCl 2 , 50 µg/ml poly(dG–dC):poly(dG–dC) (Amersham Pharmacia Biotech, Little Chalfont, UK) and 1 mM EDTA. In our competition studies, a 150 M excess of unlabeled oligonucleotide competitors was added. The DNA–protein complexes were separated in a 4% polyacrylamide gel using a running buffer containing 50 mM Tris–HCl (pH 7.8), 380 mM lysine and 1 mM EDTA.

Yeast one-hybrid assay
The Matchmaker one-hybrid system from Clontech (Palo Alto, CA) was used according to the manufacturer's instructions. Briefly, the double-stranded oligonucleotides with the 5'-(AAAGGGAGGAGC)x4-3' (for F2-3-binding protein) and 5'-(AATTTGTTTGCC)x3-3' (for F4-3-binding protein) fragments were subcloned into the pHISi-1 and pLacZi. These plasmids (F2-3-EL/pHISi-1 and F2-3-EL/pLacZi for the F2-3-binding protein and F4-3-EL/pHISi-1 and F4-3-EL/pLacZi for the F4-3-binding protein) were successively introduced into yeast YM4271, and the appropriate transformants were selected by testing ß-galactosidase expression and 3-aminotriazole sensitivity according to the manufacturer's instructions. These yeast reporter clones were transformed with DNA from the cDNA library (pACT2) made from human placenta cells (Clontech). We screened ~5x10 5 clones in each assay. Yeast cells with His + ß-gal + phenotypes were selected. The cDNA (prey) plasmids from His + ß-gal + yeast colonies were isolated and sequenced.

Cell lines
The THP-1 human macrophage-like cell line, Raji human Burkitt lymphoma cell line, HSB-2 human T leukemic cell line and JEG-3 human choriocarcinoma cell line were maintained in DMEM medium containing 10% FBS (IBL, Gunma, Japan), 100 µg/ml streptomycin and 100 U/ml penicillin ( 2224 ).

Reporter gene assays
Cells were transfected with the plasmid by the DEAE–dextran method (Stratagene) or the calcium phosphate method (Stratagene) according to the manufacturer's instructions. THP-1 and Raji cells (10 7 cells/tube) were treated with 10 µg of test plasmid in the presence of 250 µg/ml DEAE–dextran sulfate. JEG-3 cells (1.5x10 5 cells/3.5 cm dish) were transfected by the calcium phosphate method. Two days later, cell lysates were prepared and assayed for chloramphenicol acetyl transferase (CAT), luciferase and ß-galactosidase activities. The CAT activity was determined by the method described by Gorman et al . ( 25 ). After the incubation period, the products were separated from unacetylated chloramphenicol by thin-layer chromatography. The radioactivity was measured using the BioImage analyzer BAS2000 (Fuji Film, Tokyo, Japan). The luciferase and ß-galactosidase activities were determined with a Luciferase constant light kit (Roche Diagnostics Japan, Tokyo, Japan) and Galacto-Star (Tropix, Bedford, MA) according to the manufacturer's instructions. We used the pSVßgal plasmid (0.2 µg) for normalizing transfection efficiencies.

Nucleotide accession
The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank as nucleotide sequence accession no. D86416.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Isolation of the 5' flanking region of the human Fc {gamma} RIIB gene and characterization of its promoter region
The structure of the human Fc{gamma}RIIB gene was determined using genomic clones isolated from the EMBL3 Japanese genomic library. Figure 1 Go shows the 5' flanking sequence of the human Fc{gamma}RIIB gene. The gene structure of the 5' boundary of the human Fc{gamma}RIIB in the present study is identical to those reported previously ( 11 , 12 ). By Harr plot analysis, the 5' flanking sequence of the human Fc{gamma}RIIB gene showed no apparent homology to that of the human Fc{gamma}RIIA gene reported by McKenzie et al . ( 16 ) (data not shown). To determine the transcription start sites, mRNAs from THP-1 and HSB-2 cells that express all three Fc{gamma}RII transcripts and no Fc{gamma}RII mRNA respectively were annealed to the primer (complementary to nucleotides 1–23 of the human Fc{gamma}RIIB structural gene) and then primer extension analysis was performed. We found two major cap sites in human THP-1 cells, while no cap site was noted in HSB-2 cells, as indicated in Fig. 2 Go .



