Abstract
BackgroundPlacenta-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.ResultsThe 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.ConclusionFrom 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.
Highlights
Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth
The E-box binding protein is composed of the transcription factors USF1 and USF2 A procedure was developed to enrich the -binding protein(s) (E-BP) from placental nuclear protein extracts
The described features of the E-BP strikingly resembled those of the transcription factors upstream stimulating factor (USF) 1 and USF2 [19,20,21]: First, E-BP and USF proteins bind to a CACATG core E-box element; second, USF proteins are the only known heat-stable E-box-binding transcription factors; and third, the molecular weights of bovine USF proteins as deduced from their cDNA sequences (NP_001001161 and 001001162, respectively) range from 33.4 to 36.9 kDa
Summary
Placenta-derived oestrogens have an impact on the growth and differentiation of the trophoblast, and are involved in processes initiating and facilitating birth. In the placenta of the cow, expression of Cyp 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 Cyp is regulated in the bovine placenta. An oestrogen peak near term triggers endocrine and paracrine processes involved in the initiation of parturition, cervical softening and dilatation, and increasing myometrial contractibility (reviewed by [2]).
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