INTRODUCTION

Natural killer (NK)¹ lymphocytes are important effector cells in the first line of immunologic defense and play a major role in immunosurveillance. NK cells have the ability to lyse tumor cells and play a crucial role in the control of viral infections. NK cells, like cytolytic T cells (CTL), respond specifically to polymorphic determinants of MHC class I molecules. NK cells express receptors that bind to these molecules. However, instead of activating the cytolytic response as in CTLs the recognition of MHC class I turns off the NK cells. Therefore, virus infected and malignant transformed cells are lysed by NK cells as a consequence of loss of MHC class I surface expression (for reviews, see Refs. 1 and 2).

In addition to the allotype MHC class I specific receptors almost all human NK cells express the low-affinity receptor for the Fc portion of IgG, FcRIII-A (CD16-A). FcRIII-A belongs to the family of immunoglobulin G receptors (FcR) involved in the clearance of immune complexes, phagocytosis of opsonized pathogens, and various forms of antibody-dependent cellular cytotoxicity (3). The FcRIII-A  chain forms a multimeric transmembrane receptor complex with homo- and heterodimers of the FcRI subunit and/or CD3 subunit (4, 5, 6). FcRIII is also highly expressed in PMN but as a single chain receptor attached to the plasma membrane by a glycosylphosphatidylinositol anchor, FcRIII-B. The transmembrane FcRIII-A receptor on NK cells mediates antibody-dependent cellular cytotoxicity and all other antibody-dependent responses (7, 8, 9). The glycosylphosphatidylinositol-linked FcRIII-B receptor on PMN is involved in cell activation but its detailed role is less clear (10, 11, 12). Other surface molecules like FcRII-A and CR3 receptors are likely to be involved in the activation process of PMN after FcRIII-B cross-linking (13, 14). The molecular basis for the expression of functionally distinct FcRIII isoforms is given by the presence of the highly homologous FcRIII-A and FcRIII-B genes (15). Transfection experiments of reporter gene constructs indicate that differences in the proximal 198/10 gene promoter regions might be crucial for directing the expression of the FcRIII-A and B receptors to NK cells and PMN, respectively (16).

The characterization of the molecular events leading to NK cell-specific FcRIII-A expression is complicated by the finding that transcripts initiating outside the 198/10 Pprox promoter exist in NK cells (16, 17). Cloning and sequencing of these FcRIII-A transcripts, designated a2/3 and a5/6, demonstrated that they encode the known FcRIII-A receptor. The coexpression of FcRIII-A transcripts with alterations in the extracellular domain were also evident but could not be linked to the distinct a2/3 and a5/6 mRNA start sites. The medial 1817/850 region of the FcRIII-A gene containing the a2/3 and a5/6 initiation sites functioned as a transcriptional regulator in the immature NK cell line YT. This control region consisted of the two independent promoters Pmed1 (942/850) and Pmed2 (1376/1123). The 93-bp 942/850 Pmed1 promoter was further characterized. It contained a cis-acting DNA element important in conferring optimal and YT cell-specific promoter activity within its first 21 bp. These results suggested that this DNA element might be a useful target for identification of YT and NK cell restricted transcription factors.

MATERIALS AND METHODS

Polymorphonuclear granulocytes (PMN), peripheral blood mononuclar cells (adherent cell fraction, MO), and NK cells were prepared according to standard conditions described elsewhere (9, 16, 18, 19). The / T cell (MK1) and cytotoxic T cell (1B3) clones derived from donors with distinct CD16 subsets from sorted fractions were generated by limiting dilution. Clones were plated at 1 cell/well onto a feeder layer with irradiated allogeneic PBL and Epstein-Barr virus-transformed B lymphoblastoid cells (Laz509) (8). Human tumor cell lines HL60 and YT used for transfection experiments were cultured in RPMI 1640 containing 10% fetal calf serum and supplements (16). HL60 is a promyelocytic cell line and YT a cell line with NK cell characteristics (20, 21). In some experiments, HL60 cells were induced to express the FcRIII-B receptor isoform upon culturing in the presence of 1.2% Me₂SO (16).