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Fig. 1. Nucleotide sequence of the human Fc{gamma}RIIB 5' flanking region. Two major transcription start sites, as determined by primer extension, are indicated by arrows. The 5' transcription start site is designated as +1. The predicted amino acid sequence, starting at the methionine (M) at +44, is overlined with large letters of the amino acid letter code. A black circle indicates the start of the intron.

 


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Fig. 2. Mapping of the human Fc{gamma}RIIB mRNA transcription initiation sites by primer extension analysis. A labeled 23-bp primer complementary to the nucleotides 1–23 of the human Fc{gamma}RIIB cDNA (structural gene) was hybridized with mRNA from THP-1 human macrophage-like cells (lane 1) or HSB-2 human T leukemic cells (lane 2). Primer was extended by reverse transcriptase, denatured and run on a urea/acrylamide gel. The sequence shown at the left was identified using the same primer except for the absence of the 5' phosphate. Arrows indicate primer elongation stop points.

 
We next determined whether the 5' flanking region of the Fc{gamma}RIIB gene has promoter activity. As shown in Fig. 3 (A)Go , 4.1-, 2.0- and 2.3-fold increases of the CAT activity of cell extracts from THP-1 cells, Raji cells and JEG-3 cells transfected with pFcRCAT were noted as compared with pSVOOCAT. The pFcRCAT-EN produced 12.0-, 8.9- and 3.9-fold increases of the CAT activity in THP-1, Raji and JEG-3 cells respectively over that produced by pCAT-EN ( Fig. 3A Go ). Promoter activity was then mapped by comparing CAT expression driven by a series of 5' deletion mutants in THP-1 cells and the results are depicted in Fig. 3 (B)Go . The pFcRCAT-EN construct and deletion mutants exhibited significant levels of CAT activity. The pFcRCAT-EN-R and pFcR-326CAT-EN, in which the promoter region is in the reverse orientation and the 5' boundary nucleotides (–326 to +21), were internally deleted respectively, did not produce a significant level of CAT activity, as shown in Fig. 3 (B)Go . Thus, the 5' boundary for the minimal human Fc{gamma}RIIB promoter lies between nucleotides –154 and +21.



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Fig. 3. Functional analysis of the promoter region of the human Fc{gamma}RIIB gene. (A) Various chimeric genes (10 µg) were each transfected into THP-1, Raji and JEG-3, and CAT activity was measured 48 h later. The cell extracts containing 33 µg of protein were incubated with [ 14 C]chloramphenicol at 37°C for 4 h. The substrate converted to the acetylated form was separated by thin-layer chromatography and CAT activities were expressed as percent conversions. Similar results were obtained in three other experiments. (B) Chimeric genes including pFcRCAT-EN, p-1181CAT-EN, p-432CAT-EN, p-154CAT-EN, pFcRCAT-EN-R, pFcR-326CAT-EN and pCAT-EN (10 µg) were each transfected into THP-1 cells. CAT activities were determined using 50 µg of cell extract with 4-h incubation. These data represent one of three experiments performed independently.

 
Characterization of nuclear factors bound to the 5' boundary (–148 to –1) of the human Fc {gamma} RIIB gene
To examine the DNA-binding factors from various types of cells that bind to the –154 to +1 bp region of the human Fc{gamma}RIIB gene, EMSA was performed using five oligonucleotides that span –148 to –1 ( Fig. 4A Go ). As shown in Fig. 4 (C and E)Go , we found that two probes [F2 (–126 to –93) and F4 (–64 to –31)] formed specific bands, because cold F2 and F4 probes inhibited formation of specific bands (bands A, B and C) respectively. The other three probes (F1, F3 and F5) did not reveal any specific band ( Fig. 4B , D and FGo ). Since the nuclear factor PU.1 recognized a purine-rich sequence (PU box) ( 26 , 27 ) and the F2 region contained a similar nucleotide sequence, we performed supershift analysis using anti-PU.1 or anti-C/EBPß antibody. As shown in Fig. 4 (G)Go , specific bands A, B and C were not supershifted in the presence of anti-PU.1 or anti-C/EBPß antibody, although anti-PU.1 and anti-C/EBPß antibodies could supershift the probes containing PU box derived from human Fc{gamma}RI gene and CCAAT homology region derived from human IL-6 gene respectively (data not shown). Figure 4 (H)Go shows that F2-binding proteins are different from F4-binding proteins, because the F4 nucleotide could not compete with the 32 P-labeled F2 probe. Similarly, the F2 nucleotide did not inhibit complex formation with the 32 P-labeled F4 probe (data not shown).



