Human leukocyte receptors for the Fc fragment of IgG (FcR) play an important role in immune responses as they link the humoral immune system with cellular effector functions. Fc receptors represent a family of cell surface glycoproteins mediating clearance and phagocytosis of immune complexes, various forms of antibody-dependent cellular cytotoxicity (ADCC) and release of inflammatory cytokines (for review, see (1) ). Fc receptors have been divided into three classes, namely a high affinity class (FcRI) and two low affinity classes (FcRII and FcRIII). All are members of the Ig gene superfamily. Within each group, multiple subtypes have been identified by molecular cloning. Structurally conserved extracellular domains are linked to divergent transmembrane and intracellular domains. Distinctive Fc receptor genes in addition to differently spliced messages from the same Fc receptor gene are expressed on different cell types in a tissue-specific manner generating diverse cellular responses upon interaction with IgG immune complexes (for review, see (2) ).

The low affinity receptor FcRIII (CD16) is a 50-80-kDa membrane glycoprotein(3) . At least two isoforms encoded by two distinct genes, FcRIII-A and FcRIII-B, have been identified(4) . An allotypic polymorphism, NA1 and NA2, has been described to be associated with the FcRIII-B receptor isoform. FcRIII-A is a transmembrane protein expressed on activated monocytes/macrophages, NK cells, and a subset of T cells(5, 6, 7) . In contrast, FcRIII-B is a glycosylphosphatidylinositol (GPI)-anchored protein that is constitutively expressed by neutrophils and after -interferon (IFN-) stimulation by eosinophils(4, 8, 9) . In addition, FcRIII-A-specific transcripts can be induced in growth-arrested human mesangial cells by IFN-(10) . While the GPI FcRIII-B isoform is expressed on the cell surface without other subunits, efficient expression of the transmembrane FcRIII-A requires the presence of the FcRI subunit and is also enhanced by co-expression of CD3 , a subunit of the T cell receptors' complex (11, 12, 13) . Through their differential requirements for these associated ``trigger'' molecules, the FcRIII isoforms mediate different functions(14) . The FcRIII-A transmembrane receptor on NK cells mediates ADCC, where it represents the only FcR(5) . The GPI-linked FcRIII-B receptor on polymorphonuclear leukocytes is certainly involved in cell activation, but its detailed role is still controversial(15, 16) .

The FcRIII receptor appears to be under complex control. First, it is highly tissue-specific and is expressed on granulocytes as well as on activated monocytes/macrophages, NK cells, and some cytolytic T cell subsets. Second, only granulocytes use the GPI-linked FcRIII-B isoform encoded by a second gene. Third, the FcRIII-B isoform can be induced during MeSO-initiated granulocytic differentiation of the HL60 human promyelocytic cell line. Fourth, depending on the cell type, both isoforms can be up-regulated by IFN-.

In this study we report the cloning of both FcRIII-A and FcRIII-B gene promoters. Sequence analysis of all the cloned transcription initiation sites demonstrates structural heterogeneity through the use of alternative 5`-UT/S1 exons from the FcRIII-A but not from the FcRIII-B gene. We have defined the 5` sequences necessary for promoter activity by transient transfection experiments with different parts of the FcRIII-A and FcRIII-B promoters in different cell lines. Both promoters (positions -198 to -10) displayed different tissue-specific transcriptional activities when coupled to a heterologous enhancer. These different tissue specificities are suggested to be due to nucleotide differences of the FcRIII-A and FcRIII-B genes identified at 10 positions in the -198/-10 region.

MATERIALS AND METHODS

Cells

()

