Chakrabarty Anindita, Roberts Michael R
Departments of Veterinary Pathobiology, University of Missouri-Columbia, Columbia, MO 65211, USA.
BMC Mol Biol. 2007 Feb 27;8:14. doi: 10.1186/1471-2199-8-14.
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.
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.
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.
滋养层Kunitz结构域蛋白（TKDPs）构成一个高表达、胎盘特异性的多基因家族，仅存在于反刍有蹄类动物中，其特征是在C末端有一个“Kunitz”结构域，之前有一个或多个独特的N末端结构域。TKDP-1与干扰素-τ具有几乎相同的表达模式，干扰素-τ是反刍动物中的“妊娠母体识别蛋白”。我们这里的目标是确定绵羊（ov）Tkdp-1和IFNT基因是否具有相似的转录密码。
已克隆并鉴定了ovTkdp-1启动子。与IFNT启动子一样，Tkdp-1启动子对Ets-2有反应，并且响应Ets-2和组成型活性形式的蛋白激酶A（PKA）的过表达，启动子驱动的报告基因活性可增加700倍以上。出乎意料的是，与IFNT不同，负责这种上调的Tkdp-1启动子元件不结合Ets-2。然而，该控制区域内的CCAAT/增强子结合元件的突变不仅降低了基础转录活性，而且阻止了Ets-2以及环磷酸腺苷（cAMP）/PKA和Ras/丝裂原活化蛋白激酶（MAPK）的反应性。体外结合实验和体内蛋白质-蛋白质相互作用分析表明CCAAT/增强子结合蛋白-β（C/EBP-β）参与上调Tkdp-1启动子活性。在cAMP类似物存在的情况下，Ets-2和C/EBP-β的组合可将最小Tkdp-1启动子的表达上调多达930倍。与CCAAT增强子相邻的一个类似AP-1的元件，它结合Jun家族成员，是该基因基础和cAMP/C/EBP-β依赖性激活所必需的，但不是Ets-2依赖性活性所必需的。
本文证明了Ets-2，一种滋养层分化和功能的关键转录因子，如何通过非常不同的机制控制两个具有相似时空表达模式的基因（Tkdp-1和IFNT）的表达。
