Maurer R A, Notides A C
Department of Physiology and Biophysics, University of Iowa, Iowa City 52242.
Mol Cell Biol. 1987 Dec;7(12):4247-54. doi: 10.1128/mcb.7.12.4247-4254.1987.
The DNA sequences which interact with the estrogen receptor and which mediate the estrogenic regulation of prolactin gene transcription have been investigated by the use of receptor-DNA-binding experiments and gene transfer studies. Nitrocellulose filter binding assays using highly purified estrogen receptor and cloned fragments of the 5'-flanking region of the rat prolactin gene demonstrate that the receptor selectively binds to DNA sequences located between nucleotides -1713 and -1532 with respect to the transcription initiation site. The binding of the estrogen receptor to this region of the prolactin gene was strongly dependent on receptor concentration, suggesting that receptor dimers may be important in DNA binding. These data demonstrate that the selective binding of purified estrogen receptor to specific sequences of the rat prolactin gene is an intrinsic property of the receptor and is not due to the interaction of receptor with other proteins. The role of specific prolactin gene sequences in mediating the estrogenic regulation of prolactin gene transcription was confirmed by the use of prolactin-chloramphenicol acetyltransferase fusion genes. These studies demonstrated that sequences upstream of position -1532 are required for estrogen responsiveness. Furthermore, the region of the prolactin gene at -1713 to -1495 was able to confer estrogen responsiveness on the thymidine kinase promoter. Exonuclease III protection experiments further localized the receptor-binding sequences to positions -1587 to -1563. Comparison of the nucleotide sequence of the region of the prolactin gene which binds the estrogen receptor with the sequence of other estrogen-responsive genes suggested the presence of the conserved sequence [sequence in text], which shows similarity to sequences thought to mediate glucocorticoid receptor effects on transcription.
通过受体 - DNA结合实验和基因转移研究，对与雌激素受体相互作用并介导催乳素基因转录雌激素调节的DNA序列进行了研究。使用高度纯化的雌激素受体和大鼠催乳素基因5'侧翼区域的克隆片段进行的硝酸纤维素滤膜结合试验表明，该受体选择性地结合于相对于转录起始位点位于核苷酸-1713至-1532之间的DNA序列。雌激素受体与催乳素基因的这一区域的结合强烈依赖于受体浓度，这表明受体二聚体在DNA结合中可能很重要。这些数据表明，纯化的雌激素受体与大鼠催乳素基因特定序列的选择性结合是该受体的固有特性，并非由于受体与其他蛋白质的相互作用。通过使用催乳素 - 氯霉素乙酰转移酶融合基因，证实了特定催乳素基因序列在介导催乳素基因转录雌激素调节中的作用。这些研究表明，-1532位点上游的序列是雌激素反应性所必需的。此外，催乳素基因-1713至-1495区域能够赋予胸苷激酶启动子雌激素反应性。核酸外切酶III保护实验进一步将受体结合序列定位到-1587至-1563位点。将结合雌激素受体的催乳素基因区域的核苷酸序列与其他雌激素反应性基因的序列进行比较，发现了保守序列[文中序列]的存在，该序列与被认为介导糖皮质激素受体对转录作用的序列相似。