Southern Blot Analysis

Figure 2: Panel A, structure of the human FcRIII cDNA pGP5. The exon organization derived from the gene map is drawn to scale (1977 bp), 5` to 3`, left to right. Exon-intron boundaries are indicated by verticalsolidlines. The signal sequences (S), extracellular domains (EC), transmembrane domain (TM), cytoplasmic region (C), and 3`-untranslated region (UT) are shown. The restriction endonuclease sites and nucleotide map units are designated for BglII (Bg), EcoRI (E), HindIII (H), KpnI (K), and SalI (S). The fragments of pGP5 utilized as hybridization probes are depicted (80 = BamHI-BglII, 178 = BglII-SalI, 380 = SalI-KpnI, 601 = KpnI-EcoRI, 670 = EcoRI-HindIII). Panel B, structural organization of the two genes for human FcRIII A and B. The exon-intron organization is presented to scale as a linear map, 5` to 3`, left to right. Exon locations (5` S1, S2, EC1, EC2, TM/C 3`) are boxed. Below the map, the two selected genomic clones 32 and 31 for human FcRIII genes A and B are shown. Restriction enzyme sites for BamHI (B), EcoRI (E), HindIII (H), and SalI (S) are indicated by verticallines. C, sequencing strategy of the 5` end of genes A and B. The restriction maps of the subcloned 5` ends of both genes (p32 = 5` end of FcRIII-A; p31 = 5` end of NA2-FcRIII-B) are designated. Restriction sites for ApaI (A), BamHI (B), BstYI (Bs), EcoRI (E), EcoRV (RV), SacI (S) and SphI (Sp) are indicated by verticallines. The sequencing strategy is indicated by arrows below the maps.

Screening and Isolation of Genomic DNA Clones Encoding Human FcRIII-A and FcRIII-B

Figure 1: Southern blots of human genomic DNA identify differences among the FcRIII-A and the NA1 and NA2 alleles of the FcRIII-B genes. Genomic DNAs isolated from peripheral blood lymphocytes from individuals of a different phenotype (designated NA2, NA1, NA1NA2) were used. Panel A, digestion was performed with BamHI (B), HindIII (H), EcoRI (E), or with the combination of two enzymes (HB) and (EB). The digested DNA was electrophoresed in an agarose gel, transferred to Hybond-N, and hybridized with the 5` 80 bp containing BamHI-BglII fragment of cDNA pGP5 (Fig. 2A). The NA2 homozygote donor demonstrates a second BamHI restriction fragment not observed in the absence of the NA2 allele. PanelB, digestion was performed with TaqI. After electrophoresis and transfer to nylon, the blot was hybridized with an EC1 oligonucleotide. Rehybridization was performed with the 30-mer oligonucleotide complementary to S1 sequences used by primer extension analysis (see ``Materials and Methods'').

Characterization and Sequence Analysis of Genomic DNA Clones Containing the 5`-Regulatory Region of the FcRIII-A and FcRIII-B Genes

Primer Extension Analysis

RNase Protection Assay

Figure 3: Nucleotide sequence comparison of 5`-flanking regions derived from the human FcRIII genes A and B. The HindIII-XhoI fragment containing the 5`-flanking region and the first three coding exons for gene A are in uppercase letters; the translated amino acid sequence is written in the three-letter code. Nucleotide sequences shown at the right are numbered starting from the ATG translation-initiation codon designated as +1. If nucleotide sequences for gene B differ from gene A, they are given below those of FcRIII-A. Dashed lines indicate nucleotide identity to gene A. Base pair substitutions are shown by the nucleotide change; nucleotide deletions are marked by asterisks.

Figure 5: Mapping the transcription-initiation sites of the human FcRIII A and B mRNA. a, schematic presentation of the promoter region of the FcRIII genes A and B, the two P-labeled antisense RNA probes, and the oligonucleotide primer used for RNase protection and primer extension assays, respectively. The sequences of genes A and B containing transcription-initiation sites are shown below the schematic diagram. The transcription-initiation sites are indicated by vertical bars; the height of each bar is roughly proportional to the amount of mRNA starting at a particular site, as estimated from the RNase protection experiments or primer extension assay. b, primer extension of the human FcRIII A and B RNAs: 50 µg of total RNA from PMN and negative control tRNA (not shown) and 10 µg of poly(A) RNA from NK cells were hybridized with a 5`-end labeled synthetic 30-mer oligonucleotide complementary to nucleotides +11 to +40 in exon S1 followed by extension using reverse transcriptase. Sizes of reverse transcribed products were determined by comparison with HaeIII digested pBR322 DNA fragments of known molecular weights as shown on the right. The numbering of the primer-extended products indicate their distance to the ATG codon designated as +1. The localization of the major RNA species relative to the ATG codon is indicated on the left for the FcRIII-Aa1 and FcRIII-Bb1 transcripts. The 167-, 156-, 153-, and 540-nucleotide bands represent the FcRIII-Aa2/a3 and the potential FcRIII-Aa4 transcripts. c, RNase protection assay of the 5` portion of FcRIII A and B RNAs: 30 µg of total RNA from negative control tRNA and from PMN and NK cells were hybridized to the riboprobe synthesized from the gene A-derived BamHI-EcoNI fragment or from the gene B-derived BstYI-EcoNI fragment. Sizes of protected fragments were determined by comparison with a sequencing reaction run in parallel. The negative numbering of the multiple transcription-initiation sites indicate their distance to the ATG codon designated as +1. Using the III-B riboprobe, the 380-nucleotide FcRIII-Aa4 protected fragment observed in NK cells with the III-A riboprobe is converted into four smaller bands, as indicated by the arrows. The protected band at -45 represents the splice site used by the FcRIII-Aa2 transcript, as highlighted by the asterisks.

