Transcriptional control of the human thromboxane synthase gene in vivo and in vitro.
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.
Yaekashiwa
Masahiro
M
Division of Hematology, Department of Internal Medicine, University of Texas, Houston, Texas 77030, USA.
Wang
Lee-Ho
LH
D001483:Base Sequence; D015139:Blotting, Southern; D002412:Cations; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002875:Chromosomes / Q000378:metabolism; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009707:Nucleosomes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D013928:Thromboxane A2 / Q000378:metabolism; D013930:Thromboxane-A Synthase / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D002412:Cations; D002843:Chromatin; D009707:Nucleosomes; D013928:Thromboxane A2; D008156:Luciferases; D013930:Thromboxane-A Synthase