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Fig. 4. Multiple nuclear factors from various types of cells bind to the –154 bp 5' flanking region of the human Fc{gamma}RIIB gene. (A) The DNA sequences of five oligonucleotides used for EMSA. (B–F) Nuclear extracts from THP-1, Raji and JEG-3 cells were mixed with the 32 P-labeled probe. An oligonucleotide containing the NF-{kappa}B motif was used as an unrelated competitor at a 150 M excess. Bound and unbound probes were separated in native polyacrylamide gel. The arrow indicates the positions of the complex. (G) Nuclear extracts were then prepared, preincubated with antibodies (2 µl/lane) against PU.1 and C/EBPß, and analyzed by EMSA. The arrow indicates the position of the complex. (H) Nuclear extracts were mixed with the 32 P-labeled F2. A 150 M excess of unlabeled oligonucleotide competitors was added. The arrow indicates the position of the complex.

 
To further determine the precise sequences responsible for DNA binding, we next synthesized three overlapping oligonucleotides of the F2 and F4 regions, and performed EMSA ( Figs 5 and 6 Go Go ). We found that multiple nuclear factors bound to the F2-3 (–109 to-93) ( Fig. 5C Go ) and F4-3 (–47 to –31) ( Fig. 6C Go ) oligonucleotides respectively. No specific bands were noted using the F2-1 ( Fig. 5A Go ), F2-2 ( Fig. 5B Go ), F4-1 ( Fig. 6A Go ) or F4-2 ( Fig. 6B Go ) probes. We then introduced mutations in the F2-3 ( Fig. 5D Go ) and F4-3 ( Fig. 6D Go ) oligonucleotides, and performed EMSA. As shown in Figs 5 (E) and 6(E)GoGo , the F2-related probes (F2-3-1, F2-3-2 and F2-3-3) and F4-related probes (F4-3-1, F4-3-2 and F4-3-3) lost the ability to bind the 32 P-labeled F2-3 and F4-3 probes respectively. Thus, these results indicate that the sequences of GGGAGGAGC (–105 to –97) and AATTTGTTTGCC (–47 to –36) within the human Fc{gamma}RIIB gene promoter are responsible for binding of multiple nuclear factors ( Figs 5E and 6E Go Go ).



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Fig. 5. Analysis of the F2 region-specific EMSA complex. (A–C) Nuclear extracts from THP-1, Raji and JEG-3 cells were mixed with the 32 P-labeled probe. An oligonucleotide containing the NF-{kappa}B motif was used as an unrelated competitor at a 150 M excess. Bound and unbound probes were separated in native polyacrylamide gel. The arrow indicates the positions of the complex. (D) The DNA sequences of four oligonucleotides used for EMSA. (E) Nuclear extracts were mixed with the 32 P-labeled F2-3. A 150 M excess of unlabeled oligonucleotide competitors was added. The arrow indicates the position of the complex.

 


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Fig. 6. Analysis of the F4 region-specific EMSA complex. (A–C) Nuclear extracts from THP-1, Raji and JEG-3 cells were mixed with the 32 P-labeled probe. An oligonucleotide containing the NF-{kappa}B motif was used as an unrelated competitor at a 150 M excess. Bound and unbound probes were separated in native polyacrylamide gel. The arrow indicates the positions of the complex. (D) The DNA sequences of four oligonucleotides used for EMSA. (E) Nuclear extracts were mixed with the 32 P-labeled F4-3. A 150 M excess of unlabeled oligonucleotide competitors was added. The arrow indicates the position of the complex.

 
Cloning of the F2-3 and F4-3 region-binding protein by one-hybrid assay
To identify nuclear proteins that bind to the F2-3 and F4-3 regions of the human Fc{gamma}RIIB promoter, we used a one-hybrid assay. As bait sequences, we used (GGGAGGAGC)x3 and (AATTTGTTTGCC)x4 for F2-3- and F4-3-binding proteins respectively. We screened ~5x10 5 clones of a cDNA library prepared from human placenta in each assay. We isolated 12 and 21 His + ß-gal + clones in the F2-3- and F4-3-binding assays respectively. We then isolated and sequenced cDNA (prey) plasmids, and found that plasmids from two clones (F2-3-binding protein) and three clones (F4-3-binding protein) contained partial sequences that are identical to those of the human zinc-finger protein 140 (ZNF140) ( 19 ) and zinc-finger protein 91 (ZNF91) ( 20 ) respectively. Other clones were not analyzed in the present study.

