Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.
Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.
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DOI：
10.1016/s0021-9258(19)50326-x
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发表时间：
1993-06
期刊：
影响因子：
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通讯作者：
J. Sirois;L. Daniel;Simmons;Joanne;Richards
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文献类型：
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作者：
J. Sirois;L. Daniel;Simmons;Joanne;Richards
促性腺激素在排卵前卵泡颗粒细胞中瞬时诱导前列腺素内过氧化物合酶亚型2（PGS-2）mRNA和蛋白表达。为了更好地了解大鼠PGS-2（rPGS-2）基因在这些细胞中的激素调节，通过用标记的小鼠PGS-2 cDNA的5 '片段筛选大鼠肝基因组文库来分离含有rPGS-2以及多达6个5'侧翼DNA的内切酶的基因组克隆。使用卵巢卵泡mRNA的引物延伸分析鉴定了位于ATG翻译起始密码子上游144个核苷酸的单个rPGS-2转录起始位点的存在。为了检测rPGS-2基因5 ′侧翼区域内的启动子活性，将基因组片段-2698/32（1 =帽位点）以及一系列5 ′缺失突变体融合到氯霉素乙酰转移酶（CAT）报告基因的上游，并转染到颗粒细胞的原代培养物中。用-2698/32PGS.CAT转染后，毛喉素（7.5 μ M）、促卵泡激素（500 ng/ml）和促黄体激素（500 ng/ml）诱导CAT活性，而促性腺激素释放激素（10（-6）M）和白细胞介素-1 β（30 ng/ml）则无影响。缺失突变体描绘了从转录起始位点的-192到-54的区域，该区域对于报告基因的基础表达和毛喉素调节的表达都是必需的。电泳迁移率变动分析表明，相同的DNA片段（-192/-54）与颗粒细胞核提取物蛋白质具有特异性结合。在暴露于促性腺激素排卵剂量的颗粒细胞制备的提取物中观察到额外的特异性条带。总的来说，这些结果提供了第一个结构和功能证据，即颗粒细胞中促性腺激素和毛喉素对大鼠PGS-2基因的转录调控涉及5 '侧翼DNA序列，特别是转录起始位点的-192和-54之间的区域。
Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.