Cloning of FcRIII-A and FcRIII-B cDNA 5` Ends by RACE/PCR

FcRIII-A and FcRIII-B Promoter Constructs and Transient Expression Assay

RESULTS

Structural Analysis of the 5` Region of the Two Human FcRIII-A and FcRIII-B Genes

The restriction pattern of TaqI after hybridization with oligonucleotides specific for the two Exons S1 and EC1 distinguishes between the FcRIII-A and the NA1 or NA2 FcRIII-B genes. Irrespective of the phenotype used, TaqI demonstrated two restriction fragments of about 6 kb (FcRIII-A, from 0.2 kb downstream to 5.8 kb upstream of the ATG) and about 2 kb (NA1/NA2-FcRIII-B, from 0.2 kb downstream to 1.8 upstream of the ATG) (Fig. 1B). The location of these TaqI sites within the first intron and the 5`-flanking regions of the FcRIII-A and the two alleles of the FcRIII-B genes were observed to be different from the corresponding region presented earlier(4) .

Cloning and Sequence Comparison of Genomic Clones Containing the 5` Regions of the Two Human FcRIII-A and FcRIII-B Genes

The nucleotide sequences beginning at the HindIII sites 1817 and 1821 bp upstream and ending at different XhoI sites 1115 and 1216 bp downstream of the first ATG present in the pGP5 cDNA sequence were determined for the FcRIII-A and FcRIII-B genes (Fig. 3). The XhoI sites are located at positions within the EC1 exon as expected from cDNA sequence analysis of NA1 or NA2 FcRIII-B and FcRIII-A transcripts(4, 23, 30, 31) . Comparison of the sequences indicates only slight differences. A total of 58 substitutions and 16 deletions are detected within the first two introns and the flanking region. From the 38 substitutions of the flanking region, 26 are found within the first 500 bp upstream of the ATG codon. The deletion of an 8-bp sequence TGGAGCCT at position -880 in the FcRIII-A gene changes the 3-fold repeating sequence GGAGCCCT present at the same position within the FcRIII-B gene. The genomic sequences upstream from the ATG codon lack the common CAAT and TATA promoter elements at their characteristic positions. In case of the FcRIII-B gene, a pyrimidine-rich initiator (Inr) sequence is present (32) . To identify putative regulatory DNA elements important for the different cell type specificities of the FcRIII-A and FcRIII-B genes, the 1.8-kb flanking regions were analyzed by the TFD (version 7.3, September 1993) data base. A total of 599 transcription factor consensus sites for FcRIII-A and of 583 for FcRIII-B was observed. No mismatch to the consensus was allowed during this search. 39 consensus sites are differentially distributed between the FcRIII-A and FcRIII-B genes, 13 of them are located near the FcRIII-Aa1 and FcRIII-Bb1 transcription start sites (see below) (see Fig. 7).

Figure 7: Different distribution of putative transcription factor binding sites to the FcRIII-A and FcRIII-B promoters. Shown are the sequences of each -198/-10 gene promoter. The positions of all nucleotide differences are indicated. All transcription factor consensus sites are boxed.