Cloning of FcRIII-A mRNA Start Sites by RACE PCR

The strategy to obtain cDNA clones for a1, a2/3, and a5/6 related transcription initiation sites was nearly identical to that described recently (16). Starting with 2 µg of poly(A)⁺ RNA from NK cells, the reverse transcription reaction was performed using 20 pmol of a FcRIII-A gene-specific primer reverse complementary to EC1 sequences from exon V. The cDNA pools were subsequently tailed with 15 units of TdT (Life Technologies, Inc.) in the presence of 0.1 mM dATP for 10 min at 37 °C. After purification of the reaction mixture, one-fifth was used for PCR amplification with 10 pmol of oligo(dT)₁₇-adaptor, 25 pmol of adaptor, and 25 pmol of a second internal EC1 primer in a total volume of 100 µl. 2 units of Taq DNA polymerase (Promega) was added and the mixture was annealed at 56 °C for 2 min. The tailed cDNA was extended at 72 °C for 30 min. PCR conditions were as described (16). Purified RACE PCR products were digested with SalI and BglII and cloned into SalI/BamHI-digested pKS+, as outlined in Fig. 2. Miniprep plasmid DNA was sequenced using the ³²P-labeled second EC1 primer.

Identification of different 5-UTR exons in the FcRIII-A gene by RACE PCR cloning the RNA initiation sites of transcript classes a1, a2/3, and a5/6.

Analysis of the distinct FcRIII transcript classes by primer extension and RNase protection assay was performed according to methods described previously (16). Total RNAs were prepared by the guanidinium thiocyanate method (22). The complementary oligonucleotide used for primer extension, 5-CTTCCTCGTGTTACCCAGGTCCTGCGGATT-3 (795 to 824), corresponds to the genomic FcRIII-A sequence relative to the translation start codon (ATG). 10-50 µg of various RNAs were annealed to this ³²P-end-labeled oligonucleotide. The extension products were sized by electrophoresis on an 8% denaturing polyacrylamide gel and visualized by autoradiography. The dideoxynucleotide sequencing ladder of a plasmid containing the sequence with the transcription start sites was used as a molecular weight marker and run in parallel. For RNase protection analysis of a2/3 and b2 transcription initiation sites, the 236-bp HincII/ApaI restriction fragment from the FcRIII-A gene (942 to 707) was inserted into the pBluescript KS(+) vector. For analysis of transcripts encoding for extracellular alterations an SalI/PvuII fragment containing the EC1/EC2 exon border (position 258 to 406) of the pGP5 derived NA1 FcRIII-B cDNA (23) was used and cloned into pKS+. The respective antisense RNA probes were synthesized using T7 RNA polymerase. All other conditions were the same as described previously (16).

RT-PCR Analysis of FcRIII-A a2/3 and a5/6 Transcript Classes

1 µg of total RNAs were reverse transcribed using 15 units of avian myeloblastosis virus reverse transcriptase (Promega). PCR for amplification of the cDNA pool was started following a denaturing step at 94 °C with the addition of Taq DNA polymerase after 10 min at 85 °C. 35 cycles were done each including a 40-s denaturing step at 94 °C, a 30-s annealing step at 52 °C or 64 °C depending on the primer pair in use, a 45-s extension step at 72 °C, followed by a final 5-min extension at 72 °C. 5 primers specific for the distinct 5-UTR of a2/3 (5-AATCCGCAGGACCTGGGTAACAC-3, 824 to 802) and a5/6 (5-TCCACCCCTAACAAGTATC-3, 1157 to 1139) transcript classes were used with the same reverse complementary 3 primer specific for the 3-UTR originally described for a1 (5-CAGAGGCCTGAGGATGATGGGGTTGC-3, +829 to +854) (15). The PCR products were analyzed on a 1.2% agarose gel and visualized by ethidium bromide staining.

