Clark A R, Wilson M E, London N J, James R F, Docherty K
Department of Medicine, University of Birmingham, Queen Elizabeth Hospital, U.K.
Biochem J. 1995 Aug 1;309 ( Pt 3)(Pt 3):863-70. doi: 10.1042/bj3090863.
A deletion analysis of the human insulin gene extending to 2 kb upstream of the transcription start site provided evidence of regulatory sequences located upstream of the insulin-linked polymorphic region (ILPR). Within this ILPR-distal region is a sequence (Ink, for insulin kilobase upstream) which contains three potential nuclear hormone-receptor half-sites, closely matching the consensus sequence AGGTCA. These sequences are arranged as a palindromic element with zero spacing over-lapping a direct repeat with 2 bp spacing. The Ink sequence was used in electrophoretic mobility-shift assays within nuclear extracts from COS-7 cells overexpressing the vitamin D, thyroid hormone or retinoic acid receptors, or from an insulin-expressing hamster cell line, HIT-T15. These studies suggest that the insulin-expressing cell line contains thyroid hormone and retinoic acid receptors at least, and that these receptors are able to recognize the Ink sequence. Three copies of the Ink sequence were placed upstream of the thymidine kinase promoter and firefly luciferase reporter gene. In COS-7 cells expressing the appropriate nuclear hormone receptor, this construct was responsive to both thyroid hormone (18-fold) and all-trans-retinoic acid (31-fold). In HIT-T15 cells the same construct responded to all-trans-retinoic acid, but not to thyroid hormone. Within the context of a 2 kb insulin gene fragment, the Ink sequence was shown to be activated by retinoic acid and by the retinoic acid receptor, but acted as a negative element in the presence of both retinoic acid and the retinoic acid receptor. Mutagenesis studies demonstrated that the palindromic sequence was important for the retinoic acid response, and for binding of complexes containing retinoic acid receptor. In human islets of Langerhans, retinoic acid was shown to stimulate insulin mRNA levels. These results demonstrate that a functional nuclear hormone-receptor-response element is located upstream of the human ILPR. As retinoic acid and thyroid hormone are frequently involved in developmental regulatory processes, it is possible that this element may be important in the process of islet cell differentiation.
对人胰岛素基因进行的缺失分析延伸至转录起始位点上游2 kb处，这为位于胰岛素连锁多态性区域（ILPR）上游的调控序列提供了证据。在这个ILPR远端区域内有一个序列（Ink，即胰岛素上游千碱基序列），它包含三个潜在的核激素受体半位点，与共有序列AGGTCA紧密匹配。这些序列排列成一个回文元件，间距为零，与一个间距为2 bp的直接重复序列重叠。Ink序列用于在过表达维生素D、甲状腺激素或视黄酸受体的COS-7细胞的核提取物中，或来自表达胰岛素的仓鼠细胞系HIT-T15的核提取物中进行电泳迁移率变动分析。这些研究表明，表达胰岛素的细胞系至少含有甲状腺激素和视黄酸受体，并且这些受体能够识别Ink序列。将三个Ink序列拷贝置于胸苷激酶启动子和萤火虫荧光素酶报告基因的上游。在表达适当核激素受体的COS-7细胞中，该构建体对甲状腺激素（18倍）和全反式视黄酸（31倍）均有反应。在HIT-T15细胞中，相同的构建体对全反式视黄酸有反应，但对甲状腺激素无反应。在2 kb胰岛素基因片段的背景下，Ink序列被证明可被视黄酸和视黄酸受体激活，但在视黄酸和视黄酸受体同时存在的情况下起负性元件的作用。诱变研究表明，回文序列对视黄酸反应以及对视黄酸受体复合物的结合很重要。在人胰岛中，视黄酸被证明可刺激胰岛素mRNA水平。这些结果表明，一个功能性核激素受体反应元件位于人ILPR的上游。由于视黄酸和甲状腺激素经常参与发育调控过程，因此这个元件可能在胰岛细胞分化过程中很重要。
