Snape A M, Jonas E A, Sargent T D
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
Development. 1990 May;109(1):157-65. doi: 10.1242/dev.109.1.157.
Nuclear extracts from embryos of Xenopus laevis were shown to contain a protein activity, KTF-1, which binds in vitro to the promoter of the embryonic, epidermis-specific keratin gene, XK81A1. Mobility shift assays, methylation interference and footprinting analysis were used to show that the KTF-1 binding site contains an imperfect, palindromic sequence, ACCCTGAGGCT. This sequence occurs once in the XK81A1 promoter, 152-162 base pairs upstream of the transcription start site. A construct of the keratin gene in which this sequence was altered so that it no longer binds KTF-1 in vitro showed severely reduced transcription levels upon injection into Xenopus embryos, but retained epidermal specificity. Addition of KTF-1 binding sites also enhanced epidermal and non-epidermal activity of a heterologous promoter, Xenopus beta-globin, in embryos. These results suggest that KTF-1 is a general activator of embryonic keratin transcription, which acts in concert with other factors to produce high levels of epidermis-specific expression.
非洲爪蟾胚胎的核提取物被证明含有一种蛋白质活性物质KTF-1,它在体外可与胚胎表皮特异性角蛋白基因XK81A1的启动子结合。采用迁移率变动分析、甲基化干扰和足迹分析表明,KTF-1结合位点含有一个不完全的回文序列ACCCTGAGGCT。该序列在XK81A1启动子中出现一次,位于转录起始位点上游152 - 162个碱基对处。一种角蛋白基因构建体,其中该序列被改变以致于在体外不再与KTF-1结合,将其注射到非洲爪蟾胚胎中后显示转录水平严重降低,但保留了表皮特异性。在胚胎中添加KTF-1结合位点也增强了异源启动子非洲爪蟾β-珠蛋白的表皮和非表皮活性。这些结果表明,KTF-1是胚胎角蛋白转录的一般激活剂,它与其他因子协同作用以产生高水平的表皮特异性表达。