The Wilms’ tumour suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A(2) receptor gene in megakaryocytes

In humans, the TPα and TPβ isoforms of the thromboxane A(2) 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 erythroleukaemia (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 tumour (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 electrophoretic mobility shift assays 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.