An intronic enhancer regulates cyclooxygenase-1 gene expression.
ABSTRACT 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.
Article: Translational regulation of PGHS-1 mRNA: 5' untranslated region and first two exons conferring negative regulation.[show abstract] [hide abstract]
ABSTRACT: Prostaglandin endoperoxide H synthase-1 gene expression is described as inducible in a few contexts such as differentiation of megakaryoblastic MEG-01 cells into platelet-like structures. In the MEG-01 cells model of PGHS-1 gene induction, we previously reported a delay in protein synthesis and identified the translational step of gene expression as being regulated. In the current study, we mapped PGHS-1 mRNA sequences regulating translational efficiency and identified an RNA binding protein. The 5'UTR and first two exons of the PGHS-1 5' mRNA decreased the synthesis of Luciferase protein by approximately 80% without significant changes in mRNA levels when compared to controls. Both the PGHS-1 5'-UTR and the first two exons were required for activity. Sucrose density gradient fractionations of cytoplasmic extracts from MEG-01 cells infected with reporter constructs, either controls or containing PGHS-1 sequence, presented a similar profile of distribution of reporter transcripts between polysomal and non-polysomal fractions. RNA/protein interaction studies revealed nucleolin binding to the 135 nt PGHS-1 sequence. Mutation of the two NRE elements located in the 5'end of PGHS-1 mRNA sequence partially reduced the negative activity of the 135 nt sequence. Stable secondary structures predicted at the 5' end of the transcript are potentially involved in translational regulation. We propose that the 5'end of PGHS-1 mRNA represses translation and could delay the synthesis of PGHS-1 enzyme.Biochimica et Biophysica Acta 03/2007; 1769(2):92-105. · 4.66 Impact Factor