Ezashi T, Ealy A D, Ostrowski M C, Roberts R M
Department of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.
Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):7882-7. doi: 10.1073/pnas.95.14.7882.
Expression of the multiple interferon-tau (IFN-tau) genes is restricted to embryonic trophectoderm of ruminant ungulate species for a few days in early pregnancy. The promoter regions of these genes are highly conserved. A proximal (bp -91 to -69) sequence has been implicated in controlling trophoblast-specific expression. Here it was used as a target for yeast one-hybrid screening of a day 13 conceptus cDNA library. Two transcription factors of the Ets family, Ets-2 and GABPalpha, were identified, consistent with the observation that active ovine IFN-tau genes contain a single 10-bp Ets motif (core: GGAA) in the proximal segment, whereas three known inactive ovine genes contain a mutated core motif (TGAA). Cotransfection of a promoter- (-126 to +50) luciferase reporter construct from an active gene (bovineIFN-tau1; boIFNT1) and an Ets-2 expression plasmid in human JAr cells provided up to a 30-fold increase in reporter expression, whereas promoters from inactive genes were not transactivated. GABPalpha alone was ineffective and had only a approximately 2-fold positive effect when coexpressed with its partner GABPbeta. Other Ets-related transcription factors, which were not detected in the genetic screen, also provided a range of lesser transactivation effects. Coexpression of Ets-2 and activated Ras failed to transactivate the IFNT promoter greater than Ets-2 alone in JAr cells. The presence of Ets-2 in nuclei of embryonic trophectoderm was confirmed immunocytochemically. Together, these data suggest that Ets-2 plays a role in the transient expression of the nonvirally inducible IFNT genes.
多种干扰素 -τ(IFN -τ)基因的表达在怀孕早期仅在反刍有蹄类动物的胚胎滋养外胚层中持续几天。这些基因的启动子区域高度保守。一个近端序列(碱基对 -91 至 -69)被认为与控制滋养层特异性表达有关。在此,它被用作酵母单杂交筛选第 13 天孕体 cDNA 文库的靶标。鉴定出了 Ets 家族的两个转录因子,Ets -2 和 GABPα,这与以下观察结果一致:活跃的绵羊 IFN -τ基因在近端片段中含有一个单一的 10 碱基对 Ets 基序(核心:GGAA),而三个已知的不活跃绵羊基因含有突变的核心基序(TGAA)。将来自活跃基因(牛 IFN -τ1;boIFNT1)的启动子 -(-126 至 +50)荧光素酶报告基因构建体与 Ets -2 表达质粒共转染到人 JAr 细胞中,报告基因表达增加了高达 30 倍,而不活跃基因的启动子未被反式激活。单独的 GABPα无效,当其与其伴侣 GABPβ共表达时仅具有约 2 倍的正向作用。在基因筛选中未检测到的其他 Ets 相关转录因子也提供了一系列较小的反式激活作用。在 JAr 细胞中,Ets -2 和激活的 Ras 共表达未能比单独的 Ets -2 更有效地反式激活 IFNT 启动子。通过免疫细胞化学证实了 Ets -2 在胚胎滋养外胚层细胞核中的存在。总之,这些数据表明 Ets -2 在非病毒诱导的 IFNT 基因的瞬时表达中起作用。