The constructs were generated by cloning FcRIII-A and FcRIII-B genomic sequences from 10 to 198, from 10 to 1817/1821, from 850/846 to 1817/1821, from 850/846 to 942/947, and from 850/846 to 921/925 into the BamHI/BglII site of the promoterless luciferase expression vector pLuc. For promoter assays in the presence of the FcRIII-A or the FcRIII-B enhancer the sequence from position +10/+712 of both genes were cloned as a PvuII subfragment into the KpnI site 3 to the luciferase gene. 5 and 3 deletion mutants were prepared from the medial 1817/850 FcRIII-A control region by the use of restriction sites at positions 1579, 1376, 1123, 942, and 921 for subcloning. Relevant deletion mutants from the corresponding FcRIII-B region as well as some hybrid constructs were similarly generated. All constructs were sequenced by the dideoxy chain termination method and purified over two rounds of centrifugation in cesium chloride/ethidium bromide gradients.

YT or HL60 cells, maintained at 1 or 2 × 10⁷ cells/ml, were electroporated with 10 or 80 µg of the various reporter plasmids at 270 V and 750 or 2400 microfarads, using a Eurogentec gene pulser followed by transfer to 15 ml of prewarmed RPMI/10% fetal calf serum medium. Twenty hours after transfection cells were harvested and washed once in PBS. Cells were extracted in 100 µl of hypotonic buffer (25 mM Tris phosphate, pH 7.8, 8 mM MgCl₂, 1 mM EDTA, 10% glycerol) by two cycles of freezing and thawing. Luciferase activity was measured in a Berthold biolumat in 22.5 mM Tris phosphate, pH 7.8, 2 mM ATP, 10 mM MgSO₄, and 0.2 mM Luciferin.

RESULTS

Mapping and Cloning of Novel FcRIII-A Transcript Classes

Functional analysis of the 5-flanking region of the human FcRIII-A gene and the respective region of the highly homologous FcRIII-B gene led to the identification of the Pprox (198/10) promoter regions which display different tissue-specific transcriptional activities in NK cells and PMN (16). In addition, RACE-PCR cloning indicated that additional FcRIII-A mRNA start sites occurring outside the Pprox region exist in NK cells. From a total analysis of 11 cloned RACE PCR products 3 cDNA clones assigned as a2/3 were identified to initiate at two sites from 860 and 849 in a separate exon spliced from position 795 to 44 (a2) or to 62 (a3) (16). We now extended the RACE PCR analysis and isolated 17 additional cDNA clones containing the 5 ends of FcRIII-A transcripts from NK cells. 7 clones contained the 5-UTR of transcript class FcRIII-A a1 (start sites at 20, 27, and 33) and 7 other clones contained a2 but not a3 starting at 865, 871, 877, 881, and 891 (summarized in Figs. 1 and 2). The remaining 3 cDNA clones contained sequences from a further upstream region of the FcRIII-A gene and represented novel transcript classes assigned as a5/6 (Fig. 2). FcRIII-Aa5 initiate at positions 1278 and 1254, whereas FcRIII-Aa6 initiated at 1273. These 5-untranslated ends of the a5/6 transcripts were encoded by exon I ending at position 900 (a5) or alternatively at 946 (a6) and spliced to 44 now recognized as the 5-border of exon III. This splice site at 44 was used by a2 as well as a5/6 transcripts. That position is the strongest protected fragment in RNase protection experiments from FcRIII-A expressing NK cells but not from FcRIII-B expressing PMN (16).

Nucleotide sequence of the proximal Pprox and medial Pmed1 and Pmed2 promoter regions of the FcRIII-A gene.

