Characterization of the human 5-lipoxygenase gene promoter.

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 tr...

The pharmacogenetics of asthma: a candidate gene approach

“…Of particular importance, Hoshiko et al 75 have identified a GϩC-rich sequence, between Ϫ212 and Ϫ88 bp relative to the translational start site, as being necessary for promoter-reporter construct activity in HeLa and HL-60 cells; these observations, which we have confirmed, led us to examine this region of the ALOX5 promoter for mutations that might alter gene transcription. We identified a family of naturally occurring mutations that alter Sp1 and Egr-1 consensus binding sites by the addition of one or the deletion of one or two -GGGCGGsequences ( Figure 7).…”

Cell-specific Transcription of Leukotriene C4Synthase Involves a Kruppel-like Transcription Factor and Sp1

“…Deletional and mutational analyses of the 5-lipoxygenase promoter in a reporter construct revealed a GC-rich region that is important for transcription (28). Gel shift assay demonstrated that a 5 tandem repeat sequence in the GC-rich region bound to transcription factors Sp1 and Egr-1 (30), both of which transactivate the 5-lipoxygenase promoter in SL2 cells (31).…”

“…Like LTC 4 S, LTA 4 hydrolase and 5-lipoxygenase are also TATA-less genes (28,29). Deletional and mutational analyses of the 5-lipoxygenase promoter in a reporter construct revealed a GC-rich region that is important for transcription (28).…”

The 5-Lipoxygenase Promoter Is Regulated by DNA Methylation

“…In Mono Mac 6 cells, the induction of 5-LO protein expression and activity by TGF␤ and 1,25(OH) 2 D 3 was accompanied by a 64-fold up-regulation of mature 5-LO mRNA and an up to 5-fold increase in 5-LO primary transcripts (7,8), whereas no significant induction of 5-LO transcription was found in nuclear run-off assays (9). The human 5-LO gene promoter was first characterized by Hoshiko et al (10). Several features of the putative 5-LO promoter region (such as the lack of TATAA or CCAAT boxes and repeated (GϩC)-rich elements) are characteristic for so-called housekeeping genes.…”

“…Several features of the putative 5-LO promoter region (such as the lack of TATAA or CCAAT boxes and repeated (GϩC)-rich elements) are characteristic for so-called housekeeping genes. Previous data suggest that the transcription factors Egr-1 and/or Sp1 are required for basal 5-LO transcription and that they functionally interact with the 5-LO promoter and activate it via repeated response elements located between positions Ϫ212 and Ϫ88 bp, relative to the translational start site (10,11). Interestingly, naturally occurring mutations were found in the 5-LO promoter consisting of the deletion of one or two, or the addition of one Sp1 binding site (12).…”