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• W2073156984 abstract "The molecular events governing the differentiation pathway of natural killer (NK) cells are not well understood. The phenotype of mature NK cells is specified by the expression of the low affinity Fc receptor for IgG (human FcγRIII, CD16) encoded by the FcγRIII-A gene. Here we report that the Pprox promoter (−198/−10) of FcγRIII-A stimulated by its own intron enhancer (+10/+712) was only one of the cis-elements that target the expression of a reporter gene in the immature NK cell line, YT. The transcription start sites of the FcγRIII-A a2/3 and a5/6 splice alternatives in NK cells were mapped to the medial −1817/−850 FcγRIII-A control region. Two promoters, Pmed1 (−942/−850) and Pmed2 (−1376/−1123) resided in this region and controlled for the initiation of these transcript classes encoding the known FcγRIII-A receptor protein. Deletion mapping studies demonstrated that the 93 base pairs −942/−850 Pmed1 sequence was sufficient to confer cell type-specific expression in YT cells. The 5′ end of Pmed1 (−942 to −921) was required for full promoter function indicating the presence of an important sequence motif recognized by a YT-specific factor. Our data suggest that this motif might be a useful tool for subsequent identification of putative transcription factors uniquely active in YT and NK cells. The molecular events governing the differentiation pathway of natural killer (NK) cells are not well understood. The phenotype of mature NK cells is specified by the expression of the low affinity Fc receptor for IgG (human FcγRIII, CD16) encoded by the FcγRIII-A gene. Here we report that the Pprox promoter (−198/−10) of FcγRIII-A stimulated by its own intron enhancer (+10/+712) was only one of the cis-elements that target the expression of a reporter gene in the immature NK cell line, YT. The transcription start sites of the FcγRIII-A a2/3 and a5/6 splice alternatives in NK cells were mapped to the medial −1817/−850 FcγRIII-A control region. Two promoters, Pmed1 (−942/−850) and Pmed2 (−1376/−1123) resided in this region and controlled for the initiation of these transcript classes encoding the known FcγRIII-A receptor protein. Deletion mapping studies demonstrated that the 93 base pairs −942/−850 Pmed1 sequence was sufficient to confer cell type-specific expression in YT cells. The 5′ end of Pmed1 (−942 to −921) was required for full promoter function indicating the presence of an important sequence motif recognized by a YT-specific factor. Our data suggest that this motif might be a useful tool for subsequent identification of putative transcription factors uniquely active in YT and NK cells. Natural killer (NK) 1The abbreviations used are: NKnatural killerbpbase pairCR3complement receptor 3CTLcytolytic T lymphocytesFcγRIIIlow-affinity receptor for Fc domain of IgGEC1/2exons coding for the extracellular domains of FcγRIIIMHCmajor histocompatibility complexNKEnatural killer elementPCRpolymerase chain reactionPMNpolymorphonuclear leukocytesRACErapid amplification of cDNA endsRLUrelative light unitsRTreverse transcriptionTKthymidine kinaseUTRuntranslated region. 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 1Robertson M.J. Ritz J. Blood. 1990; 76: 2421-2438Crossref PubMed Google Scholar and 2Gumperz J.E. Parham P. Nature. 1995; 378: 245-248Crossref PubMed Scopus (229) Google Scholar). natural killer base pair complement receptor 3 cytolytic T lymphocytes low-affinity receptor for Fc domain of IgG exons coding for the extracellular domains of FcγRIII major histocompatibility complex natural killer element polymerase chain reaction polymorphonuclear leukocytes rapid amplification of cDNA ends relative light units reverse transcription thymidine kinase untranslated region. 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, FcγRIII-A (CD16-A). FcγRIII-A belongs to the family of immunoglobulin G receptors (FcγR) involved in the clearance of immune complexes, phagocytosis of opsonized pathogens, and various forms of antibody-dependent cellular cytotoxicity (3Van de Winkel J.G.J. Capel P.J.A. Immunol. Today. 1993; 14: 215-221Abstract Full Text PDF PubMed Scopus (628) Google Scholar). The FcγRIII-A α chain forms a multimeric transmembrane receptor complex with homo- and heterodimers of the FcϵRIγ subunit and/or CD3ζ subunit (4Kurosaki T. Ravetch J.V. Nature. 1989; 342: 805-807Crossref PubMed Scopus (147) Google Scholar, 5Anderson P. Caliguri M. O'Brien C. Manley T. Ritz J. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2274-2278Crossref PubMed Scopus (154) Google Scholar, 6Gessner J.E. Radeke H.H. Uciechowski P. Resch K. Schmidt R.E. Immun. Infekt. 1995; 23: 67-69PubMed Google Scholar). FcγRIII is also highly expressed in PMN but as a single chain receptor attached to the plasma membrane by a glycosylphosphatidylinositol anchor, FcγRIII-B. The transmembrane FcγRIII-A receptor on NK cells mediates antibody-dependent cellular cytotoxicity and all other antibody-dependent responses (7Werfel T. Uciechowski P. Tetteroo P.A.T. Kurrle R. Deicher H. Schmidt R.E. J. Immunol. 1989; 142: 1102-1106PubMed Google Scholar, 8Uciechowski P. Werfel T. Leo R. Gessner J.E. Schubert J. Schmidt R.E. Immunobiology. 1992; 185: 28-40Crossref PubMed Scopus (17) Google Scholar, 9Uciechowski P. Gessner J.E. Schindler R. Schmidt R.E. Eur. J. Immunol. 