Proc. Nadl. Acad. Sci. USA Vol. 87, pp. 9073-9077, December 1990 Biochemistry
Characterization of the human 5-lipoxygenase gene promoter (leukotrienes/transcriptional regulation/nuclear protein)
SHIGERU HoSHIKO*, OLOF RADMARK, AND BENGT SAMUELSSON Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden
Contributed by Bengt Samuelsson, July 5, 1990
ABSTRACT Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Spl binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Spl could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts. The enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes transformation of arachidonic acid to (5S)-hydroperoxy-6-trans-6,11,14-ciseicosatetraenoic acid (5-HPETE) and further to leukotriene A4 [(5S)-6-oxido-7,9,11-trans-14-cis-eicosatetraenoic acid]. The enzymes participating in leukotriene biosynthesis have been the subject for several recent studies, and cDNA clones corresponding to 5-lipoxygenase have been isolated (for review, see ref. 1). Also, the human 5-lipoxygenase gene has been isolated and characterized (2). Several features of the putative promoter region were characteristic for so-called housekeeping genes (3) (no TATAA or CCAAT, G+C-rich, multiple GGGCGG sequences). This paper describes further studies regarding the 5-lipoxygenase gene promoter.t In particular, a sequence containing five GGGCGG repeats was required for efficient transcription of chloramphenicol acetyltransferase (CAT) gene constructs, and gel-shift assays showed that transcription factor Spl could bind to DNA containing this G+C-rich region.
MATERIALS AND METHODS Plasmid Constructions. Plasmid pUCOCAT was constructed from pCAT3M (4) and pUC19 (5). 5-Lipoxygenase gene promoter DNA was excised from recombinant phage 1x12A [5.9 kilobase (kb) Kpn I-BstEII fragment] (2). Insertion into pUCOCAT provided 5LO5900CAT, from which several deletion derivatives were prepared (compare Fig. 2). A detailed description of the plasmid constructions can be obtained from the authors. Cell Culture and DNA Transfection. HeLa and HeLaS3 cells (maintained in this institute) were cultivated in Dulbecco's modified Eagle's medium supplemented with 5% (vol/ vol) fetal calf serum. HL-60 cells (American Type Culture Collection) and K-562 were maintained in RPMI 1640 medium containing 10% (vol/vol) fetal calf serum. HepG2 and
RBL1 cells were maintained in Eagle's medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% (vol/vol) fetal calf serum. Plasmids for DNA transfection were prepared by alkaline lysis and purified by CsCI centrifugation. For transfections the following methods were used: HeLa and HepG2, calcium phosphate precipitation followed by 15% (vol/vol) glycerol shock (2 min) (6); HL-60 and RBL1, DEAE-dextran (250 ,ug/ml) followed by chloroquine (0.1 or 0.2 mM, 60 min) (7); and K-562, electroporation [230 V, 960 ,uF, in phosphate-buffered saline (PBS)] (8). Cells were harvested within 48 hr after transfection, washed with PBS, and suspended in 100 p.1 of 0.25 M Tris HCl, pH 7.8, for sonication. After centrifugation (15,000 x g for 10 min), the supernatants were assayed for protein (9), as well as for CAT activity. CAT and Luciferase Assays. CAT activity was measured as described (10). To estimate transfection efficiencies of HeLa cells, the firefly luciferase containing plasmid pSV2AL 5' was cotransfected, and the luciferase activity (11) was taken as an estimation of the transfection efficiency. CAT assay results from different transfection experiments could thus be normalized and compared. RNA Preparation and S1 Nuclease Protection Assays. For preparation of RNA -108 HeLa cells (10 to 14 15-cm dishes) were transfected with 100,ug of plasmid DNA per dish. After 36 hr, RNA was extracted (12). Poly(A)+ RNA was purified on oligo(dT)-cellulose. The probe was prepared from 5LO292CAT by digestion with Bgl II, labeling of the 5' end, and digestion with EcoRI. The labeled DNA probe was purified by PAGE. Poly(A)+ RNA (40 ,ug) was hybridized with probe (3 x 105 cpm, 30 ng) in a solution containing 80% (vol/vol) formamide overnight at 53°C. Subsequently S1 nuclease digestion was performed (2). DNA Sequence Analysis. This technique was performed as described (13). Preparation of Nuclear Proteins and Detection of DNABinding Proteins. HeLaS3 and HL-60 nuclear extracts were prepared, and gel-retardation assay was performed as described (14, 15). Oligonucleotides. 5'-GGGGCGGGGCTGCA-3' and 5'GCCCCGCCCCTGCA-3' were hybridized, after phosphorylation ligation provided concatemers. The monomer and concatemer forms were used as Spl competitors in gelretardation analysis.
RESULTS Identification of the Promoter Region. To identify DNA sequences regulating 5-lipoxygenase gene expression, the region (-5900 to +20) was excised from the genomic clone 1x12A (2) and inserted into a plasmid already containing the CAT reporter gene (+1 is adenine in the ATG start codon). Abbreviations: CAT, chloramphenicol acetyltransferase; PMA, phorbol 12-myristate 13-acetate. *Permanent address: Pharmaceutical Laboratories, Meiji Seika Kaisha Ltd., Kohoku-ku, Yokohama 222, Japan. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M38191).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9073
9074
Biochemistry: Hoshiko
et
al.