To confirm the binding specificity of the two zinc-finger proteins, we transformed yeasts carrying either F2-3-EL/pHISi-1 and F2-3-EL/pLacZi or F4-3-EL/pHISi-1 and F4-3-EL/pLacZi with pZNF140/pACT2, pZNF91/pACT2 or pACT2. Colonies were tested for ß-galactosidase activity. Figure 7 (A)Go shows the typical results. ZNF140/pACT2 and ZNF91/pACT2 could specifically bind to the F2-3 and F4-3 elements respectively in yeast, so that ß-galactosidase activity was detected. Conversely, ZNF140/pACT2 and ZNF91/pACT2 did not interact with the F4-3 and F2-3 elements respectively. Figure 7 (B)Go shows that the full lengths of ZNF140 and ZNF91 consisted of 457 and 1191 amino acid residues respectively. We sequenced the inserts from F2-3- and F4-3-binding clones, and found that the inserts encoded zinc-finger structures of the fragments of ZNF140 and ZNF91 proteins respectively ( Fig. 7B Go , open rectangles). As shown previously ( 19 , 20 ), these transcripts were ubiquitously expressed on various types of cells (data not shown).



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Fig. 7. Cloning and function of the human ZNF140 and ZNF91. (A) Yeast strains containing F2-3-EL/pLacZi and F2-3-EL/pHISi-1 or F4-3-EL/pLacZi and F4-3-EL/pHISi-1 were transformed with pACT2, ZNF140/pACT2 or ZNF91/pACT2. The colony was tested for ß-galactosidase assay. (B) Structure of the ZNF140 and ZNF91 proteins. Open rectangles indicate the portions of ZNF140 and ZNF91 proteins encoded by the inserts from the F2-3- and F4-3-binding clones identified by a one-hybrid assay. (C) Functional assay of ZNF140 and ZNF91. Ten micrograms of each of pcDNA3.1, pcDNA3.1-ZNF140, pCAGGS, pCAGGS-ZNF91 and pFcR2-154/pGL3EN together with 0.2 µg of pSVßgal were transfected into JEG-3 cells. After a 48-h incubation period, cell lysates were prepared and then assayed for luciferase and ß-galactosidase activities. Transfection efficiency was normalized by ß-galactosidase assay. Data of group 2, 4 and 6 were relative to those of group 1, 3 and 5 respectively. These data represent one of three experiments performed independently.

 
ZNF140 and ZNF91 function as repressors in –154 human Fc {gamma} RIIB promoter-mediated luciferase expression
Having established that two zinc-finger proteins can specifically bind to the F2-3 and F4-3 region respectively, it is of great interest to determine the function of these proteins. We constructed expression plasmids and transfected them into JEG-3 cells with the reporter plasmid (pFcR2-154/pGL3EN). As shown in Fig. 7 (C)Go , when either human ZNF140 or ZNF91 was expressed, both proteins inhibited luciferase activity. Furthermore, the two proteins demonstrated an additive suppressive effect when they were simultaneously expressed. We also obtained the similar results using human 293T and HepG2 cell lines (data not shown).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In humans, three Fc{gamma}RII genes (Fc{gamma}RIIA, Fc{gamma}RIIB and Fc{gamma}RIIC) are differentially regulated in a variety of cells. In the present study, we found that the promoter regions of the human Fc{gamma}RIIA and Fc{gamma}RIIB genes are different. First, we found that a Harr plot between the Fc{gamma}RIIA and Fc{gamma}RIIB promoter sequences indicated no apparent homology (data not shown). Second, two different transcription start sites were found in the human Fc{gamma}RIIA promoter. One transcription start site is located at a 5'-UT exon ~1 kb 5' to the ATG translation initiation codon, while a second start site was mapped near the ATG codon. On the other hand, we found that the single exon continuous with the ATG codon contains at least two transcription start sites in the human Fc{gamma}RIIB gene, the major one 42 bp 5' to ATG and a minor one 22 bp 5' to ATG in THP-1 cells ( Figs 1 and 2 Go Go ). We could not detect any transcription start site 5' to the 42 bp 5' to the ATG codon by primer extension analysis (data not shown), thus indicating that the human Fc{gamma}RIIB gene does not contain a discrete 5'-UT region. Our results are similar to those for the mouse Fc{gamma}RIIIA gene ( 28 ) and the human Fc{gamma}RI gene ( 2931 ), both of which have multiple transcription start sites mapped near the ATG codon. Third, we found that various elements found in the Fc{gamma}RIIA and Fc{gamma}RIIB gene promoters are not identical. Thus, all of these findings indicate that the expression profiles of the Fc{gamma}RIIA and Fc{gamma}RIIB genes are differentially regulated.

