Transcriptional Control of the Human Thromboxane Synthase Genein Vivo and in Vitro

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.

A role for Rab27b in NF-E2-dependent pathways of platelet formation

“…20 Transcriptional targets of NF-E2 must serve essential roles in molecular mechanisms of platelet biogenesis and release. 5,36,49 Some pathways that seem to function downstream of NF-E2, such as prostaglandin metabolism 37,50 and inside-out integrin signaling, 38,39 are as yet not directly implicated in proplatelet formation or platelet release, although ␤1 tubulin is both down-regulated in NF-E2-null MKs 32 and required for optimal platelet production. 51 Here we identify Rab27b as a new component in NF-E2-dependent pathways of platelet assembly.…”

MafG Sumoylation Is Required for Active Transcriptional Repression

“…To directly examine the effects of MafG transgenes on MARE-dependent transcription, we quantified mRNA encoding TXS, a well-characterized target gene of the p45-MafG heterodimer (36). The TXS expression levels correlated well with the PPF ratios, indicating that MafG represses MAREdependent transcription but that MafG K14R does not (Fig.…”

“…Similarly, mafG-null mutant mice and, with increased severity, mafG Ϫ/Ϫ ::mafK ϩ/Ϫ compound mutant mice display defective proplatelet formation (PPF). Expression of the thromboxane synthetase (TXS) gene, a well-characterized megakaryocytic MARE-dependent target gene (36), was dramatically suppressed in megakaryocytes of p45-null mice (4), as well as in small maf mutant mice (20). Thus, a synergy between p45 and small Mafs was strongly implicated from these studies.…”

Transgenic expression of BACH1 transcription factor results in megakaryocytic impairment

“…[24][25][26][27] ChIP studies have demonstrated recruitment of p45 to the MAREs within the promoters of the p45 targets in a cell line with a megakaryocytic lineage. 28 Small maf mutant mice, as well as mafG and mafG::mafK compound mice also showed mild and severe thrombocytopenia, respectively, demonstrating that p45 and small Maf proteins function cooperatively in vivo. 29,30 On the other hand, we previously reported that BACH1 is expressed in megakaryocytes as well as in erythrocytes.…”