Next, we performed primer extension and RNase protection to determine the a2/3 start sites more precisely. In the RNase protection experiments the FcRIII-A specific riboprobe ranging from 942 to 707 covering the 3-border of exon II at 795 from the ATG was used, as described under "Materials and Methods." This analysis was done using RNA from various cells types. Multiple bands were observed preferentially in NK cells and 1B3 T cells, in very reduced amounts in culture activated peripheral blood monocytes but not in U937, HL60, and YT cell lines (Fig. 3). As shown on the right side of Fig. 3 one major protected fragment which mapped to position 887, as well as several minor products ranging from 865 to 889 were observed in NK cells. Consistent with these data, primer extension experiments located the a2/3 transcription initiation sites to the same region, with the major site identified at position 865 and minor sites at 867, 871, 872, 873, 878, 880, and 884.

Mapping the FcRIII-Aa2/3 mRNA start sites by RNase protection and primer extension.

We also analyzed transcription initiation from the same region of the FcRIII-B gene using RNA from Me₂SO-treated HL60 cells and PMN (lanes 3, 9, and 13 in Fig. 3). Differentiation of HL60 cells by Me₂SO resulted in no induction of FcRIII-Bb2 transcript initiation. In contrast, induction of FcRIII-Bb1 transcript initiation is evident (16). Both primer extension and RNase protection identified the major FcRIII-Bb2 mRNA start site in PMN at position 875 from the ATG translation start codon. That the 875 start site in PMN is specifically driven by the FcRIII-B and not the FcRIII-A gene was assessed by RT-PCR cloning and sequence analysis from PMN of two NA-2 homozygous donors. Specificity was demonstrated for several independent cDNA clones by the presence of nucleotides T and C at positions 147 and 141 within the exon V encoding the EC1 domain, as shown in Fig. 4, left side.

RNase protection assay demonstrating the expression of exon V/VI FcRIII-A mRNA variants.

The Alternative FcRIII-A Transcripts a2/3 and a5/6 Encode the Same FcRIII-A Receptor Isoform in NK Cells

The heterogeneity and the level of transcription of the distinct FcRIII-A as well as FcRIII-B mRNAs suggested the presence of simultaneously active but separate transcriptional control regions within each FcRIII gene in their respective cell types. Alternative FcRIII promoters could regulate the tissue-specific expression of transcripts encoding for additional FcRIII-A or FcRIII-B gene derived receptor-related isoforms within a single cell. As a first step testing this possibility we performed RNase protection experiments with a riboprobe containing a partially overlapping sequence from exons V/VI encoding the two extracellular EC1 and EC2 domains of FcRIIIA. For the synthesis of the riboprobe a 147-bp portion of the cDNA pGP5 from the SalI site in exon V to the PvuII site in exon VI was subcloned in sense orientation upstream of the phage T7 promoter within the pKS+ plasmid to generate pEC1-2, as described under "Materials and Methods." Analysis was done by hybridizing the EC1/EC2 spanning riboprobe made from pEC1-2 to various FcRIII-A positive and negative RNAs. As expected, in the negative HL60, U937, Jurkat, and YT cell lines and with the control yeast tRNA no FcRIII-A specific fragments protected by the riboprobe were detected. Interestingly, NK cells, 1B3 cytotoxic T cells, MK1 / T cells, and culture activated monocytes, all of them encoding the FcRIII-A receptor on the cell surface, demonstrated coexpression of full-length transcripts along with transcripts containing alterations within the EC1/EC2 domains. As shown in Fig. 4, two protected fragments of 73/74 and of 147 nucleotides can be distinguished. The 147-nucleotide fragment matched the used EC1/EC2 overlap. The 73/74-nucleotide fragment was mainly protected from exon V derived EC1 sequences. Using an exon IV/V overlapping riboprobe only one protected fragment was observed (data not shown). These results indicated the effective transcription of normal FcRIII-A as well as splice alternatives beyond exon V. Whether such splice variants led to the production of FcRIII-A related receptors remains to be addressed.