In the present study, we found that the 5' boundary for the minimal human Fc{gamma}RIIB promoter lies between nucleotides –154 and +21 in our assay system. This result is similar to those for the human Fc{gamma}RI ( 3034 ) and mouse Fc{gamma}RIIIA ( 28 ) genes, whose promoters have minimal structures (~150 bp upstream of the cap site) with an IFN-{gamma}-responsive region (GRR) and a PU box, and a PU box and myeloid-restricted region (MRR) respectively. Mutation analysis of the F2-3 and F4-3 regions indicated that the sequences of GGGAGGAGC (–105 to –97) and AATTTGTTTGCC (–47 to –36) within the human Fc{gamma}RIIB promoter participated in the binding by nuclear factors. The sequence of the F2-3 region is similar to that of the PU.1-responsive element, but the PU.1 nuclear factor did not bind to the F2-3 region, since anti-PU.1 antibody did not supershift ( Fig. 4G Go ). This result is in contrast to that for the human Fc{gamma}RI gene promoter, since the PU.1 was shown to bind to the –107 to –74 region of the human Fc{gamma}RI promoter ( 32 , 33 ). Thus, these results indicate that the expression profiles of the human Fc{gamma}RIIB and Fc{gamma}RI genes are also differently regulated.

We also isolated the F2-3- and F4-3-binding proteins from the human placenta library by a one-hybrid assay. We found that the human ZNF140 and ZNF91 were the F2-3 and F4-3 region-binding proteins respectively ( Fig. 7 Go ). These ZNF140 and ZNF91 genes were previously isolated by PCR with zinc-finger motifs as primers ( 19 , 20 , 35 ). In humans, it has been estimated that there are 300–700 different zinc-finger protein genes and the vast majority of zinc-finger proteins contained a Kruppel-associated box (KRAB), such as the C 2 H 2 type ( 36 , 37 ). These genes are well conserved and were distributed from Drosophila to humans ( 35 , 36 ). Drosophila Kruppel and hunchback ZNF proteins are involved in embryonic pattern formation, while mouse Krox-20 in both patterning of the hindbrain and control of cell proliferation ( 38 , 39 ). Most zinc-finger proteins have been shown to be involved in development, but their other functions remain obscure. Vissing and his group reported that ZNF140, ZNF133, ZNF136 and ZNF141—each of which contains a KRAB box—function as transcription repressors when fused to a heterologous DNA-binding domain from the yeast GAL4 protein ( 40 , 41 ). We extended the previous findings by indicating that ZNF140 functioned as a transcription repressor when the human Fc{gamma}RIIB natural promoter region was connected to the luciferase gene ( Fig. 7C Go ). We also found that ZNF91 was also a transcription repressor, although the function of this protein is not yet known. These results indicate that ZNF140 and ZNF91 function as transcription repressors in human Fc{gamma}RIIB gene expression. Furthermore, T lymphoid cells do not express all three Fc{gamma}RII transcripts, but high amounts of ZNF91 transcripts are expressed in human T lymphoid cells and T leukemic cell lines (data not shown), HSB-2 and CEM, as found by Bellefroid et al . ( 20 ). The fact that ZNF91 functions as a repressor in the Fc{gamma}RIIB transcription suggests that ZNF91 may repress the Fc{gamma}RIIB transcription in T lymphoid cells, leading to no expression of the human Fc{gamma}RIIB. However, we do not yet know the precise mechanisms by which ZNF91 represses transcription.

