Mélin F, Miranda M, Montreau N, DePamphilis M L, Blangy D
CNRS, UPR 272, Laboratoire Virus et Différenciation de l'Université Pierre et Marie Curie, Villejuif, France.
EMBO J. 1993 Dec;12(12):4657-66. doi: 10.1002/j.1460-2075.1993.tb06154.x.
In an effort to identify transcriptional elements that are recognized at different stages of early mouse development, polyomavirus (PyV) enhancer mutations were selected for their ability to support PyV transcription and replication in various mouse undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cell lines. Several of these enhancer mutations were then isolated, sequenced and tested for their ability to stimulate the PyV early gene promoter in plasmid DNA that was either transfected into EC, ES and fibroblast cell lines, or injected into the nuclei of mouse 1-cell and 2-cell embryos. EC, ES and fibroblast cell lines showed clear preferences for different enhancer configurations, and cleavage-stage embryos (2- to 8-cell stage) strongly preferred the same enhancer configuration favored by ES cells. This 'embryo responsive' (ER) enhancer configuration was characterized by a tandem duplication of the region containing a single point mutation that created a DNA binding site for Transcription Enhancer Factor-1 (TEF-1). ER enhancers stimulated the PyV promoter up to 350-fold in embryos, and were up to 74-fold more active than the wild-type PyV enhancer. Most of the activity from PyER enhancers could be duplicated in 2-cell embryos by synthesizing only the tandemly repeated sequence. Comparison of these synthetic enhancers with ER enhancers confirmed that TEF-1 DNA binding sites were highly preferred in ES cells and cleavage-stage embryos, and suggested that ER enhancer activity resulted primarily from cooperative interaction between either two closely spaced TEF-1 DNA binding sites or two TEF-1 DNA binding sites separated by a third, as yet unidentified, transcription factor binding site. These results provide a prototype of a mammalian embryo responsive enhancer, and suggest that TEF-1 plays an important role in activation of gene expression at the beginning of mammalian development.
为了鉴定在小鼠早期发育不同阶段被识别的转录元件，选择多瘤病毒（PyV）增强子突变体，是因为它们能够在各种小鼠未分化胚胎癌（EC）和胚胎干细胞（ES）系中支持PyV转录和复制。然后分离出其中几个增强子突变体，进行测序，并测试它们在质粒DNA中刺激PyV早期基因启动子的能力，该质粒DNA要么转染到EC、ES和成纤维细胞系中，要么注射到小鼠单细胞和二细胞胚胎的细胞核中。EC、ES和成纤维细胞系对不同的增强子构型表现出明显的偏好，而卵裂期胚胎（2至8细胞期）强烈偏好ES细胞所青睐的相同增强子构型。这种“胚胎反应性”（ER）增强子构型的特征是包含单个点突变的区域发生串联重复，该点突变产生了转录增强因子-1（TEF-1）的DNA结合位点。ER增强子在胚胎中刺激PyV启动子的活性高达350倍，比野生型PyV增强子的活性高74倍。通过仅合成串联重复序列，PyER增强子的大部分活性可以在二细胞胚胎中重现。将这些合成增强子与ER增强子进行比较证实，TEF-1 DNA结合位点在ES细胞和卵裂期胚胎中高度优先，这表明ER增强子活性主要源于两个紧密间隔的TEF-1 DNA结合位点之间或由第三个尚未鉴定的转录因子结合位点隔开的两个TEF-1 DNA结合位点之间的协同相互作用。这些结果提供了一个哺乳动物胚胎反应性增强子的原型，并表明TEF-1在哺乳动物发育开始时基因表达的激活中起重要作用。
