Upstream stimulating factors 1 and 2 enhance transcription from the placenta-specific promoter 1.1 of the bovine cyp19 gene

Abstract

Background: Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth. The enzyme that converts androgens into oestrogens, aromatase cytochrome P450 (P450arom), is encoded by the Cyp19 gene. In the placenta of the cow, expression of Cyp19 relies on promoter 1.1 (P1.1). Our recent studies of P1.1 in vitro and in a human trophoblast cell line (Jeg3) revealed that interactions of placental nuclear protein(s) with the E-box element at position -340 are required for full promoter activity. The aim of this work was to identify and characterise the placental E-box (-340)-binding protein(s) (E-BP) as a step towards understanding how the expression of Cyp19 is regulated in the bovine placenta.

Results: The significance of the E-box was confirmed in cultured primary bovine trophoblasts. We enriched the E-BP from placental nuclear extracts using DNA-affinity Dynabeads and showed by Western blot analysis and supershift EMSA experiments that the E-BP is composed of the transcription factors upstream stimulating factor (USF) 1 and USF2. Depletion of the USFs by RNAi and expression of a dominant-negative USF mutant, were both associated with a significant decrease in P1.1-dependent reporter gene expression. Furthermore, scatter plot analysis of P1.1 activity vs. USF binding to the E-box revealed a strong positive correlation between the two parameters.

Conclusion: From these results we conclude that USF1 and USF2 are activators of the bovine placenta-specific promoter P1.1 and thus act in the opposite mode as in the case of the non-orthologous human placenta-specific promoter.

Figure 1

Schematic representation of the bovine placenta-specific promoter P1.1. The promoter is shown as black horizontal line with the E-box (-340) shown as a black box. The transcription start site is marked by +1 and an arrow. Exons 1.1 and 2 are drawn as thick black lines, the ~20 kb intron between them is indicated. The translation start site within exon 2 is marked by the ATG codon.

Figure 2

E-box (-340) is essential for transcriptional activity of P1.1 in primary bovine trophoblast cells. Cells were transiently transfected with a promoterless luciferase reporter gene plasmid (pGL3), which served as a control, or with luciferase reporter gene plasmids containing the proximal sequence of P1.1 and part of the untranslated exon 1 (base pairs -404 to +113) with either a wild type (P1.1wt) or a mutated E-box (-340) motif (Emut), along with plasmid CMV-lacZ. Activities of the luciferase and lacZ reporter genes were measured 24 h after transfection. The quotient of the luciferase and β-galactosidase activities was calculated to normalise for transfection efficiency. Results are expressed as the mean ± S.E.M of n = 3 experiments. Different letters above the columns indicate significant differences (p < 0.05).

Figure 3

The E-box binding factor is composed of USF1 and USF2. A) Western blot analysis of proteins eluted from immobilised target DNA containing a wild type (Ewt), or a mutated E-box motif (Emut). Protein samples were subjected to SDS-PAGE alongside the nuclear extract (NE). Gels were blotted and incubated with commercial anti USF1- and anti USF2-IgG. Both antibodies interacted with nuclear extracts and eluates from the wild type, but not from the mutated target oligonucleotides. B) EMSA experiments with the labelled E-box probe and bovine placenta-derived nuclear extracts. With nuclear extract, a complex (shift) formed (lane 1). Specificity of the complex was demonstrated by competition with a 100-fold excess of the unlabelled probe (lane 2) or a mutated probe (lane 3). Addition of anti USF1 antibodies strongly reduced the complex (lane 4). In the presence of anti USF2 antibodies, a supershift complex formed (lane 5). There is no evidence for more than one specific complex, suggesting that the probe is bound by USF1/2 heterodimers.

Figure 4

siRNA-mediated double knockdown of USF1 and USF2 reduces P1.1 activity. A) To examine the transcript specificity of knockdowns, Jeg3 trophoblast cells were transfected with pools of siRNAs targeting USF1 (siUSF1), USF2 (siUSF2) or a siControl pool, which does not target any known transcript. Mock-transfected cells treated only with the transfection vehicle were used to normalise measurements. USF transcripts were quantified by real-time PCR 24 h after transfection. The relative transcript abundance was calculated by dividing measurements from siRNA treated cells with measurements from mock-transfected cells. Mean values ± S.E.M. of n = 3 experiments are shown. B) To analyse the effect of USF knockdown on P1.1-dependent transcription, Jeg3 cells were transiently transfected with the P1.1-luciferase reporter gene plasmid along with siRNAs (siUSF1, siUSF2 or siUSF1+USF2). Jeg3 cells transfected with a non-targeting siRNA (siControl), or without siRNA served as controls. Luciferase activities were measured 72 h after transfection. Results (relative light units, rlu) are shown as means ± S.E.M. of at least n = 3 experiments. Only the double knockdown resulted in a significant reduction of the reporter gene activity (p < 0.05), as indicated by an asterisk.

Figure 5

P1.1-mediated gene expression is positively correlated with USF binding to the E-box (-340). A) Transient cotransfection assays in Jeg3 cells of the P1.1-luciferase reporter gene plasmid in the presence of the indicated amounts of the dominant-negative A-USF construct. The luciferase activities were measured 24 h after transfection. To normalise measurements, the results from analogous cotransfection experiments with the empty expression plasmid were arbitrarily set to 1. Mean values ± S.E.M. of n = 3 experiments are shown. Differences between A-USF transfected cells and cells transfected with the empty expression vector were significant, as indicated by asterisks (* p < 0.05; ** p < 0,001). B) representative EMSA experiments with the labelled E-box probe and Jeg3 cell extracts left from reporter gene analyses. Jeg3 cells were transiently transfected with the P1.1-luciferase reporter gene plasmid plus 5 ng of the empty expression vector (- A-USF) or 5 ng of the A-USF expression plasmid (+ A-USF). In the presence of A-USF, binding of USF to the E-box probe is impaired. C) Scatter plot of luciferase activity vs. USF binding. Data points represent reporter gene and USF binding activities of individual extracts from reporter gene experiments with different amounts of A-USF- or empty expression plasmids. USF binding was quantified by applying the Image Quant software to EMSA experiments similar to the one shown in panel B. Data were statistically analysed with the Pearson product moment test. The correlation coefficient, r, and the p value are indicated.