Fig. 4.

ZEB1, which is induced by P₄ (27, 56) and inhibited by E₂ treatment, binds to the Dnm3os promoter and stimulates expression of the miR-199a–3p/miR-214 cluster at the transcriptional level. A and B, hTERT-HM human myometrial cells were infected with recombinant adenoviruses expressing either ZEB1 or β-gal, as a control. Overexpression of ZEB1 significantly increased miR-199a–3p and miR-214 expression after 48 h (n = 3). Data shown in A were normalized to U6 and expressed relative to those of cells infected with the β-gal adenovirus, which were assigned a value of 1. C and D, hTERT-HM cells were transfected with either ZEB1 siRNA or nontargeting control siRNA. Knockdown of ZEB1 decreased expression of miR-199a and miR-214 after 72 h (n = 3). Data shown in C were normalized to U6 and expressed relative to those of cells transfected with a nontargeting siRNA, which were assigned a value of 1. E and F, ChIP and quantitative PCR were used to quantify relative binding activity of endogenous ZEB1 to the Dnm3os promoter in pregnant mouse myometrium at 15.5 dpc vs. labor (19.0 dpc). E, Schematic of the Dnm3os promoter containing E-boxes previously shown to bind Twist1 (35). Primers used to amplify genomic DNA containing the putative ZEB1 binding site(s) are indicated by the arrows. F, Binding of endogenous ZEB1 was determined by quantitative PCR, normalized to input, and expressed as fold-increase over binding using nonimmune IgG in place of antibodies to ZEB1. In myometrial tissues from pregnant mice, endogenous ZEB1 binding to the Dnm3os promoter was significantly decreased at 18.5 dpc, when compared with 15.5 dpc. Data shown are the mean ± sem from three independent experiments, each conducted in triplicate. G and H, Ovariectomized, nonpregnant mice were injected with E₂ or vehicle. ZEB1 mRNA (G) and protein (H) expression were decreased in uterine tissues after 24 h estrogen treatment. Data shown in bar graph (G) are the mean ± sem (n = 5 mice per treatment group). ZEB1 mRNA levels were analyzed by qRT-PCR, normalized to GAPDH, and expressed relative to the vehicle-injected control values, which were assigned a value of 1. I, Luciferase activity of COS-7 cells infected with recombinant adenoviruses expressing ZEB1 or β-gal (Control) and transfected with luciferase reporter constructs containing the Dnm3os promoter (−640 to 0 bp) or a truncated Dnm3os promoter (−640 to −357 bp) lacking critical E-boxes shown in E. Enhancing ZEB1 expression induced expression of the luciferase reporter containing the intact Dnm3os promoter but had no effect on the reporter lacking the putative ZEB1 binding sites. Data are the mean ± sem of values from three independent experiments, conducted in triplicate and expressed relative to cells transfected with an empty luciferase vector. J, Overexpression of ZEB1 in hTERT-HM cells induced expression of the primary transcript of miR-199a/miR214, pri-miR-199a, when compared with cells treated with the control adenovirus expressing β-gal (n = 3). *, P < 0.05. Pri-miR-199a values were normalized to 18S RNA and expressed relative to those of cells infected with the β-gal adenovirus, which were assigned a value of 1.