To determine the potential correlation between exon V/VI splice variants and separate clusters of transcription initiation we performed RT-PCR analysis using RNA from NK cells with distinct a2/3 or a5/6 specific 5-UTR primers and a single 3 end primer complementary to known FcRIII-A 3-UTR sequences, as described under "Materials and Methods." Using this strategy of amplification, a2/3 and a5/6 main products of 928/946 and 1156/1110 bp were generated indicating no gross alteration within exons V and VI encoding for the extracellular domains (Fig. 5). This was also verified by sequence analysis (data not shown). Therefore, the transcript classes a2/3 and a5/6 encode for the same FcRIII-A receptor in NK cells as originally demonstrated for a1 (15).

RT-PCR analysis of FcRIII-A a2/3 and a5/6 specific mRNA from NK cells.

Proximal and Medial FcRIII-A Control Regions Act as YT Cell-specific Transcriptional Regulators

Differences in the cell type specific activities of the proximal 198/10 FcRIII-A and FcRIII-B Pprox gene promoters were most evident in combination with enhancer elements. Such elements might be provided by intronic sequences from +10 to +712 between exon III and IV or the more upstream regions from 1817 or 1821 to 198 in both genes, as described recently (16, 17). The newly identified FcRIII-A and -B transcription initiation clusters now suggest that differential promoter activities would not be restricted to the proximal 198/10 regions but could also be located to the stimulatory 1817/1821 to 198 upstream regions.

To examine this possibility we first linked the 1817/850 and 1821/846 fragments from the FcRIII-A/B upstream regions covering with their most 3 ends the a2/3 or b2 mRNA start sites to the promoterless luciferase gene. The reporter plasmids pIII-A(1817/850) + (intr.A)Luc and pIII-B(1821/846) + (intr.B)Luc which also contain their respective intron enhancers cloned downstream to the luciferase gene were then transfected into HL60 and YT cells. These two cell lines were used in all our functional studies. Although presenting a more immature phenotype they can serve as model systems for PMN and NK cells, as described earlier (16). As shown in Fig. 6, the 1817/850 region of FcRIIIA produced a strong luciferase activity in the immature NK cell line YT. The amount of activity observed in HL60 cells was strongly reduced and a weak expression was seen only in the presence of the intron enhancer. Compared with the complete 1817/10 and the proximal 198/10 regions the 1817/850 sequence caused a significant higher promoter activity but a similar cell type specificity with preferential expression in YT cells. Such promoter activities were not detected in transfected Jurkat T cells and myeloid U937 cells (data not shown). From these data we conclude that in addition to the 198/10 FcRIII-A Pprox promoter the 1817/850 upstream region acts as an YT specific transcriptional regulator. This region will be referred to as the medial control region of the FcRIII-A gene.

Functional analysis of the 5-flanking region of human FcRIII-A and comparison with the homologous FcRIII-B gene.

Surprisingly, the same 1821/846 medial region from the FcRIIIB gene did not produce significant luciferase activity in HL60 cells even in the presence of its endogenous intron enhancer. Very low activity could be detected in YT cells. As shown in Fig. 6, this was in sharp contrast to the results when using the complete 1821/10 and the proximal 198/10 FcRIII-B regions. Reporter plasmids containing these latter sequences produced very strong promoter activities specific for HL60 cells. Our data indicate that HL60 cells lack some factors necessary for proper function of the medial but not the proximal FcRIII-B promoters. Based on these observations we focused in our subsequent analysis mainly on the FcRIII-A medial control region.

The FcRIII-A Medial Control Region Consists of the Two Separate Promoters Pmed1 and Pmed2

After establishing the contribution of the FcRIII-A medial control region in YT cell-specific expression we addressed the question whether the cis-acting sequences from this region were sufficient and responsible for constitutive promoter activity and cell type specificity. Two series of 5 and 3 deletion mutants were generated and cloned upstream to the luciferase gene into reporter plasmids lacking the endogenous intron enhancer and tested by transfection into YT cells. This approach provided evidence that the FcRIII-A medial control region consists of two separate promoters, termed Pmed1 and Pmed2. These two promoter activities appeared to be YT cell specific. All of the deletion mutants were almost negative for luciferase expression when transfected into HL60 cells (data not shown).

