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- PublicationRegulated expression of the α isoform of the human thromboxane A2 receptor during megakaryocyte differentiation : a coordinated role for WT1, Egr1 & Sp1Thromboxane plays an essential role in haemostasis, regulating platelet aggregation and vessel tone. In humans, it signals through the TPalpha and TPbeta isoforms that are transcriptionally regulated by distinct promoters, Prm1 and Prm3, respectively. Herein, the consequence of megakaryocytic differentiation on Prm1-directed TPα expression was investigated. Phorbol ester (PMA) treatment substantially increased TPα mRNA and Prm1-directed gene expression in human erythroleukemia (HEL) and K562 cells. Deletional analyses localized the major responsive element(s) to the upstream -8500 and -7504 region while mutation of four WT1/Egr1/Sp1 cis-elements therein established that each contribute to the induction. Moreover, PMA increased Egr1, but not WT1 or Sp1, expression while the NAB1 co-repressor impaired PMA-induction of Egr1 and Prm1-directed gene expression. Chromatin immunoprecipitations established that WT1 is predominantly bound in vivo to the 5’ Prm1 region in non-differentiated HEL cells. In response to PMA, there was initial induction in Egr1 and associated reduction in WT1 binding to Prm1 in vivo which was displaced by Sp1 following sustained treatment. Collectively, data establish that regulated WT1 followed by sequential Egr1 and Sp1 binding to elements within Prm1 mediate repression and subsequent induction of TPα during differentiation into the megakaryocytic phenotype, shedding significant insights into factors regulating TPa expression therein.
274Scopus© Citations 14
- PublicationThe Wilms’ tumor suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A2 receptor gene in megakaryocytesIn humans, TPα and TPβ isoforms of the thromboxane A2 receptor are transcriptionally regulated by distinct promoters, designated Prm1 and Prm3. Previous investigations identified two upstream repressor regions (URR) 1 and URR2 within Prm1. Herein, it was sought to characterize Prm1, identifying the factor(s) regulating URR1 and URR2 in human erythroleukemia (HEL) 92.1.7 cells. Genetic reporter assays and 5’ deletions confirmed the presence of URR1 and URR2 but also identified a third repressor, designated RR3, within the proximal “core” promoter. Bioinformatic analysis revealed several GC elements representing putative sites for Egr1/Sp1/Wilms tumor (WT)1 within URR1, URR2 and RR3. While mutation of three GC elements within URR1 and of an adjacent GC element suggested that repressor binding occurs through a cooperative mechanism, repressors binding to the single GC elements within URR2 and RR3 act independently to regulate Prm1. While EMSAs and supershift assays demonstrated that each of the GC elements can bind Egr1 and WT1 in vitro, chromatin immunoprecipitations established that WT1 is the factor predominantly bound to each of the repressor regions in vivo. Additionally, ectopic expression of -KTS isoforms of WT1 decreased Prm1-directed gene expression and TPα mRNA expression. Collectively, these data establish WT1 as a critical repressor of Prm1, suppressing TPα expression in the platelet progenitor megakaryoblastic HEL cells.
280Scopus© Citations 8