Slater E P, Rabenau O, Karin M, Baxter J D, Beato M
Mol Cell Biol. 1985 Nov;5(11):2984-92. doi: 10.1128/mcb.5.11.2984-2992.1985.
In this study DNA-binding and gene transfer experiments were performed to examine a potential glucocorticoid regulatory element (GRE) in the human growth hormone gene. As assayed by nitrocellulose filter binding, only two regions of the human growth hormone gene, the 5'-flanking sequences and a fragment containing part of the first intron, were retained preferentially by purified glucocorticoid-receptor complexes. The relative binding by the transcribed sequences was three times greater than the relative binding by the 5'-flanking sequences, but less than the relative binding by a fragment containing the human metallothionein-IIA gene GRE. The intron, but not the 5'-flanking sequences, generated a "footprint" when the receptor complex was used to protect the segments against exonuclease III digestion; the protected sequence spanned nucleotides +86 to +115 in the first intron and contained a structure homologous in 14 of 16 nucleotides to a 16-nucleotide consensus GRE. The hexanucleotide 5'-TGTCCT-3', thought to be important for GRE activity, not only was found in this sequence and in the 5'-flanking region, but also was present twice in the 3' end of the gene that did not show specific receptor binding. The latter results suggest that the hexanucleotide alone is not sufficient to generate specific receptor binding tight enough to be assayed in this way. To test the biological activity of the intron binding site, a fragment containing these sequences was fused 5' to the human metallothionein-IIA gene promoter depleted of its GRE and linked to the structural sequences of the herpes simplex virus thymidine kinase (TK) gene. When this hybrid gene was transfected into Rat 2 TK- cells, its expression was induced threefold by the glucocorticoid dexamethasone, as assessed by transfection efficiency and RNA blotting analyses. Expression of the same gene without the human growth hormone gene segment was not affected by the steroid, whereas the wild-type human metallothionein-IIA gene promoter containing its GRE responded to the hormone by a sixfold increase in thymidine kinase mRNA. These results indicate that the human growth hormone gene contains a structure within its first intron that can function as a GRE.
在本研究中，进行了DNA结合和基因转移实验，以检测人生长激素基因中潜在的糖皮质激素调节元件（GRE）。通过硝酸纤维素滤膜结合分析，纯化的糖皮质激素受体复合物仅优先保留人生长激素基因的两个区域，即5'侧翼序列和包含部分第一内含子的片段。转录序列的相对结合比5'侧翼序列的相对结合大3倍，但小于包含人金属硫蛋白-IIA基因GRE的片段的相对结合。当受体复合物用于保护片段免受核酸外切酶III消化时，内含子而非5'侧翼序列产生了“足迹”；受保护的序列跨越第一内含子中的核苷酸+86至+115，并且在16个核苷酸中的14个核苷酸中包含与16核苷酸共有GRE同源的结构。被认为对GRE活性很重要的六核苷酸5'-TGTCCT-3'不仅存在于该序列和5'侧翼区域中，而且在未显示特异性受体结合的基因3'端中也出现了两次。后一结果表明，仅六核苷酸不足以产生足够紧密的特异性受体结合，从而无法以这种方式进行检测。为了测试内含子结合位点的生物学活性，将包含这些序列的片段与缺失其GRE的人金属硫蛋白-IIA基因启动子5'端融合，并与单纯疱疹病毒胸苷激酶（TK）基因的结构序列相连。当将该杂合基因转染到大鼠2 TK-细胞中时，通过转染效率和RNA印迹分析评估，其表达被糖皮质激素地塞米松诱导了3倍。不含人生长激素基因片段的相同基因的表达不受类固醇的影响，而包含其GRE的野生型人金属硫蛋白-IIA基因启动子对该激素的反应是胸苷激酶mRNA增加6倍。这些结果表明，人生长激素基因在其第一内含子中包含一个可以作为GRE发挥作用的结构。