Comparison with promoter sequences of other Fc receptor genes reveals a significant conservation in one region of these promoters. We termed this region the FcR motif. The FcR motif has the sequence TTCCTTCCTCTTTT homologous to the PU-box and is found in the human FcRI-A/-B/-C, FcRII-A, and the mouse FcRI, FcRIII genes within the first 100 bp of the ATG(33, 34, 35, 36) . A similar FcR motif is also present in the human FcRIII-A/-B (positions -80 to -67, Fig. 3) and the rat and mouse FcRI genes(37, 38) .

Mapping and Cloning of the Transcription Initiation Sites of the Human FcRIII-A and FcRIII-B Genes

Figure 4: Structure of the 5`-end of the human FcRIII-A and FcRIII-B genes and nucleotide sequence comparison of distinct transcripts derived from the two FcRIII genes. a, 5`-end exon-intron organization of the two genes for FcRIII. Exon locations (5`-UT, 5`-UT/S1, S2, EC1) are indicated by boxes. There are multiple transcription initiation sites shown by the arrows. The positions of the start sites cloned by RACE/PCR are indicated as the distance to the ATG codon. The distinct transcripts are designated as b1 in the case of the FcRIII-B gene, and as a1, a2, a3, and a4 in the case of the FcRIII-A gene. Positions -113/-111/-107 represent the main starting site used by FcRIII-Bb1 transcripts. Use of the more 5`-sites at -860/-849 by the FcRIII-Aa2 and FcRIII-Aa3 transcripts are associated with alternative splicing of the 3` end of the first intron, as indicated by the lines below the FcRIII-A gene. A FcRIII-Aa4 cDNA clone starting at -333 is also shown, but it should be noted that it does not represent a full-length a4 transcript. b, nucleotide sequence comparison of distinct transcripts derived from the two FcRIII genes. RACE/PCR products resulting from amplification with a reverse complement EC1 primer, followed by a SalI/BglII digest were cloned into pBluescript KS+, as described under ``Material and Methods.'' Sequence analysis was performed with the oligonucleotide reverse complementary to the underlined nucleotides. Four types of transcripts originating from the FcRIII-A gene and one transcript type originating from the FcRIII-B gene can be distinguished. The transcript names are listed at the left. With the exception of the FcRIII-Aa4 transcript, all types of transcripts (a1, a2, a3, and b1) are associated with multiple initiation sites. For reasons of clarity, these sites are not presented here but are shown under a (see above). The positions from the ATG codon (typed in boldface) of the most 5`-site used by each type of transcripts and from the 5`-UT/5`UT-S1 exon borders of the FcRIII-Aa2/a3 transcripts are shown above the respective nucleotide sequence.

In order to verify the heterogeneity of the cloned transcription-initiation sites from the different types of FcRIII-A and FcRIII-B transcripts, primer-extension analysis and RNase protection experiments were performed. The results obtained by both methods are summarized in Fig. 5a (lowerpart). Due to the absence of a single defined major transcription initiation site common to both genes, the numbers of nucleotide positions in the 5`-flanking regions were assigned on the basis of the first nucleotide of the ATG translation start codon as +1. Therefore, the size of the observed protected bands or reverse transcribed products is shown by negative numbering estimated by their distance to the ATG codon. For primer-extension analysis, the P-end labeled oligonucleotide reverse complementary to nucleotides +11 to +40 of both genes (Fig. 5a) was used, as described under ``Materials and Methods.'' By using RNA from NK cells and PMN, multiple bands were observed that were absent with yeast tRNA. The starting sites at 20, 27, and 33 bp upstream of the first ATG codon (Fig. 5b) were used equally by FcRIII-Aa1 in NK cells and FcRIII-Bb1 in PMN. In NK cells, the bands corresponding to the putative -45, -54, -63 transcripts were hardly detectable compared with PMN. Compared with the RACE/PCR data, the 167-, 156-, and 153-nucleotide reverse transcribed products represented the FcRIII-Aa2/a3 transcripts (Fig. 4b). In the RNase protection experiments shown in Fig. 5c, two FcRIII-A and FcRIII-B specific riboprobes, from -348 to +60 relative to ATG, as diagrammed at the top of Fig. 5a were used. Consistent with the primer extension data, RNase protection located the transcription-initiation sites common to both genes at -19, -27, -33 but with different intensities to -45, -54, -63 upstream of the first ATG codon (Fig. 5c). In NK cells, the protected fragment at -45 is much more prominent than the corresponding reverse transcribed product from the same position, as indicated by the asterisks in Fig. 5c compared with Fig. 5B. Therefore, this band represents the frequently used splice site found in the cloned FcRIII-Aa2 transcripts (Fig. 4b).

