Transcriptional activation of the human hematopoietic prostaglandin D synthase gene in megakaryoblastic cells. Roles of the oct-1 element in the 5'-flanking region and the AP-2 element in the untranslated exon 1.
The human hematopoietic prostaglandin D synthase (H-PGDS) gene is highly expressed in human megakaryoblastic cells, in which phorbol ester induces its expression. We characterized the promoter activity of the 5'-flanking region and the untranslated exon 1 (-1044 to +290) of the human H-PGDS gene in human megakaryoblastic Dami cells. Transient expression analysis using the luciferase reporter gene revealed that the 5'-flanking region and the untranslated exon 1 were sufficient for efficient expression of the H-PGDS gene in Dami cells, but not in monocytic U937 cells. Deletion and site-directed mutagenesis of the Oct-1 element in the 5'-flanking region decreased the promoter activity by approximately 30% compared with that of the entire region from -1044 to +290. An electrophoretic mobility shift assay demonstrated that Oct-1 specifically bound to the promoter region. Interestingly, even only untranslated exon 1 (+1 to +290) showed approximately 60% of the promoter activity of the entire region from -1044 to +290. Site-directed mutagenesis of the AP-2 element within the untranslated exon 1 abolished the basal promoter activity as well as its phorbol ester-mediated up-regulation. In AP-2-deficient HepG2 cells, the H-PGDS promoter activity was enhanced by coexpression with AP-2alpha. These findings indicate that the Oct-1 element in the 5'-flanking region acts as a positive cis-acting element and that the AP-2 element in the untranslated exon 1 is crucial for both basal and phorbol ester-mediated up-regulation of human H-PGDS gene expression in megakaryoblastic Dami cells.
Fujimori
K
K
Department of Molecular Behavioral Biology and the Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.
Kanaoka
Y
Y
Sakaguchi
Y
Y
Urade
Y
Y
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000201:enzymology*; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons; D051863:Host Cell Factor C1; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D054834:Lipocalins; D008533:Megakaryocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050811:Octamer Transcription Factor-1; D012045:Regulatory Sequences, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D020506:Untranslated Regions; D015854:Up-Regulation / Q000187:drug effects
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D054834:Lipocalins; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; C493673:TFAP2A protein, human; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D020506:Untranslated Regions; D004247:DNA; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D013755:Tetradecanoylphorbol Acetate