Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element
Isolation of human type 2 deiodinase gene promoter and characterization of a functional cyclic adenosine monophosphate response element
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DOI：
10.1210/en.141.5.1804
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发表时间：
2000-05-01
期刊：
影响因子：
4.8
通讯作者：
Centanni, M
中科院分区：
文献类型：
--
作者：
Canettieri, G;Celi, FS;Centanni, M
我们分析了人类2型脱碘酶(HD2)基因5‘侧翼区的结构和功能。在ATG的-470/-474处确定了两个主要的转录起始点。在HEK 293细胞中，以荧光素酶为报告基因，HD2基因5‘侧翼区可以有效地指导瞬时基因的转录。通过从ATG中删除包含-766/-759的cAMP反应元件(CRE)的区域，基础转录活性显著降低。Forsklin处理显著增加了含有Cre的构建体细胞中的荧光素酶活性。在不包含Cre或包含诱变的Cre的构建物中，这种效应被取消。Northern印迹分析显示，cAMP激动剂刺激JEG-3细胞后，HD2信使RNA明显增加。用HD2-Cre探针和HEK293核提取液进行的滋养层迁移率改变分析表明，存在DNA-蛋白质复合体，该复合体被特定的未标记寡核苷酸竞争，并被抗CREB和抗Cre调节剂-1抗体超位。CREB的显性负性抑制剂A-CREB可完全抑制Forsklin对HD2启动子的诱导作用。在对cAMP不敏感的F9畸胎癌细胞中，CREB蛋白与HD2启动子Construction：和PKA共转染后，能够刺激HD2基因的转录，这些结果表明HD2基因5‘侧翼区存在一个功能性启动子，其特征是存在一个CRE。CREB在cAMP介导的HD2基因启动子诱导中的特异性参与也被证明。
We analyzed the structure and function of the 5' flanking region of the human type 2 deiodinase (hD2) gene. Two major transcription start sites were identified at -470/- 474 from the ATG. The 5' flanking region of hD2 gene efficiently directed transcription in transient transfection studies, using luciferase as reporter gene, in HEK 293 cells. Basal transcriptional activity was significantly reduced by deleting the region containing a canonical cAMP-responsive element (CRE) located -766/-759 from ATG. Forskolin treatment significantly increased luciferase activity in cells transfected with CRE-containing constructs. This effect was abolished in constructs that did not contain CRE or contained the mutagenized CRE. Northern blot analysis in JEG-3 cells revealed that the hD2 messenger RNA was markedly increased after stimulation with cAMP agonist. The trophoretic mobility shift assay with hD2-CRE probe and HEK 293 nuclear extract showed the occurrence of a DNA-protein complex, which was competed by specific unlabeled oligonucleotides and supershifted by the anti-CREB and anti-CRE modulator-1 antibodies. A-CREB, a dominant negative inhibitor of CREB, completely inhibited forskolin induction of the hD2 promoter. CREB protein, once cotransfected with hD2 promoter construct: and pKA in F9 teratocarcinoma cells, which are unresponsive to cAMP, was able to stimulate the hD2 gene transcription.These results indicate the existence of a functional promoter within the 5' flanking region of hD2 gene which is characterized by the presence of a CRE. The specific involvement of CREB in the cAMP-mediated hD2 gene promoter induction also has been demonstrated.