Abstract
BackgroundThe 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.ResultsThe 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-β) as involved in up-regulating the Tkdp-1 promoter activity. A combination of Ets-2 and C/EBP-β 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-β-dependent activation of the gene, but not for Ets-2-dependent activity.ConclusionThis 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.
Highlights
The trophoblast Kunitz domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a Cterminal "Kunitz" domain, preceded by one or more unique N-terminal domains
Expression patterns of the ovTKDP-1 and IFN-τ messages during pregnancy TKDP-1 and IFN-τ mRNA are expressed within a comparable window of early pregnancy [7], side by side comparisons have not been made
The expression patterns for ovTKDP-1 and ovIFN-τ mRNAs were similar both in magnitude and over developmental time (Fig. 1A). Both transcripts were detectable in sheep conceptuses at day of pregnancy, with concentrations peaking at day and subsequently declining after day 17 to become almost undetectable by day 25 relative to the amplified product derived from the endogenous control, ribosomal protein S25
Summary
The trophoblast Kunitz domain proteins (TKDPs) constitute a highly expressed, placenta-specific, multigene family restricted to ruminant ungulates and characterized by a Cterminal "Kunitz" domain, preceded by one or more unique N-terminal domains. The Kunitz domain is an evolutionary conserved module, existing in C. elegans, D. melanogaster, and all known vertebrates, that generally functions as a serine peptidase inhibitor, functions unrelated to peptidase inhibition have been documented [3,4,5,6]. This independently folding unit is preceded by one or more "repeat" sequences of ~80 amino acids of unknown function, which have similar but not identical sequences [1,2]. The function of these proteins remains unclear, their unusually high expression at the interface between the maternal and fetal systems and the presence of conserved Kunitz units at their carboxyl termini strongly suggest an important role during pregnancy that relates to the evolution of the non-invasive placental type encountered in ruminant species
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