The Wilms’ tumour suppressor protein WT1 acts as a key transcriptional repressor of the human thromboxane A₂ receptor gene in megakaryocytes

Abstract

In humans, the 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 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.

Regulation of the human prostacyclin receptor gene in megakaryocytes: Major roles for C/EBPδ and PU.1

“…As previous, real-time QT-PCR analysis of this data indicates that products were generated from anti-C/EBP , anti-C/EBP and anti-PU.1 immunoprecipitates in the -1761 to -1577 region, but their relative abundance is substantially reduced relative to products derived from the -1528 to -1327 region, in the absence or presence of PMA (Fig 6F(i & ii)). Also as previous, as a further negative control, QT-PCR analysis using primers specific to a region of Prm3 of the TP gene [47] generated amplicons from the input chromatin but not from the anti-C/EBP , anti-C/EBP , anti-PU.1, IgG immunoprecipitates or the no 1 o antibody controls (Supplemental Fig 1B(i & ii)). In parallel with this, immunoblot analysis established that while the level of PU.1 expression in HEL cells was not altered following PMA treatment, the actual level of C/EBP expression was substantially reduced at 16 and 24 hr post-incubation (Fig 6G & 6H), while immunoblotting for the ubiquitously expressed HDJ-2 confirmed uniform protein loading (Fig 6I).…”

“…However, Real-time QT-PCR analysis of this data indicates that products were generated from anti-C/EBP , anti-C/EBP and anti-PU.1 immunoprecipitates in the -1761 to -1577 region, but their relative abundance is substantially reduced relative to those products derived from the -1528 to -1327 region (Fig 2C(ii)). As a further negative control, QT-PCR analysis using primers which were previously described by us [47] and are specific to a region of Prm3 of the thromboxane receptor (TP) gene generated amplicons from the input chromatin but not from the anti-C/EBP , anti-C/EBP , anti-PU.1, IgG immunoprecipitates or the no 1 o antibody control (Supplemental Fig 1B(i)). Endogenous expression of C/EBP , C/EBP and PU.1 in HEL cells was confirmed by immunoblot analysis, where secondary immunoblotting for the ubiquitously expressed HDJ-2 confirmed uniform protein loading (Fig 2D (i-iii)).…”

Regulated Expression of the α Isoform of the Human Thromboxane A2 Receptor during Megakaryocyte Differentiation: A Coordinated Role for WT1, Egr1, and Sp1

“…As stated, WT1 binds to GC elements at -8345, -8281 and -8146 within URR1 and to another adjacent GC element at -7831 within UAR1 in quiescent HEL cells to repress transcriptional activity of Prm1 22 . Since the GC elements at -8345, -8281, -8146 and -7831 consist of putative binding sites for WT1/Egr1/Sp1 (Table I) , suggests that each elements at -8345, -8281, -8146 and -7831 acts independently to contribute to increased Prm1-directed gene expression in response to PMA.…”

Interaction of angio-associated migratory cell protein with the TPα and TPβ isoforms of the human thromboxane A2 receptor

“…TPα and TPβ are identical for their N-terminal 328 residues but differ exclusively within their carboxyl-terminal (C-tail) domains [25][26][27][28]. In humans, TPα and TPβ arise through alternative splicing of a common 1 o transcript but display distinct patterns of expression throughout the vasculature, being transcriptionally regulated by distinct promoters within the single TP gene [29][30][31][32][33][34][35][36].…”