Blake M C, Azizkhan J C
Lineberger Cancer Research Center, Department of Pediatrics, University of North Carolina, Chapel Hill 27599-7295.
Mol Cell Biol. 1989 Nov;9(11):4994-5002. doi: 10.1128/mcb.9.11.4994-5002.1989.
The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.
二氢叶酸还原酶（DHFR）基因编码一种对代谢和细胞生长很重要的酶。我们在体外已发现仓鼠DHFR基因启动子内存在多种DNA-蛋白质相互作用。这些相互作用发生在两种真核转录因子的共有结合位点上。即Sp1和E2F。DHFR的E2F共有位点具有二重对称，且其位置在主要转录起始位点的紧下游3'处，十分独特。在HeLa细胞核提取物中已检测到E2F与DHFR启动子的相互作用，通过使用部分纯化的E2F得以证实，并通过DNA-蛋白质相互作用的酶法和化学分析法对其进行了表征。E2F识别序列的突变消除了E2F与DHFR启动子的结合，导致体外转录活性降低两到五倍，在瞬时表达试验中DHFR启动子活性降低五倍。因此，E2F与DHFR启动子的相互作用是DHFR基因高效表达所必需的。