Calame and her group reported that the ZF5 zinc-finger protein activated the HIV-1 long terminal repeat promoter, and repressed the ß-actin, c- myc and herpes simplex thymidine kinase promoters ( 42 , 43 ). ZF5 zinc-finger protein thus can both activate and repress in the context of different natural promoters. In the Drosophila system, Sauer and Jackle ( 44 ) reported that Kruppel ( Kr ), required for normal thorax and abdominal development, itself acts as a concentration-dependent positive and negative regulator of transcription. In the present study, we found that multiple nuclear factors could bind to the same F2-3 and F4-3 regions. Thus, other unknown positive factors might regulate the transcription of the human Fc{gamma}RIIB gene. Alternatively, the ZNF140 and ZNF91 might work as positive regulators at the optimal concentrations, as suggested by Sauer and Jackle ( 44 ). Furthermore, THP-1, Raji and JEG-3 cell lysates gave different band patterns in the EMSA assay, indicating that various types of cells might contain different nuclear factors for these regions. Taken collectively, these results indicate that the regulation of the human Fc{gamma}RIIB gene expression might be complex in various types of cells. Identification of all promoter-binding proteins will resolve these issues.


    Acknowledgments
 
We thank T. Iwata and A. Tamori for technical assistance. We also thank D. Mrozek and T. Matsui for their comments. This work was partially supported by Japan Health Sciences Foundation.


    Abbreviations
 
CAT chloramphenicol acetyl transferase
EMSA electrophoretic mobility shift assay
Fc{gamma}RI high-affinity Fc receptor I for IgG
Fc{gamma}RII low-affinity Fc receptor II for IgG
KRAB Kruppel-associated box
UT untranslated