1992; 22: 1635-1638Crossref PubMed Scopus (26) Google Scholar). The glycosylphosphatidylinositol-linked FcγRIII-B receptor on PMN is involved in cell activation but its detailed role is less clear (10Hundt M. Schmidt R.E. Eur. J. Immunol. 1992; 22: 811-816Crossref PubMed Scopus (104) Google Scholar, 11Hoffmeyer F. Witte K. Gebhardt U. Schmidt R.E. J. Immunol. 1995; 155: 4016-4023PubMed Google Scholar, 12Tamm A. Kister A. Nolte K.U. Gessner J.E. Schmidt R.E. J. Biol. Chem. 1996; 271: 3659-3666Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Other surface molecules like FcγRII-A and CR3 receptors are likely to be involved in the activation process of PMN after FcγRIII-B cross-linking (13Zhou M. Brown E.J. J. Cell. Biol. 1994; 125: 1407-1416Crossref PubMed Scopus (284) Google Scholar, 14Stöckl J. Majdic O. Pickl W.F. Rosenkranz A. Prager E. Gschwantler E. Knapp W. J. Immunol. 1995; 154: 5452-5463PubMed Google Scholar). The molecular basis for the expression of functionally distinct FcγRIII isoforms is given by the presence of the highly homologous FcγRIII-A and FcγRIII-B genes (15Ravetch J.V. Perussia B. J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (486) Google Scholar). 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 FcγRIII-A and B receptors to NK cells and PMN, respectively (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The characterization of the molecular events leading to NK cell-specific FcγRIII-A expression is complicated by the finding that transcripts initiating outside the −198/−10 Pprox promoter exist in NK cells (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Gessner J.E. Grussenmeyer T. Schmidt R.E. Immunobiol. 1995; 193: 341-355Crossref PubMed Scopus (15) Google Scholar). Cloning and sequencing of these FcγRIII-A transcripts, designated a2/3 and a5/6, demonstrated that they encode the known FcγRIII-A receptor. The coexpression of FcγRIII-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 FcγRIII-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. Polymorphonuclear granulocytes (PMN), peripheral blood mononuclar cells (adherent cell fraction, MO), and NK cells were prepared according to standard conditions described elsewhere (9Uciechowski P. Gessner J.E. Schindler R. Schmidt R.E. Eur. J. Immunol. 1992; 22: 1635-1638Crossref PubMed Scopus (26) Google Scholar, 16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18Phillips J.H. Chang C. Lanier L.L. Eur. J. Immunol. 1991; 21: 895-899Crossref PubMed Scopus (25) Google Scholar, 19Radeke H.H. Gessner J.E. Uciechowski P. Mägert H.-J. Schmidt R.E. Resch K. J. Immunol. 1994; 153: 1281-1292PubMed Google Scholar). The γ/δ 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) (8Uciechowski P. Werfel T. Leo R. Gessner J.E. Schubert J. Schmidt R.E. Immunobiology. 1992; 185: 28-40Crossref PubMed Scopus (17) Google Scholar). Human tumor cell lines HL60 and YT used for transfection experiments were cultured in RPMI 1640 containing 10% fetal calf serum and supplements (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). HL60 is a promyelocytic cell line and YT a cell line with NK cell characteristics (20Collins S.J. Blood. 1987; 70: 1233-1244Crossref PubMed Google Scholar, 21Yodoi J. Teshigawara K. Nikaido T. Fukui K. Noma T. Honjo T. Takigawa M. Sasaki M. Minato N. Tsudo M. Uchiyama T. Maeda M. J. Immunol. 1985; 134: 1623-1630PubMed Google Scholar). In some experiments, HL60 cells were induced to express the FcγRIII-B receptor isoform upon culturing in the presence of 1.2% Me2SO (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The strategy to obtain cDNA clones for a1, a2/3, and a5/6 related transcription initiation sites was nearly identical to that described recently (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Starting with 2 μg of poly(A)+ RNA from NK cells, the reverse transcription reaction was performed using 20 pmol of a FcγRIII-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 (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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. Analysis of the distinct FcγRIII transcript classes by primer extension and RNase protection assay was performed according to methods described previously (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Total RNAs were prepared by the guanidinium thiocyanate method (22Chirgwin J.W. Przybyla A.E. McDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16610) Google Scholar). The complementary oligonucleotide used for primer extension, 5′-CTTCCTCGTGTTACCCAGGTCCTGCGGATT-3′ (−795 to −824), corresponds to the genomic FcγRIII-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 FcγRIII-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 FcγRIII-B cDNA (23Peltz G.A. Grundy H.O. Lebo R.V. Yssel H. Barsh G.S. Moore K.