Abstract
Thromboxane A(2), a potent mediator of vasoconstriction and platelet aggregation, is synthesized from prostaglandin H(2) by thromboxane synthase (TXAS). We report here on promoter analyses of human TXAS using in vitro transcription and in vivo transfection methods. The 39-bp core promoter, containing both TATA and initiator elements, accurately initiates transcription in an orientation-dependent manner in a cell-free transcription system. Mutation of either TATA or initiator abolished transcriptional activity, but the upstream sequence had no effect on TXAS promoter activities in vitro, suggesting that the core promoter is sufficient for transcriptional activity from a naked DNA template. In contrast, mutation of both elements drastically decreased the promoter activity in vivo. Furthermore, TXAS proximal promoter containing the nucleotides -90 to -56 relative to the transcriptional start site was necessary for promoter transactivation in vivo. Transcriptional activities from 5'-deletion mutants indicated that the effects of the nucleotides -90/-56 were more pronounced in stably transfected cells (a 150-fold difference) than in the transiently transfected cells (an 8-fold difference), reflecting the effects of TXAS transcriptional activation from replicating and nonreplicating DNA templates. Partial micrococcal nuclease digestion indicated that the sequence -90/-56, where the NF-E2 site is located, is associated with alterations of nucleosomal structure at the regions of promoter and reporter gene but not the region further downstream. Mutagenesis and forced expression studies demonstrated a critical role of p45 NF-E2 in controlling TXAS expression under native chromatin conditions. Band shifting and chromatin immunoprecipitation assays indicated that p45 NF-E2 was bound to the TXAS promoter in vitro and in vivo. Indirect end labeling and ligation-mediated PCR analyses further demonstrated that the occupation of TXAS promoter NF-E2 site was associated with disruption of nucleosomal structure. Collectively, these results indicate that binding of NF-E2 is critical both for alteration of the nucleosomal structure and for activation of the TXAS promoter, whereas the TATA and initiator elements are essential for transcription.
Highlights
Logic and pathologic processes including hemostasis/thrombosis, cardiovascular disease [1, 2], pulmonary diseases [3, 4], and glomerulonephritis [5]
Mutation of either TATA or initiator abolished transcriptional activity, but the upstream sequence had no effect on thromboxane synthase (TXAS) promoter activities in vitro, suggesting that the core promoter is sufficient for transcriptional activity from a naked DNA template
In Vitro Transcription of the Human TXAS Gene Promoter—In initial experiments, we used the core promoter of the TXAS gene linked to a 377-bp G-free cassette vector, pC2AT, as a template (pC2AT-TX36(ϩ)) to examine TXAS gene expression
Summary
Materials—The G-free cassette vectors, pC2AT and pMLC2AT190, were kindly provided by Drs Michele Sawadogo and Ming-Jer Tsai [33, 34]. 5 g of nuclear extracts were incubated with a 32P-end-labeled probe (TXAS promoter nucleotides Ϫ121 to ϩ5) in a binding reaction buffer containing 20 mM HEPES, pH 7.9, 0.2 M EDTA, 15% glycerol, 1 g of poly(dI-dC), 100 mM KCl, 2.5 mM MgCl2, and 1 mM dithiothreitol for 20 min at room temperature. 3 ϫ 107 cells were washed with phosphate-buffered saline, and chromatin-DNA was cross-linked by incubation of cells with 1% formaldehyde at room temperature for 20 min [40] before being washed twice with ice-cold phosphate-buffered saline and resuspended in 6 ml of suspension buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 0.1 mM EGTA, 15 mM NaCl, 50 mM KCl, 0.15 mM spermine, and 0.5 mM spermidine) containing 0.2% Nonidet P-40 and 5% sucrose.
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