An intronic enhancer regulates cyclooxygenase-1 gene expression.
To identify cis-elements regulating PMA-induced prostaglandin H synthase-1 (PGHS-1) gene expression in the human megakaryoblast cell line, MEG-01, we performed promoter reporter assays with a luciferase reporter vector containing the -2030/-22 region of the human PGHS-1 gene. PMA treatment for 24 h increased PGHS-1 promoter activity by twofold. Mutagenesis studies of the promoter revealed a single Sp1 site essential for PMA-inducible transcription. Insertion of a highly conserved 100 bp sequence cloned from intron 8 into the -2030/-22 reporter plasmid enhanced PMA-dependent transcription 10-fold. Mutation of either a consensus AP-1 site within intron 8 or the Sp1 site in the promoter reduced PMA-induced activity by 80-100%. Gel shift assays using the intron 8 AP-1 sequence demonstrated the formation of an AP-1-specific DNA-protein complex. Our results suggest that inducible PGHS-1 gene expression involves the coordinate functioning of a Sp1 site in the promoter and an AP-1 site in intron 8.
DeLong
Cynthia J
CJ
Department of Biological Chemistry, University of Michigan Medical School, 5416 Medical Science I, 1301 Catherine St., Ann Arbor, MI 48109-0606, USA.
Smith
William L
WL
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics; D051545:Cyclooxygenase 1 / Q000096:biosynthesis / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006801:Humans; D007438:Introns / Q000502:physiology*; D008533:Megakaryocytes / Q000166:cytology / Q000201:enzymology; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D016329:Sp1 Transcription Factor / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000502:physiology; D015854:Up-Regulation / Q000235:genetics
D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D051545:Cyclooxygenase 1