Transcripts at positions -77/-81 and clustering around -113 were observed by both methods in PMN but not in NK cells (Fig. 5, b and c). The cluster is the main FcRIII-Bb1 transcription initiation site not only in PMN but also in MeSO-differentiated HL60 cells (data not shown). Immediately upstream, there is a region with homology to the functional active interferon responsive region of the FcRI receptor gene(39) . Not only in NK cells but also in activated monocytes and the FcRIII-A positive T cell clone 1B3(7) , the transcripts from -77/-81 were not initiated (data not shown). All of these cell types express the FcRIII-A gene-encoded isoform of the receptor. A sequence closely resembling the initiator element (Inr) encompasses the transcription start site at -77/-81. At position -75 within this Inr motif, a nucleotide exchange T to C was detected between the FcRIII-A and FcRIII-B genes.

A major protected fragment of about 380 nucleotides corresponding to a putative FcRIII-Aa4 -338 transcript is seen in NK cells only using the FcRIII-A derived riboprobe (Fig. 5c, closedarrow). After hybridization with the FcRIII-B derived riboprobe, this specific band was converted to four main protected fragments of 236, 204, 151, and 137 nucleotides caused by improper FcRIII-A:FcRIII-B RNA:RNA pairing recognized by RNaseA (Fig. 5c, open arrows). This pattern of protected fragments in NK cells was also observed by using a FcRIII-B riboprobe from -707 to +60 relative to ATG (data not shown). Reverse transcriptase PCR analysis with the primer extension oligonucleotide and a second primer complementary to positions -300/-280 but not to -370/-350 yields a FcRIII-Aa4 product of the expected size in NK cells, which is absent in PMN (data not shown). Results of primer extension did not predict a start site at -338 but showed a 540-nucleotide extended product in NK cells (Fig. 5b, openarrow). Therefore, it is very likely that the position at -338 represents a FcRIII-Aa4 specific splice acceptor site.

Functional Activity of the Promoter Regions of the Two Human FcRIII-A and FcRIII-B Genes

Figure 6: Cell type specific activity of the FcRIII A and B promoters after transfection into different cell lines. a, genomic material of the FcRIII A and B genes extending to the upstream positions -33, -198, and -1817/-1821 from the ATG codon were cloned into the luciferase reporter plasmid pLuc (left part). Transfections were performed using electroporation of logarithmically growing U937, HL60, YT, and Jurkat cells as described under ``Materials and Methods.'' 20 h after electroporation, cell extracts were prepared and assayed for luciferase activity in a luminometer. An SV40 luciferase chimeric gene was used as a positive control to monitor the efficiency of transfection, as indicated by the gray bars. White bars represent activities from constructs containing promoter sequences of the FcRIII-A gene, dark bars represent results from the B gene. At least five independent experiments were performed with each construct. The promoter activities of a representative experiment measured in relative light units (RLU) are shown in the right part for all cell types analyzed. b, YT, HL60, and U937 cells were transfected with the 198 promoter fragment of the FcRIII-A and FcRIII-B genes in the presence or absence of the heterologous human lysozyme enhancer cloned 3` to the luciferase reporter gene into the same plasmid. Dark and white bars represent luciferase activities dependent on FcRIII-B and FcRIII-A gene sequences, respectively. Results are depicted as described above