Proc. Natl. Acad. Sci. USA 87 (1990)
The resulting plasmid (5L05900CAT) was transiently introduced to HeLa and HepG2 cells, as well as to myeloid cell lines HL-60, K-562, and RBL1. Although several methods were tried, transfection efficiencies for the myeloid cells were low (see Materials and Methods for optimal procedure for each cell line). Nevertheless, the 5900-base pair (bp) DNA fragment clearly had promoter activity in all cells tested (Fig. 1). Before further analysis of the 5-lipoxygenase gene promoter, it was confirmed that the CAT expressions seen after transfection with 5LOCAT derivatives were truly controlled by the 5-lipoxygenase gene promoter. For this purpose poly(A)+ RNA was prepared from HeLa cells transfected with three different SLOCAT constructs, and the transcriptional initiation sites for the chimeric RNA species were determined by S1 nuclease protection assay (Fig. 2A). RNADNA hybrids originating between nucleotides -65 and -62
found. This is practically identical to the initiation of 5-lipoxygenase gene transcription in human leukocytes, showing that observed CAT activities were controlled by the 5-lipoxygenase gene promoter. Expression of endogenous 5-lipoxygenase in these cell lines was determined by Northern (RNA) blots. HeLa and HepG2 cells were negative, whereas K-562 (weak), RBL1, and HL-60 (particularly after treatment with dimethyl sulfoxide) cells were positive (data not shown). For further studies we choose the human epithelial cell line HeLa (for ease of transfection) and also the promyelocytic cell line HL-60, which expresses the 5-lipoxygenase gene endogenously. Some aspects regarding transfection of HL-60 cells should be mentioned. Thus, when HL-60 cells were transfected with the negative control plasmid pUCOCAT (lacking a promoter before the CAT gene), unexpectedly high background CAT activity was obtained (-80% of the CAT activity after transfection with positive control plasmid pSV2CAT containing simian virus 40 early promoter). For HeLa cells, the
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Characterization of the human 5-lipoxygenase gene promoter (leukotrienes/transcriptional regulation/nuclear protein)
SHIGERU HoSHIKO*, OLOF RADMARK, AND BENGT SAMUELSSON Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden
Contributed by Bengt Samuelsson, July 5, 1990
ABSTRACT Nucleotide sequences that direct transcription of the human 5-lipoxygenase gene have been examined by ligation to the chloramphenicol acetyltransferase gene and determination of chloramphenicol acetyltransferase activity in transfected HeLa and HL-60 cells. Various lengths of 5'flanking sequences up to 5.9 kilobase pairs 5' of the transcriptional initiation sites were tested. Two positive and two negative apparent regulatory regions were seen. Part of the promoter sequence (-179 to -56 from ATG), which includes five repeated GC boxes (the putative Spl binding sequence) was essential for transcription in both HeLa and HL-60 cells. Gel-shift assays (using the DNA fragment -212 to -88) revealed that the transcriptional factor Spl could bind to this region of the 5-lipoxygenase promoter. Furthermore, HL-60 nuclear extracts contained specific nuclear factor(s) binding to 5-lipoxygenase promoter DNA, which could not be detected in HeLa cell nuclear extracts. The enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes transformation of arachidonic acid to (5S)-hydroperoxy-6-trans-6,11,14-ciseicosatetraenoic acid (5-HPETE) and further to leukotriene A4 [(5S)-6-oxido-7,9,11-trans-14-cis-eicosatetraenoic acid]. The enzymes participating in leukotriene biosynthesis have been the subject for several recent studies, and cDNA clones corresponding to 5-lipoxygenase have been isolated (for review, see ref. 1). Also, the human 5-lipoxygenase gene has been isolated and characterized (2). Several features of the putative promoter region were characteristic for so-called housekeeping genes (3) (no TATAA or CCAAT, G+C-rich, multiple GGGCGG sequences). This paper describes further studies regarding the 5-lipoxygenase gene promoter.t In particular, a sequence containing five GGGCGG repeats was required for efficient transcription of chloramphenicol acetyltransferase (CAT) gene constructs, and gel-shift assays showed that transcription factor Spl could bind to DNA containing this G+C-rich region.