    Notes
 
Transmitting editor: T. Kurosaki

Received 9 April 2001, accepted 16 May 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Fanger, M. W., Shen, L., Grazione, R. F. and Guyre, P. M. 1989. Cytotoxicity mediated by human Fc receptors for IgG. Immunol. Today 10:92.[ISI][Medline]
  2. Ravetch, J. V. and Kinet, J.-P. 1991. Fc receptors. Annu. Rev. Immunol. 9:457.[ISI][Medline]
  3. van de Winkel, J. G. J. and Anderson, C. L. 1991. Biology of human Immunoglobulin G Fc receptors. J. Leuk. Biol. 49:511.[ISI][Medline]
  4. Anderson, C. L., Shen, L., Eicher, D. M., Wewers, M. D. and Gill, J. K. 1990. Phagocytosis mediated by three distinct F{gamma} receptor classes on human leukocytes. J. Exp. Med. 171:1333.[Abstract]
  5. Leslie, R. C. Q. 1985. Complex aggregation: a critical event in macrophage handling of soluble immune complexes. Immunol. Today 6:183.[ISI]
  6. Nathan, C. and Cohn, Z. 1980. Role of oxygen-dependent mechanisms in antibody-induced lysis of tumor cells by activated macrophages. J. Exp. Med. 152:198.[Abstract/Free Full Text]
  7. Kipps, T. J., Parham, P., Punt, J. and Herzenberg, L. A. 1985. Importance of immunoglobulin isotype in human antibody-dependent cell-mediated cytotoxicity directed by murine monoclonal antibodies. J. Exp. Med. 161:1.[Abstract]
  8. Ferreri, N. R., Howland, W. C. and Spiegelberg, H. L. 1986. Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE. J. Immunol. 136:4188.[Abstract/Free Full Text]
  9. Bich Thuy, L. T. and Revillard, J. P. 1982. Selective suppression of human B lymphocyte differentiation into IgG-producing cells by soluble Fc{gamma} receptors. J. Immunol. 129:150.[Abstract/Free Full Text]
  10. Yamamoto, K. and Johnston, R., Jr. 1984. Dissociation of phagocytosis from stimulation of the oxidative metabolic burst in macrophages. J. Exp. Med. 159:405.[Abstract]
  11. Brooks, D. G., Qiu, W. Q., Luster, A. D. and Ravetch, J. V. 1989. Structure and expression of human IgG FcR II (CD32) Functional heterogeneity is encoded by the alternatively spliced products of multiple genes. J. Exp. Med. 170:1369.[Abstract]
  12. Qiu, W. Q., de Bruin, D., Brownstein, B. H., Pearse, R. and Ravetch, J. V. 1990. Organization of the human and mouse low-affinity Fc{gamma}R genes: duplication and recombination. Science 248:732.[ISI][Medline]
  13. Ravetch, J. V. and Anderson, C. L. 1990. Fc{gamma}R family: proteins, transcripts, and genes. In Metzger, H., ed., Fc Receptors and the Action of Antibodies, p. 211. American Society for Microbiology, Washington, DC.
  14. Cassel, D. L., Keller, M. A., Surrey, S., Schwartz, E., Schreiber, A. D., Rappaport, E. C. and McKenzie, S. E. 1993. Differential expression of Fc{gamma}RIIA, Fc{gamma}RIIB and Fc{gamma}RIIC in hematopoietic cells: analysis of transcripts. Mol. Immunol. 30:451.[ISI][Medline]
  15. Stuart, S. G., Simister, N. E., Clarkson, S. B., Kacinski, B. M., Shapiro, M. and Mellman, I. 1989. Human IgG Fc receptor (hFcRII; CD32) exists as multiple isoforms in macrophages, lymphocytes and IgG-transporting placental epithelium. EMBO J. 8:3657.[Abstract]
  16. McKenzie, S. E., Keller, M. A., Cassel, D. L., Schreiber, A. D., Schwartz, E., Surrey, S. and Rappaport, E. F. 1992. Characterization of the 5'-flanking transcriptional regulatory region of the human Fc{gamma} receptor gene, Fc{gamma}RIIA. Mol. Immunol. 29:1165.[ISI][Medline]
  17. Niwa, H., Yamamura, K. and Miyazaki, J. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193.[ISI][Medline]
  18. Stenegelin, S., Stamenkovic, I. and Seed, B. 1988. Isolation of cDNAs for two distinct human Fc receptors by ligand affinity cloning. EMBO J. 7:1053.[Abstract]
  19. Tommerup, N. and Vissing, H. 1995. Isolation and fine mapping of 16 novel human zinc finger-encoding cDNAs identify putative candidate genes for developmental and malignant disorders. Genomics 27:259.[ISI][Medline]
  20. Bellefroid, E. J., Marine, J.-C., Ried, T., Lecocq, P. J., Riviere, M., Amemiya, C., Poncelet, D. A., Coulie, P. G., de Jong, P., Szpirer, C., Ward, D. C. and Martial, J. A. 1993. Clustered organization of homologous KRAB zinc-finger genes with enhanced expression in human T lymphoid cells. EMBO J. 12:1363.[Abstract]
  21. Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475.[Abstract]
  22. Kurata, N., Akiyama, H., Taniyama, T. and Marunouchi, T. 1989. Dose-dependent regulation of macrophage differentiation by mos mRNA in a human monocytic cell line. EMBO J. 8:457.[Abstract]
  23. Taniyama, T., Yoshida, K. and Furuta, T. 1988. Demonstration of a novel tumor killing factor secreted from human macrophage-monocyte hybridomas. J. Immunol. 141:4061.[Abstract/Free Full Text]