In the promyelocytic HL60 cells, the complete 5`-flanking region of the FcRIII-B gene, pIII-B(-1821)Luc, shows a strong promoter activity compared with a reduced activity using the same fragment of the FcRIII-A gene (Fig. 6a). Similar results are obtained using HL60 cells expressing FcRIII-B after MeSO treatment (data not shown). Using the promonocytic U937 cells, results are less pronounced, and without a heterologous enhancer only basal and almost equal levels of transcription can be observed (see below). In contrast, in the NK-like YT cell line, the FcRIII-A gene construct pIII-A(-1817)Luc is much more active than the corresponding fragment of the FcRIII-B gene (Fig. 6a). Thus, the differential promoter activities of the two 1.8-kb 5`-flanking regions in the YT and HL60 ± MeSO cells reflect the expected gene activities of FcRIII-A and FcRIII-B in NK cells and PMN, respectively. The 198-bp promoter fragments of gene A termed pIII-A(-198)Luc and of gene B termed pIII-B(-198)Luc, each containing all respective starting sites of the FcRIII-Aa1 and FcRIII-Bb1 transcripts (Fig. 4a) conferred only a reduced activity compared with the complete 1.8-kb sequences. This indicates that additional more distal located elements enhance the activity of the 198-bp FcRIII-A and FcRIII-B promoters. The activities of these two 198-bp promoters were also stimulated in U937, HL60, and YT cells through the heterologous, human lysozyme enhancer within the combined pIII-A(-198) + (LysE)Luc and pIII-B(-198) + (LysE)Luc constructs (Fig. 6b). In YT cells, the enhancer-dependent stimulation was selective for the FcRIII-A 198-bp promoter inducing a differential activity specific for NK cells. On the other hand, a selective enhancement of the FcRIII-B promoter was found in the myeloid U937 and HL60 cells. Therefore, sequences that direct a different cell type specificity of FcRIII-Aa1 versus FcRIII-Bb1 transcription are located to the first 198 bp from the ATG codon within the respective FcRIII-A and FcRIII-B promoters.

DISCUSSION

These studies establish the initial characterization of the FcRIII-A and FcRIII-B genes with emphasis on the structure of their promoter regions. Both genes have nearly identical restriction maps but can be distinguished through different gene-specifically associated TaqI restriction fragments. Differences in the location of the TaqI sites within the 5`-flanking regions of the FcRIII-A and FcRIII-B genes proved to be useful in cloning the respective gene promoter regions. Here, we demonstrate differences in the sites of transcription initiation as well as in the 5` end gene organization of both genes. Regulatory regions within their 5`-flanking sequences contributing to the cell type specificity of FcRIII-A and FcRIII-B gene expression were identified.

DNA sequence analysis of the 1.8-kb FcRIII-A and FcRIII-B promoters reveals an overall identity of >95%. Most of the nucleotide differences between FcRIII-A and FcRIII-B are within the first 500 bp of the ATG. From the substitutions identified between FcRIII-A and FcRIII-B, 11 generate the dinucleotide CpG, a candidate for methylation. In the murine system, thymoma cells express the low affinity receptors FcRII and FcRIII only after pretreatment with 5`-azacytidine, indicating a possible role for CpG methylation in controlling Fc gene activity(40, 41) . The methylation status of the transcriptionally active region of the human FcRIII-A and FcRIII-B receptors is currently under investigation.

Differential experimental strategies were necessary to map the transcription initiation sites, to identify a more 5` end heterogeneity in FcRIII-A mRNA than in FcRIII-B mRNA, and to determine at least four distinct types of FcRIII-A transcripts, a1-a4. The 5` end of the FcRIII-B gene is organized in a single 5`-UT/S1 exon continuous with the ATG codon. The 5` end of the FcRIII-A gene can be encoded by alternative 5`-UT and 5`-UT/S1 exons. Therefore, compared with the organization of other human Fc receptor genes, the 5` end of FcRIII-B is similar to that of FcRI(39) , whereas the 5` end of FcRIII-A is related to FcRII-A(34) . At least four distinct types of FcRIII-A transcripts could be identified, distinguished by 5`-UT ends of different sizes and sequences. Each type of transcripts uses multiple starting sites. FcRIII-Aa1 transcripts start from -33, -27, and -20 upstream from the ATG. These three sites are also used by the FcRIII-Bb1 transcripts. Initiation of FcRIII-Aa2 occurs at -860 and -849 in a discrete 5`-UT exon. The sequence is colinear with FcRIII-A genomic sequence until a 5`-GT splice site (position -794) and continues through a 3`-AG splice site (position -45) into the 5`-UT/S1 exon. The related type of FcRIII-Aa3 transcripts uses an alternative 3`-consensus AG splice site located at -63. The protection pattern of RNA obtained from NK cells with different riboprobes demonstrates an FcRIII-Aa4 transcript at about -338 from ATG. Results of primer extension did not predict a start site at -338 but showed a 540-nucleotide extended product. A potential intron 3`-splice acceptor site is present near the -338 site. These observations are indicative for the existence of another discrete exon mapped further upstream, which is encoded only by the FcRIII-Aa4. The a4 transcript derived by RACE/PCR cloning from NK cells shows a continuous sequence from -333 to the ATG identical with FcRIII-A gene sequences. Northern blot analysis with exon-specific FcRIII cDNA probes derived from pGP5 (Fig. 2A) demonstrates no variation in molecular weight; only a single 2.2-kb FcRIII-A mRNA is detectable. Therefore, we speculate that the elongation of the 5` end has to be compensated by a shortening at another region within the FcRIII-Aa4 transcript. It will be necessary to obtain full-length cDNA clones corresponding to FcRIII-Aa4.

