Characterization and functional analysis of the 5′-flanking region of the mouse 20α-hydroxysteroid dehydrogenase gene
Characterization and functional analysis of the 5′-flanking region of the mouse 20α-hydroxysteroid dehydrogenase gene
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DOI：
10.1042/bj20040276
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发表时间：
2004-09-15
影响因子：
4.1
通讯作者：
Nishihara, M
中科院分区：
文献类型：
--
作者：
Hirabayashi, K;Ishida, M;Nishihara, M
20 α -羟基类固醇脱氢酶（20 α - hsd）在小鼠和大鼠的黄体中将黄体酮代谢为非活性类固醇，但在人类中没有，被认为在缩短这些啮齿动物的发情周期中起着至关重要的作用。我们确定了小鼠20a-HSD基因5'侧区域的核苷酸序列，并使用大鼠黄体化颗粒细胞培养检测了其启动子活性。利用不同长度的5'侧翼区域的报告基因结构进行的报告基因分析显示，转录起始位点上游- 83至60 bp之间的区域对转录活性至关重要。此外，突变分析表明，该区域的Sp1位点对报告基因的表达至关重要。电泳迁移位移试验表明，大鼠黄体化颗粒细胞核提取物中蛋白质与该区域的相互作用被启动子中具有野生型Sp1序列的竞争对手抑制，而Sp1序列不具有突变。Supershift分析证实Sp1和Sp3存在于这些细胞的核提取物中，并且这些因子与元素结合。最后，启动子活性通过Sp1表达载体和Sp3表达载体的共同转染而提高，在较小程度上，进一步支持这些因子参与20 α - hsd基因的表达。
20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD), which metabolizes progesterone to an inactive steroid in the corpus luteum of mice and rats but not of humans, is thought to play a crucial role in shortening the oestrous cycles in these rodent species. We determined the nucleotide sequence of the 5'-flanking region of the mouse 20a-HSD gene, and examined its promoter activity using a rat luteinized granulosa cell culture. A reporter assay, using reporter constructs of various lengths of the 5'-flanking region, revealed that the region between - 83 and 60 bp upstream of the transcription start site was essential for transcriptional activity. Furthermore, mutational analysis demonstrated that a putative Sp1 site in this region was critical to the expression of the reporter gene. Electrophoretic mobility-shift assays showed that the interaction of proteins in a nuclear extract from rat luteinized granulosa cells with this region was inhibited by a competitor having the wildtype Sp1 sequence in its promoter, but not a mutated Sp1 sequence. Supershift analysis confirmed that Sp1 and Sp3 were present in the nuclear extract of these cells, and that these factors bound to the element. Finally, promoter activity was elevated by the co-transfection of an Sp1 expression vector, and, to a lesser extent, by an Sp3 expression vector, supporting further the involvement of these factors in the expression of the 20alpha-HSD gene.