As shown in Fig. 7, different FcRIII-A 5 deletion mutants originating at position 1817 produced variable amounts of luciferase activities in YT cells suggesting the presence of compensatory enhancer, repressor as well as promoter elements. The original reporter plasmid pIII-A(1817/850)Luc produced luciferase activities in the range of 15 to 23 ×10³ relative light units (RLU) which were set as 100% relative activity. As an internal standard we used the pTK luciferase control plasmid. Deletion of the most upstream region from 1817 to 1579 drastically reduced activity indicating the presence of a stimulatory element. Further deletion to position 1376 caused an increase to full luciferase expression. This suggested that negative regulatory sequences reside in the 1579 to 1376 region which might be compensated by the enhancer element from the most upstream region. To analyze whether the 1579 to 1376 region acted as a silencer, this part of the FcRIII-A gene was cloned upstream to the thymidine kinase (TK) promoter into the pTK luciferase control plasmid and transfected into YT cells. A reduction in the expression levels down to 26% was achieved indicating that this property of repression is transferable to a heterologous promoter (data not shown).

Extensive 5 and 3 end deletion mapping studies from the medial control region of the FcRIII-A gene to identify two separate promoters Pmed1 (942/850) and Pmed2 (1376/1123).

Luciferase activity continued in more extensive 5 deletion mutants containing the 942 to 850 sequence covering all of the mapped a2/3 mRNA start sites. The 942/850 sequence within the FcRIII-A medial control region will be referred to as the Pmed1 promoter. A further deletion by 21 bp with the pIII-A(921/850)Luc plasmid resulted in a strong decrease in Pmed1 promoter activity. This suggested that the sequence between 942 and 921 contains an YT cell-specific promoter element.

The importance of the 21-bp 942/921 region for YT specific activity of the Pmed1 promoter was also observed in functional studies using 3 end deletion mutants. The deletion of the first 70 bp from the Pmed1 promoter within the pIII-A(1817/921)Luc plasmid retained almost complete luciferase expression. A further deletion of the remaining 21-bp Pmed1 sequences reduced the relative activity of the pIII-A (1817/942) 3 deletion mutant to levels of about 25-30%. Most importantly, these data also suggested that additional promoter sequences within the FcRIII-A medial control region upstream from the Pmed1 promoter account for this residual luciferase expression. To define the region responsible for this remaining promoter activity we generated more extensive 3 end deletion mutants as well as constructs containing nonoverlapping fragments. As shown in Fig. 7, all the constructs containing the 1376 to 1123 sequence were able to confer activity to the luciferase reporter gene. In the absence of the upstream repressing element of the 1579 to 1376 region the pIII-A(1376/1123) reporter construct produced the highest amounts of luciferase activity comparable to what has been observed with the Pmed1 promoter. This region also contains the 5 ends of the cloned a5/6 RACE PCR products at 1278, 1270, and 1254 and will be referred to as the FcRIII-A Pmed2 promoter.

