Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites
Fibroblast growth factor-8 expression is regulated by intronic engrailed and Pbx1-binding sites
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DOI：
10.1074/jbc.274.9.6020
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发表时间：
1999-02-26
影响因子：
4.8
通讯作者：
MacArthur, CA
中科院分区：
文献类型：
--
作者：
Gemel, J;Jacobsen, C;MacArthur, CA
成纤维细胞生长因子 8 (FGF8) 在脊椎动物发育中发挥着关键作用，在脊椎动物胚胎的时间和空间限制区域中正常表达。我们现在报道了 Fgf8 区域的鉴定，这些区域对其在小鼠 ES 细胞衍生的胚状体中的转录调控至关重要。使用人生长激素报告基因稳定转染 ES 细胞，用于鉴定具有启动子/增强子活性的 Fgf8 基因区域。 Fgf8 的 2 千碱基 5' 区域显示出含有启动子活性。发现源自 Fgf8 大内含子的 0.8 KB 片段能够以方向依赖性方式将 Fgf8 启动子表达的人生长激素增强 3-4 倍。内含子片段包含 AP2、Pbx1 和 Engrailed 转录因子的 DNA 结合位点。凝胶转移和蛋白质印迹实验记录了 ES 细胞胚状体核提取物中这些转录因子的存在。 Engrailed 或 Pbx1 位点的体外诱变表明这些位点调节内含子片段的活性。此外，Engrailed 和 Pbx1 位点的体外诱变表明，其他未鉴定的位点负责观察到的内含子片段的转录增强。
Fibroblast growth factor-8 (FGF8) plays a critical role in vertebrate development and is expressed normally in temporally and spatially restricted regions of the vertebrate embryo. We now report on the identification of regions of Fgf8 important for its transcriptional regulation in murine ES cell-derived embryoid bodies. Stable transfection of ES cells, using a human growth hormone reporter gene, was employed to identify regions of the Fgf8 gene with promoter/enhancer activity. A 2-kilobase 5' region of Fgf8 was shown to contain promoter activity. A 0.8-kilobase fragment derived from the large intron of Fgf8 was found to enhance human growth hormone expressed from the Fgf8 promoter 3-4-fold in an orientation dependent manner. The intronic fragment contains DNA-binding sites for the AP2, Pbx1, and Engrailed transcription factors. Gel shift and Western blot experiments documented the presence of these transcription factors in nuclear extracts from ES cell embryoid bodies. In vitro mutagenesis of the Engrailed or Pbx1 site demonstrated that these sites modulate the activity of the intronic fragment. In addition, in vitro mutagenesis of both Engrailed and Pbx1 sites indicated that other unidentified sites are responsible for the transcriptional enhancement observed with the intronic fragment.