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1013-1017Crossref PubMed Scopus (105) Google Scholar) 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 (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 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) (15Ravetch J.V. Perussia B. J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (486) Google Scholar). The PCR products were analyzed on a 1.2% agarose gel and visualized by ethidium bromide staining. The constructs were generated by cloning FcγRIII-A and FcγRIII-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 FcγRIII-A or the FcγRIII-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 FcγRIII-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 FcγRIII-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. Functional analysis of the 5′-flanking region of the human FcγRIII-A gene and the respective region of the highly homologous FcγRIII-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 (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In addition, RACE-PCR cloning indicated that additional FcγRIII-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) (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We now extended the RACE PCR analysis and isolated 17 additional cDNA clones containing the 5′ ends of FcγRIII-A transcripts from NK cells. 7 clones contained the 5′-UTR of transcript class FcγRIII-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 Fig. 1, Fig. 2). The remaining 3 cDNA clones contained sequences from a further upstream region of the FcγRIII-A gene and represented novel transcript classes assigned as a5/6 (Fig. 2). FcγRIII-Aa5 initiate at positions −1278 and −1254, whereas FcγRIII-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 FcγRIII-A expressing NK cells but not from FcγRIII-B expressing PMN (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Next, we performed primer extension and RNase protection to determine the a2/3 start sites more precisely. In the RNase protection experiments the FcγRIII-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. We also analyzed transcription initiation from the same region of the FcγRIII-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 FcγRIII-Bb2 transcript initiation. In contrast, induction of FcγRIII-Bb1 transcript initiation is evident (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Both primer extension and RNase protection identified the major FcγRIII-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 FcγRIII-B and not the FcγRIII-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. The heterogeneity and the level of transcription of the distinct FcγRIII-A as well as FcγRIII-B mRNAs suggested the presence of simultaneously active but separate transcriptional control regions within each FcγRIII gene in their respective cell types. Alternative FcγRIII promoters could regulate the tissue-specific expression of transcripts encoding for additional FcγRIII-A or FcγRIII-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 FcγRIIIA. 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 FcγRIII-A positive and negative RNAs. As expected, in the negative HL60, U937, Jurkat, and YT cell lines and with the control yeast tRNA no FcγRIII-A specific fragments protected by the riboprobe were detected. Interestingly, NK cells, 1B3 cytotoxic T cells, MK1 γ/δ T cells, and culture activated monocytes, all of them encoding the FcγRIII-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 FcγRIII-A as well as splice alternatives beyond exon V. Whether such splice variants led to the production of FcγRIII-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 FcγRIII-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 FcγRIII-A receptor in NK cells as originally demonstrated for a1 (15Ravetch J.V. Perussia B. J. Exp. Med. 1989; 170: 481-497Crossref PubMed Scopus (486) Google Scholar). Differences in the cell type specific activities of the proximal −198/−10 FcγRIII-A and FcγRIII-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 (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Gessner J.E. Grussenmeyer T. Schmidt R.E. Immunobiol. 1995; 193: 341-355Crossref PubMed Scopus (15) Google Scholar). The newly identified FcγRIII-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 FcγRIII-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 (16Gessner J.E. Grussenmeyer T. Kolanus W. Schmidt R.E. J. Biol. Chem. 1995; 270: 1350-1361Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). As shown in Fig. 6, the −1817/−850 region of FcγRIIIA 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 FcγRIII-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 FcγRIII-A gene. Surprisingly, the same −1821/−846 medial region from the FcγRIIIB 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 FcγRIII-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 FcγRIII-B promoters. Based on these observations we focused in our subsequent analysis mainly on the FcγRIII-A medial control region. After establishing the contribution of the FcγRIII-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 FcγRIII-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 FcγRIII-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 FcγRIII-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). 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 FcγRIII-A med" @default.
• W2073156984 created "2016-06-24" @default.
• W2073156984 creator A5015512878 @default.
• W2073156984 creator A5019579516 @default.
• W2073156984 creator A5028557050 @default.
• W2073156984 creator A5036911759 @default.
• W2073156984 date "1996-11-01" @default.
• W2073156984 modified "2023-10-14" @default.
• W2073156984 title "Separate Promoters from Proximal and Medial Control Regions Contribute to the Natural Killer Cell-specific Transcription of the Human FcγRIII-A (CD16-A) Receptor Gene" @default.
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