To examine whether nucleotide differences were responsible for repressed FcRIII-B medial promoter activity, some of the FcRIII-A deletion mutants were compared with their corresponding FcRIII-B sequences in the functional studies. To test the contribution of each Pmed1 and Pmed2 region, analysis were also done using A/B hybrid constructs. All constructs lacked the intron structures to avoid possible neutralizing effects due to promoter/enhancer competition. As shown in Fig. 8, the complete 1821/846 medial control region of the FcRIII-B gene was inactive in its respective HL60 cells and produced some residual activity about 8-fold lower compared to the FcRIII-A derived sequences in YT cells. Slightly higher levels of luciferase expression were produced by the pIII-B(947/846)Luc plasmid, 4-fold lower than observed for the corresponding FcRIII-A Pmed1 promoter region. The deletion of the 947/846 region in the pIII-B(1821/947)Luc construct resulted in a complete loss of promoter activity, whereas the pIII-A(1817/942)Luc containing the FcRIII-A Pmed2 promoter was still active (data not shown). Similar results were obtained using Pmed1/Pmed2 hybrid constructs from both genes (Fig. 8). Therefore, the residual FcRIII-B activity resides within the Pmed1 but not the Pmed2 region. Total repression or strong reduction in the FcRIII-B Pmed2 and Pmed1 activities in YT cells appeared to be mediated by differences in the nucleotide sequence. Comparison of the 1380/1127 FcRIII-B and 1376/1123 FcRIII-A Pmed2 sequences revealed a single nucleotide exchange of A to G at position 1341 in FcRIII-B creating a Sp1 consensus site in case of the FcRIII-A gene (boxed in Fig. 1). As outlined in Fig. 9, the sequence of the FcRIII-A Pmed1 promoter differed through a truncation of the 8-bp motif GGAGCCCT which is three times repeated in the respective FcRIII-B region. For both genes the main a2/3 or b2 mRNA start sites were mapped to positions 887 and 865 in NK cells or 875 in PMN directly downstream to this motif (Fig. 3).

Functional comparison of FcRIII-A and FcRIII-B gene subfragments demonstrates the 942 to 921 region of the FcRIII-A Pmed1 promoter as the most important element to confer YT cell-type specificity.

Nucleotide sequence of the FcRIII-A 942/850 Pmed1 promoter and the comparative FcRIII-B 947/846 gene region.

DISCUSSION

The human Fc receptors with low affinity for IgG (FcRIII, CD16) are encoded by two genes (III-A and III-B) resulting in differential tissue-specific expression of alternative transmembrane or glycosylphosphatidylinositol-anchored isoforms in NK cells and PMN, respectively. Sequence conservation of about 97% identity have been described between both coding (15) and flanking (16) regions of each gene. Reconstitution studies of the distinct FcRIII cell type specificities in transgenic mice have indicated that the cis-elements sufficient for NK or PMN restriction might locate to the same 5.8-kilobase fragment containing about 4.5 kilobase pairs of the 5-flanking sequence and the first intron in each gene (24). In vitro transfection analyses have demonstrated that the first 0.2-kilobase (198/10) of 5-flanking FcRIII sequences (16) enhanced by their first introns contribute in directing the expression of a reporter gene to their respective YT (NK-like) or HL60 (PMN-like) cell types (17). The data presented here define transcription initiation upstream of the 198/10 sequences from both genes, suggesting additional control through alternative promoters. In the case of FcRIII-B the respective upstream sequences are rather inactive, most likely due to the premature phenotype of HL60 cells used as PMN substitutes (Fig. 6). Our deletion studies indicate that the YT/NK cell-specific expression of FcRIII-A is dependent on a complex arrangement of transcriptional regulatory regions including a putative enhancer from 1817 to 1579, a suppressor from 1579 to 1376 as well as the two adjacent promoter elements Pmed2 from 1376 to 1123 and Pmed1 from 942 to 850 (Fig. 7).