MATERIALS AND METHODS Plasmid Constructions. Plasmid pUCOCAT was constructed from pCAT3M (4) and pUC19 (5). 5-Lipoxygenase gene promoter DNA was excised from recombinant phage 1x12A [5.9 kilobase (kb) Kpn I-BstEII fragment] (2). Insertion into pUCOCAT provided 5LO5900CAT, from which several deletion derivatives were prepared (compare Fig. 2). A detailed description of the plasmid constructions can be obtained from the authors. Cell Culture and DNA Transfection. HeLa and HeLaS3 cells (maintained in this institute) were cultivated in Dulbecco's modified Eagle's medium supplemented with 5% (vol/ vol) fetal calf serum. HL-60 cells (American Type Culture Collection) and K-562 were maintained in RPMI 1640 medium containing 10% (vol/vol) fetal calf serum. HepG2 and
RBL1 cells were maintained in Eagle's medium and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% (vol/vol) fetal calf serum. Plasmids for DNA transfection were prepared by alkaline lysis and purified by CsCI centrifugation. For transfections the following methods were used: HeLa and HepG2, calcium phosphate precipitation followed by 15% (vol/vol) glycerol shock (2 min) (6); HL-60 and RBL1, DEAE-dextran (250 ,ug/ml) followed by chloroquine (0.1 or 0.2 mM, 60 min) (7); and K-562, electroporation [230 V, 960 ,uF, in phosphate-buffered saline (PBS)] (8). Cells were harvested within 48 hr after transfection, washed with PBS, and suspended in 100 p.1 of 0.25 M Tris HCl, pH 7.8, for sonication. After centrifugation (15,000 x g for 10 min), the supernatants were assayed for protein (9), as well as for CAT activity. CAT and Luciferase Assays. CAT activity was measured as described (10). To estimate transfection efficiencies of HeLa cells, the firefly luciferase containing plasmid pSV2AL 5' was cotransfected, and the luciferase activity (11) was taken as an estimation of the transfection efficiency. CAT assay results from different transfection experiments could thus be normalized and compared. RNA Preparation and S1 Nuclease Protection Assays. For preparation of RNA -108 HeLa cells (10 to 14 15-cm dishes) were transfected with 100,ug of plasmid DNA per dish. After 36 hr, RNA was extracted (12). Poly(A)+ RNA was purified on oligo(dT)-cellulose. The probe was prepared from 5LO292CAT by digestion with Bgl II, labeling of the 5' end, and digestion with EcoRI. The labeled DNA probe was purified by PAGE. Poly(A)+ RNA (40 ,ug) was hybridized with probe (3 x 105 cpm, 30 ng) in a solution containing 80% (vol/vol) formamide overnight at 53°C. Subsequently S1 nuclease digestion was performed (2). DNA Sequence Analysis. This technique was performed as described (13). Preparation of Nuclear Proteins and Detection of DNABinding Proteins. HeLaS3 and HL-60 nuclear extracts were prepared, and gel-retardation assay was performed as described (14, 15). Oligonucleotides. 5'-GGGGCGGGGCTGCA-3' and 5'GCCCCGCCCCTGCA-3' were hybridized, after phosphorylation ligation provided concatemers. The monomer and concatemer forms were used as Spl competitors in gelretardation analysis.
RESULTS Identification of the Promoter Region. To identify DNA sequences regulating 5-lipoxygenase gene expression, the region (-5900 to +20) was excised from the genomic clone 1x12A (2) and inserted into a plasmid already containing the CAT reporter gene (+1 is adenine in the ATG start codon). Abbreviations: CAT, chloramphenicol acetyltransferase; PMA, phorbol 12-myristate 13-acetate. *Permanent address: Pharmaceutical Laboratories, Meiji Seika Kaisha Ltd., Kohoku-ku, Yokohama 222, Japan. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M38191).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9073
9074
Biochemistry: Hoshiko
et
al.
Proc. Natl. Acad. Sci. USA 87 (1990)
The resulting plasmid (5L05900CAT) was transiently introduced to HeLa and HepG2 cells, as well as to myeloid cell lines HL-60, K-562, and RBL1. Although several methods were tried, transfection efficiencies for the myeloid cells were low (see Materials and Methods for optimal procedure for each cell line). Nevertheless, the 5900-base pair (bp) DNA fragment clearly had promoter activity in all cells tested (Fig. 1). Before further analysis of the 5-lipoxygenase gene promoter, it was confirmed that the CAT expressions seen after transfection with 5LOCAT derivatives were truly controlled by the 5-lipoxygenase gene promoter. For this purpose poly(A)+ RNA was prepared from HeLa cells transfected with three different SLOCAT constructs, and the transcriptional initiation sites for the chimeric RNA species were determined by S1 nuclease protection assay (Fig. 2A). RNADNA hybrids originating between nucleotides -65 and -62
found. This is practically identical to the initiation of 5-lipoxygenase gene transcription in human leukocytes, showing that observed CAT activities were controlled by the 5-lipoxygenase gene promoter. Expression of endogenous 5-lipoxygenase in these cell lines was determined by Northern (RNA) blots. HeLa and HepG2 cells were negative, whereas K-562 (weak), RBL1, and HL-60 (particularly after treatment with dimethyl sulfoxide) cells were positive (data not shown). For further studies we choose the human epithelial cell line HeLa (for ease of transfection) and also the promyelocytic cell line HL-60, which expresses the 5-lipoxygenase gene endogenously. Some aspects regarding transfection of HL-60 cells should be mentioned. Thus, when HL-60 cells were transfected with the negative control plasmid pUCOCAT (lacking a promoter before the CAT gene), unexpectedly high background CAT activity was obtained (-80% of the CAT activity after transfection with positive control plasmid pSV2CAT containing simian virus 40 early promoter). For HeLa cells, the
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