Two main differences have been observed in the initiation of FcRIII-Aa1 and FcRIII-Bb1 transcripts. First, the major transcription initiation sites of FcRIII-Bb1 in freshly isolated PMN and in PMN-differentiated HL60 cells (data not shown) are clustered around -113 from ATG and are absent in FcRIII-Aa1 expressing NK cells. Immediately upstream there is a region of homology to the functional active interferon responsive region present within the promoter of the high affinity FcRI receptor gene (Fig. 7)(39) . INF-induced FcRIII-B transcription in eosinophils and INF-induced FcRIII-A transcription in glomerular mesangial cells has been described(9, 10) . Second, FcRIII-Bb1, but not FcRIII-Aa1 transcripts, can be initiated at -77 and -81 from ATG mediated by a pyrimidine-rich initiator (Inr) of the sequence TACTCCCT also found within the adenovirus major late promoter (TFII-I-ML-Inr2)(42) . At position -75 within this Inr motif, a nucleotide exchange of C to T was detected between the FcRIII-B and FcRIII-A genes resulting in the alteration of the Inr consensus YAYTCYYY to YAYTTYYY(42) . Based on recent in vitro findings, this alteration by itself cannot be sufficient for a nonfunctional initiator in case of FcRIII-A(43) . The factor TFII-I is binding not only to some Inr elements but also to upstream USF (E box) sites. It was also suggested that TFII-I and USF interact cooperatively at both Inr and E box sites(42) . In this respect, it is interesting to note that a potential USF binding site of the sequence GGCAGGTGAC is present at position -122 within the FcRIII-B gene (44) . This E box is mutated by substituting the CA to a TG at position -120/-119 in case of FcRIII-A. Therefore, it might be possible that the mutations both at positions -120/-119 and -75 are necessary to account for the observation that the -77/-81 transcriptional start site is not utilized in the FcRIII-A gene.

FcRIII-A and FcRIII-B do not possess typical TATA and CAAT boxes, a finding consistent with the presence of multiple initiation sites. Most but not all of the different initiated FcRIII-Aa1 and FcRIII-Bb1 transcripts can be explained by the presence of the potential interferon responsive region and the Inr/E-box sequences. Several putative binding sites of transcription factors are differentially distributed between the FcRIII-A and FcRIII-B gene promoters as marked in Fig. 7. A Sp1 consensus sequence at -97(45) , two AP-2 sites at -98 and -181(46) , one AP-1 site at -155(47) , one CDP (CCAAT displacement protein) site at -125(48) , one NF-B site at -182(44) , an Ets-1 element at -78(44) , and a potential site for the ets family member PU.1 (GAGGAA) in inverse orientation at -76 (49) are present in case of FcRIII-A only. PU.1 binds to the PU box and is expressed in myeloid cells like U937 and HL60 (49) but not in NK cells and the YT cell line (data not shown). The sequence around the putative PU.1 site (TTACTTCCTCCTGT) in the FcRIII-A gene is nearly identical to a 14-bp promoter element TTCCTTCCTCTTTT conserved among the human FcRI-A/-B/-C, FcRII-A, and the mouse FcRI, FcRIII genes(33, 34, 35, 36) . A similar region of homology (TTCCTTCTCCTTYG) is also present in the rat and mouse FcRI genes (37, 38) . A common feature of the Fc receptor genes is the location of this conserved element, the FcR motif, centered at the transcription initiation units within the first 100 bp from the ATG codon. This FcR motif has been characterized to be the DNA target of myeloid specific activators in the FcRI-A gene(50) . Preliminary analysis suggests an interaction of nuclear proteins from myeloid but not NK cells with an oligonucleotide representing the FcRIII-A promoter region from bp -95 to -54 containing the putative PU.1 site (data not shown). Interestingly, the myeloid-specific shifted complex can be competed with this FcR motif. Thus, we speculate that the conserved FcR motif most likely recognized by PU.1 might be involved in the expression of Fc receptors in myeloid cells.