The engagement of transcription initiation factors by both Pmed1 and Pmed2 regions is supported by the finding that the mRNA start sites of transcript classes a2/3 and a5/6 mapped to both elements in mature NK cells. In all functional studies both Pmed1 and Pmed2 promoters are much more active than the recently characterized (198/10) Pprox promoter in YT cells. This is in accordance with RNase protection experiments which identifies the 44 splice site present in a2 and a5/6 as the prominent protected fragment (16). An important question addressed in these studies is whether the distinct transcript classes a2/3 and a5/6 compared to a1 are controlled by alternative promoters and are involved in the tissue-specific expression of more than a single FcRIII-A receptor isoform in NK cells. The coexpression of mRNAs with or without alterations in exons V/VI encoding the extracellular EC1/EC2 domains (Fig. 4) strongly inferred that alternative splicing participate in FcRIII-A transcript heterogeneity. On the other hand, RT-PCR amplification with NK cell-derived mRNA yields a pattern of a1, a2/3, and a5/6 products indicative for a single FcRIII-A receptor (Fig. 5 and data not shown). In addition, characterization of nine cDNA clones isolated from two independent libraries constructed to size-fractionated lymphokine-activated killer or NK cell mRNA identified two clones containing exon II sequences specific for a2 which are otherwise identical to the FcRIII-A coding sequence (data not shown). We propose that evolvement of the separate Pprox, Pmed1, and Pmed2 promoters does not necessarily correlate with the events of alternative splicing within the extracellular domain of the FcRIII-A receptor. Whether other transcript classes like FcRIII-Aa4 (16) express for modified FcRIII-A products remains to be addressed. Based on earlier experiments it is most likely that a4 contains additional 5 end sequences distinct from a1, a2/3, and a5/6, suggesting that the FcRIII-Aa4 initiate by a more upstream transcriptional regulator different from Pmed1 and Pmed2 (16, 17).

Results from our functional studies showed high promoter activity within the 92-bp segment (residues 942 to 850) of the FcRIII-A gene, termed the Pmed1 promoter. Comparison with other parts of the III-A and III-B genes in YT and HL60 cells revealed that differential cell type specificities are due to this region (Fig. 8). An 8-bp repeat motif differed between Pmed1 and the relative FcRIII-B gene region. This motif, GGAGCCCT, is repeated three times in FcRIII-B but affected in FcRIII-A Pmed1 by a C to T exchange in the second repeat and absent in the third repeat (Fig. 9). In YT cells the truncated version present in Pmed1 shows strong improved activity over the FcRIII-B sequence. Attempts to identify YT-specific DNA-binding proteins in gel retardation assays recognizing this sequence difference were not successful. The same pattern of binding were observed for both sequences (data not shown). Therefore, the different organization of the repeat motif might influence binding affinity or oligomerization of a transcription factor rather than destroying binding capability. Possibly, a functional cooperation between the distinct repeat motifs and a further upstream element common to both genes could render only the FcRIII-A Pmed1 to be specifically active in YT cells. The 21-bp sequence from residue 942 to 921 on the 5 end of the Pmed1 is required for full promoter function. Deleting this region resulted in almost inactive Pmed1 activity. It is reasonable to assume a cis-element within this region to cooperate with the repeat motif. Sequence comparison revealed a consensus site for Ets proteins, GGAA/T, within this 21-bp segment. Several Ets family members have been shown to be involved in the differential expression of T-cell specific genes, such as the TCR, interleukin-2, and perforin (25, 26, 27). The 15-bp NKE motif, CCCACTTCCTGGCCA, bearing the core Ets site is nearly identical to the mPfp CTL-specific element (residues 508 to 494) (Fig. 9). The trans-acting factor NF-P2 exclusively expressed in cytolytic lymphocytes and specifically modulated upon activation has been identified to interact with the mPfp 15-mer sequence (24). The coexpression of perforin and FcRIII-A in some subsets of CTLs, / T cells as well as NK cells, suggests that NF-P2 or related proteins may contribute to the FcRIII-A Pmed1 specificity.

The molecular basis for NK and YT cell-specific transcription is completely unknown. Our data describe for the first time some of the relevant FcRIII-A cis-acting gene elements including Pmed1 and Pmed2. A YT cell-specific sequence motif (NKE) located within the first 21 bp in the Pmed1 promoter element. We propose that a functional cooperation between the NKE and the so-called repeat motif contributes to the YT-specificity of the FcRIII-A Pmed1 promoter. The identification and characterization of NK cell-specific DNA elements will allow targeting gene expression to NK cells clarifying the role of these important effector cells in the first line of immunologic defense. In addition, NK and YT cell-specific sequence motifs can be used to isolate transcription factors uniquely active in these cell types. Finally, elucidation of the factors involved in the expression of NK cell-specific genes as FcRIII-A will give insight into the control of NK cell differentiation and development.