Boissier F, Augé-Gouillou C, Schaeffer E, Zakin M M
Laboratoire d'Expression des Gènes Eucaryotes Institut Pasteur, Paris, France.
J Biol Chem. 1991 May 25;266(15):9822-8.
We previously identified a 300-base pair long enhancer, located 3.6 kilobases upstream of the cap site of the human transferrin gene. A 5' deletion up to position 86 of the enhancer resulted in complete loss of the enhancer activity. Here we show by competition footprint analysis, gel retardation assays, and transient expression studies in hepatoma and HeLa cells that the enhancer is composed of two distinct structural and functional domains, A (nucleotides 1-86) and B (nucleotides 87-291). Each domain is a proto-enhancer of a different type. Domain A is a proto-enhancer that, when multimerized, is able by itself to stimulate transcription from the heterologous SV40 promoter, both in Hep3B and HeLa cells. It contains the octanucleotide TGTTTGCT sequence and is the binding site of two liver-specific nuclear factors and of a different HeLa nuclear factor. Domain B contains four binding sites interacting with several liver nuclear proteins. In order to bind, any of these proteins requires the presence of all the others. This domain is able to block the activity of a downstream negative element, but it has no enhancer activity by itself. In the presence of the transferrin promoter, full enhancer activity requires the association of the two domains A and B.
我们先前鉴定出一个长300个碱基对的增强子，它位于人转铁蛋白基因帽位点上游3.6千碱基处。对该增强子进行5'端缺失直至第86位，导致增强子活性完全丧失。在此我们通过竞争足迹分析、凝胶阻滞试验以及在肝癌细胞和HeLa细胞中的瞬时表达研究表明，该增强子由两个不同的结构和功能结构域组成，即A结构域（核苷酸1 - 86）和B结构域（核苷酸87 - 291）。每个结构域都是不同类型的原增强子。结构域A是一种原增强子，当多聚化时，它自身能够在Hep3B细胞和HeLa细胞中刺激来自异源SV40启动子的转录。它包含八聚体TGTTTGCT序列，是两种肝脏特异性核因子以及一种不同的HeLa核因子的结合位点。结构域B包含四个与几种肝脏核蛋白相互作用的结合位点。为了结合，这些蛋白质中的任何一种都需要其他所有蛋白质的存在。该结构域能够阻断下游负调控元件的活性，但它自身没有增强子活性。在存在转铁蛋白启动子的情况下，完整的增强子活性需要结构域A和B的联合作用。