Cells expressing the FcRIII-A or FcRIII-B receptor, namely culture-activated monocytes, granulocytes, and NK cells are refractory to transfection. Therefore, we have utilized the NK cell-like YT line, the promonocytic U937 cells, and the promyelocytic HL60 cells(21, 18, 19) . These cells are easy to transfect, but they present a premature state of FcRIII-A and FcRIII-B expression. The NK-like YT cells have been described to express FcRIII only at low levels. U937 and HL60 cells are normally negative for FcRIII-A or FcRIII-B expression. However, it should be noted that the expression of the FcRIII-B receptor can be induced after granulocytic differentiation of HL60 cells by MeSO(20) . Sequences of both genes from positions -198 to -10 encoding for all of the mapped and cloned initiation sites of FcRIII-Aa1 and FcRIII-Bb1 transcripts behave like a typical promoter in these cells: they direct transcription in an orientation-dependent manner (data not shown) and are stimulated by a heterologous enhancer (Fig. 6b). Furthermore, using YT and HL60 cells, the -198/-10 promoters are stimulated in a cell type-specific manner by the human lysozyme enhancer as well as with homologous sequences up to about 1800 bp. For example in the NK-like YT cells, the FcRIII-A promoter is much more active. In contrast, in HL60 cells, the FcRIII-B promoter is most active. In accordance with this observation, granulocytic differentiation of HL60 cells using MeSO results in FcRIII-B specific promoter activity (not shown). This differential activity agrees to the in vivo specificity of the complete FcRIII-A and FcRIII-B genes in NK cells versus PMN. Thus, important tissue-specific elements are located in the -198/-10 regions. A total of 10 nucleotide exchanges predicted by sequence analysis exists within the FcRIII-A and FcRIII-B -198/-10 promoters. 8 of the 10 nucleotide differences are of special interest able to generate different consensus sites for well characterized transcription factors (summarized in Fig. 7). Preliminary analysis suggests that cell type specificity cannot simply be mapped and attributed to just one nucleotide difference only. ()For both cell type specificities, several elements are likely to work together in a cooperative fashion. Therefore, different combinations of mutated FcRIII-A and FcRIII-B hybrid promoters need to be constructed and tested for their ability to change the promoter activities and specificities. With HL60 and YT cells, there is now an excellent model available to identify and characterize mutations sufficient and necessary for expression of FcRIII-A and FcRIII-B in NK cells versus PMN, respectively.

FOOTNOTES

*

This work was supported in part by Grants SFB265/B1 and Schm 596/3-2 of the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z46222 [GenBank]and Z46223[GenBank].

§

Supported by a postgraduate grant from the state of Niedersachsen.

¶

To whom correspondence should be addressed: Dept. of Immunology, Hannover Medical School, Konstanty-Gutschowstrasse 8, 30625 Hannover, Germany. Tel.: 49-511-532-3623; Fax: 49-511-532-5648.

()

The abbreviations used are: ADCC, antibody dependent cellular cytotoxicity; bp, base pair(s); FcRIII, low affinity receptor for Fc domain of IgG; GPI, glycosylphosphatidylinositol; kb, kilobase pair(s); PCR, polymerase chain reaction; PMN, polymorphonuclear leukocytes; RACE, rapid amplification of cDNA ends; NK, natural killer; USF, upstream stimulatory factor; UT, untranslated.

()

J. Yodoi, personal communication.

()

T. Grussenmeyer, unpublished observations.

ACKNOWLEDGEMENTS

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