title: Cell-specific transcription of leukotriene C(4) synthase involves a Kruppel-like transcription factor and Sp1.
abstract: Leukotriene C(4) synthase (LTC(4)S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5'-flanking region of the human LTC(4)S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides -122 and -56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides -120 and -115 or the Inr (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gene by approximately 60%, whereas double mutations decreased the expression by approximately 80%. The incubation of nuclear extracts from THP-1 and K562 cells with a (32)P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides -165 to -125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides -149 to -145 and -139 to -135). An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a -228 to -3 LTC(4)S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by approximately 66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the -228 construct in COS cells but not its CACCC mutant construct. These findings indicate the involvement of Sp1 and an Inr in non-cell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC(4)S gene. These are the first such analyses of a member of a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.
authors.affiliation: Department of Medicine, Harvard Medical School, and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Zhao
authors.forename: K F
authors.initials: KF
authors.lastname: Austen
authors.forename: B K
authors.initials: BK
authors.lastname: Lam
mesh_terms: D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors; D005982:Glutathione Transferase; D003850:Deoxyribonuclease I; C041116:leukotriene-C4 synthase