Ets-2 and C/EBP-beta are important mediators of ovine trophoblast Kunitz domain protein-1 gene expression in trophoblast

Background: The trophoblast Kunitz domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a C-terminal "Kunitz" domain, preceded by one or more unique N-terminal domains. TKDP-1 shares an almost identical expression pattern with interferon-tau, the "maternal recognition of pregnancy protein" in ruminants. Our goal here has been to determine whether the ovine (ov) Tkdp-1 and IFNT genes possess a similar transcriptional code. Results: The ovTkdp-1 promoter has been cloned and characterized. As with the IFNT promoter, the Tkdp-1 promoter is responsive to Ets-2, and promoter-driven reporter activity can be increased over 700-fold in response to over-expression of Ets-2 and a constitutively active form of protein Kinase A (PKA). Unexpectedly, the promoter element of Tkdp-1 responsible for this up-regulation, unlike that of the IFNT, does not bind Ets-2. However, mutation of a CCAAT/enhancer binding element within this control region not only reduced basal transcriptional activity, but prevented Ets-2 as well as cyclic adenosine 5'-monophosphate (cAMP)/PKA and Ras/mitogen-activated protein kinase (MAPK) responsiveness. In vitro binding experiments and in vivo protein-protein interaction assays implicated CCAAT/enhancer binding protein-beta (C/EBP-beta) as involved in up-regulating the Tkdp-1 promoter activity. A combination of Ets-2 and C/EBP-beta can up-regulate expression of the minimal Tkdp-1 promoter as much as 930-fold in presence of a cAMP analog. An AP-1-like element adjacent to the CCAAT enhancer, which binds Jun family members, is required for basal and cAMP/C/EBP-beta-dependent activation of the gene, but not for Ets-2-dependent activity. Conclusion: This paper demonstrates how Ets-2, a key transcription factor for trophoblast differentiation and function, can control expression of two genes (Tkdp-1 and IFNT) having similar spatial and temporal expression patterns via very different mechanisms.