Anderson G M, Freytag S O
Molecular Biology Research Program, Henry Ford Hospital, Detroit, Michigan 48202.
Mol Cell Biol. 1991 Apr;11(4):1935-43. doi: 10.1128/mcb.11.4.1935-1943.1991.
Many eucaryotic promoters contain multiple binding sites for sequence-specific DNA-binding proteins. In some cases, these proteins have been shown to interact synergistically to activate transcription. In this study, we address the possibility that the transcription factor Sp1 can synergistically activate a native human promoter in a cellular context that closely resembles that of a single-copy gene. Using DNase I footprinting with affinity-purified Sp1, we show that the human argininosuccinate synthetase (AS) promoter contains three sites that bind Sp1 with different affinities. These binding sites were mutated to abolish Sp1 binding, individually and in all possible combinations, to generate a series of AS promoter-chloramphenicol acetyltransferase (CAT) expression constructs. Mutations designed to increase Sp1 binding were also introduced at each site. The in vivo transcriptional activity of these mutant AS promoter-CAT constructs was then measured in stably transfected human RPMI 2650 cell lines. Our results show that each of the three Sp1-binding sites contributes to full activation of the human AS promoter and that the relative contribution of each site correlates well with its in vitro affinity for Sp1. More importantly, we find that the three Sp1-binding sites when present in the same promoter activate transcription to a level that is 8 times greater than would be expected given their individual activities in the absence of the other two sites. Thus, we provide direct evidence that Sp1-binding sites in their native context in a human promoter can interact synergistically in vivo to activate transcription. The ability to activate transcription synergistically may be the reason that many cellular promoters have multiple Sp1-binding sites arranged in tandem and in close proximity.
许多真核生物启动子含有多个序列特异性DNA结合蛋白的结合位点。在某些情况下，这些蛋白已被证明能协同相互作用以激活转录。在本研究中，我们探讨了转录因子Sp1在与单拷贝基因极为相似的细胞环境中协同激活天然人类启动子的可能性。通过使用亲和纯化的Sp1进行DNase I足迹分析，我们发现人类精氨琥珀酸合成酶（AS）启动子含有三个以不同亲和力结合Sp1的位点。这些结合位点被单独或组合突变以消除Sp1结合，从而产生一系列AS启动子-氯霉素乙酰转移酶（CAT）表达构建体。还在每个位点引入了旨在增加Sp1结合的突变。然后在稳定转染的人类RPMI 2650细胞系中测量这些突变的AS启动子-CAT构建体的体内转录活性。我们的结果表明，三个Sp1结合位点中的每一个都对人类AS启动子的完全激活有贡献且每个位点的相对贡献与其体外对Sp1的亲和力密切相关。更重要的是，我们发现当三个Sp1结合位点存在于同一启动子时，其激活转录的水平比在没有其他两个位点时其各自活性预期的水平高8倍。因此，我们提供了直接证据表明人类启动子中天然环境下的Sp1结合位点在体内可协同相互作用以激活转录。协同激活转录的能力可能是许多细胞启动子串联且紧密排列有多个Sp1结合位点的原因。
