CHARACTERIZATION OF A COMPLEX GLUCOCORTICOID RESPONSE UNIT IN THE PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE
CHARACTERIZATION OF A COMPLEX GLUCOCORTICOID RESPONSE UNIT IN THE PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE
复制标题
DOI：
10.1128/mcb.10.9.4712
复制
发表时间：
1990-09-01
影响因子：
5.3
通讯作者：
GRANNER, DK
中科院分区：
文献类型：
--
作者：
IMAI, E;STROMSTEDT, PE;GRANNER, DK
定义了 H4IIE 大鼠肝癌细胞中糖皮质激素诱导磷酸烯醇丙酮酸羧激酶 (PEPCK) 基因所需的最小 DNA 序列。这种新型糖皮质激素反应单元 (GRU) 跨越约 110 个碱基对 (bp)，包括两个受体结合元件和两个辅助因子结合元件。纯化的糖皮质激素受体与相对于转录起始位点位于-395 和-349 bp 之间的两个区域（GR1 和GR2）结合。粗制大鼠肝核提取物中的因子与 DNA 的 -455 至 -431 和 -420 至 -403 bp 区域结合，分别称为辅助因子 1 (AF1) 和辅助因子 2 (AF2) 元件。凝胶延迟分析表明，至少有两种蛋白质与 AF1 结合，并且它们与与 AF2 结合的蛋白质不同。将 GR1、GR2、AF1 和 AF2 的各种组合与氯霉素乙酰转移酶 (CAT) 报告基因融合，并与糖皮质激素受体表达质粒 (pSVGR1) 共转染至 H4IIE 细胞中，以鉴定功能性 GRU。单独的糖皮质激素受体结合区和辅助因子结合区都不足以赋予糖皮质激素反应性。糖皮质激素受体结合区的两个组成部分独立发挥作用，并且如果存在辅助因子元件，则各自占最大反应的一半。类似地，AF1 或 AF2 的缺失使 PEPCK 基因的糖皮质激素诱导减少至最大值的大约一半。我们认为复杂的 PEPCK 基因 GRU 提供了肝脏中关键酶所需的严格调节。
The minimal DNA sequence required for glucocorticoid induction of the phosphoenolpyruvate carboxykinase (PEPCK) gen in H4IIE rat hepatoma cells was defined. This novel glucocorticoid response unit (GRU) spans about 110 base pairs (bp) and includes two receptor-binding elements plus two accessory factor-binding elements. Purified glucocorticoid receptor bound to two regions (GR1 and GR2) between -395 and -349 bp relative to the transcription start site. Factors in crude rat liver nuclear extract bound to DNA in the regions -455 to -431 and -420 to -403 bp, which are designated accessory factor 1 (AF1) and accessory factor 2 (AF2) elements, respectively. Gel retardation analysis revealed that at least two proteins bound to AF1 and that they were distinct from the protein(s) that bound to AF2. Various combinations of GR1, GR2, AF1, and AF2 were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and cotransfected with a glucocorticoid receptor expression plasmid (pSVGR1) into H4IIE cells to identify the functional GRU. Neither the glucocorticoid receptor binding region nor the accessory factor binding region alone was sufficient to confer glucocorticoid responsiveness. The two components of the glucocorticoid receptor binding region functioned independently, and each accounted for half of the maximal response, provided the accessory factor elements were present. Similarly, deletion of either AF1 or AF2 diminished glucocorticoid induction of the PEPCK gene to approximately half of the maximum. We propose that the complex PEPCK gene GRU provides the strigent regulation required of the critical enzyme in liver.