  24. Tsuchiya, S., Yamabe, M., Yamaguchi, Y., Kobayashi, Y., Konno, T. and Tada, K. 1980. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int. J. Cancer 26:171.[ISI][Medline]
  25. Gorman, C. M., Moffat, L. F. and Howard, B. H. 1982. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044.[ISI][Medline]
  26. Klemsz, M. J., McKercher, S. R., Celada, A., Beveren, C. V. and Maki, R. A. 1990. The macrophage and B cell-specific transcription factor PU.1 is related to the ets oncoprotein. Cell 61:113.[ISI][Medline]
  27. Ray, D., Bosselut, R., Ghysdale, J., Mattei, M.-G., Tavitian, A. and Moreau-Gachelin, F. 1992. Characterization of Spi-B, a transcription factor related to the putative oncoprotein Spi-1/PU.1. Mol. Cell. Biol. 12:4297.[Abstract]
  28. Feinman, R., Qiu, W. Q., Pearse, R. N., Nikolajczyk, S., Sen, R., Sheffery, M. and Ravetch, J. V. 1994. PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc{gamma}RIIIA promoter. EMBO J. 13:3852.[Abstract]
  29. van de Winkel, J. G. J., Ernst, L. K., Anderson, C. L. and Chiu, I.-M. 1991. Gene organization of the human high affinity receptor for IgG, Fc{gamma}RI (CD64). J. Biol. Chem. 266:13449.[Abstract/Free Full Text]
  30. Pearse, R. N., Feinman, R. and Ravetch, J. V. 1991. Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: transcriptional induction by {gamma}-interferon is mediated through common DNA response elements. Proc. Natl Acad. Sci. USA 88:11305.[Abstract]
  31. Benech, P. D., Sastry, K., Iyer, R. R., Eichbaum, Q. G., Raveh, D. P. and Ezekowitz, R. A. B. 1992. Definition of interferon {gamma}-response elements in a novel human Fc{gamma} receptor gene (Fc{gamma}RIb) and characterization of the gene structure. J. Exp. Med. 176:1115.[Abstract]
  32. Eichbaum, Q. G., Iyer, R., Raveh, D. P., Mathieu, C. and Ezekowitz, R. A. B. 1994. Restriction of interferon {gamma} responsiveness and basal expression of the myeloid human Fc{gamma}gR1b gene is mediated by a functional PU.1 site and a transcription initiator consensus. J. Exp. Med. 179:1985.[Abstract]
  33. Perez, C., Wietzerbin, J. and Benech, P. D. 1993. Two cis-DNA elements involved in myeloid-cell-specific expression and gamma interferon (IFN-{gamma}) activation of the human high-affinity Fc{gamma} receptor gene: a novel IFN regulatory mechanism. Mol. Cell. Biol. 13:2182.[Abstract]
  34. Perez, C., Coeffier, E., Moreau-Gachelin, F., Wietzerbin, J. and Benech, P. D. 1994. Involvement of the transcription factor PU.1/Spi-1 in myeloid cell-restricted expression of an interferon-inducible gene encoding the human high-affinity Fc{gamma} receptor. Mol. Cell. Biol. 14:5023.[Abstract]
  35. Bellefroid, E. J., Poncelet, D. A., Lecocq, P. J., Revelant, O. and Martial, J. A. 1991. The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc. Natl Acad. Sci. USA 88:3608.[Abstract]
  36. Bellefroid, E. J., Lecocq, P. J., Benhida, A., Poncelet, D. A., Belayew, A. and Martial, J. A. 1989. The human genome contains hundreds of genes coding for finger proteins of the Kruppel type. DNA 8:377.[ISI][Medline]
  37. Crossley, P. H. and Little, P. F. R. 1991. A cluster of related zinc finger protein genes is deleted in the mouse embryonic lethal mutation tw18. Proc. Natl Acad. Sci. USA 88:7923.[Abstract]
  38. Tautz, D., Lehmann, R., Schnurch, H., Schuh, R., Seifert, E., Kienlin, A., Jones, K. and Jackle, H. 1987. Finger protein of novel structure encoded by hunchback, a second member of the gap class of Drosophila segmentation genes. Nature 327:383.[ISI]
  39. Chavrier, P., Lemaire, P., Revelant, O., Bravo, R. and Charnay, P. 1988. Characterization of a mouse multigene family that encodes zinc finger structure. Mol. Cell. Biol. 8:1319.[ISI][Medline]
  40. Margolin, J. F., Friedman, J. R., Meyer, W. K.-H., Vissing, H., Thiesen, H.-J. and Raucher, I. F. J. 1994. Kruppel-associated boxes are potent transcriptional repression domains. Proc. Natl Acad. Sci. USA 91:4509.[Abstract]
  41. Vissing, H., Meyer, W. K., Aagaard, L., Tommerup, N. and Thiesen, H. J. 1995. Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins. FEBS Lett. 369:153.[ISI][Medline]
  42. Kaplan, J. and Calame, K. 1997. The ZiN/POZ domain of ZF5 is required for both transcriptional activation and repression. Nucleic Acids Res. 25:1108.[Abstract/Free Full Text]
  43. Numoto, M., Niwa, O., Kaplan, J., Wong, K.-K., Merrell, K., Kamiya, K., Yanagihara, K. and Calame, K. 1993. Transcriptional repressor ZF5 identifies a new conserved domain in zinc finger proteins. Nucleic Acids Res. 21:3767.[Abstract]
  44. Sauer, F. and Jackle, H. 1991. Concentration-dependent transcriptional activation or repression by Krupple from a single binding site. Nature 353:563.[ISI][Medline]




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