Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues.
Steroidogenic factor 1, an orphan nuclear receptor, regulates the expression of the rat aromatase gene in gonadal tissues.
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DOI：
10.1210/mend.7.6.8395654
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发表时间：
1993-06
影响因子：
--
通讯作者：
J. Lynch;D. Lala;J. Peluso;W. Luo;K. Parker;B. White
中科院分区：
文献类型：
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作者：
J. Lynch;D. Lala;J. Peluso;W. Luo;K. Parker;B. White
在一个协调一致的分析的基因编码的三个小鼠类固醇羟化酶，我们确定和表征的转录调控蛋白，命名为类固醇生成因子1（SF-1），这有助于在肾上腺皮质细胞中的协调表达。SF-1是核受体家族的孤儿成员，与类固醇羟化酶上游的PyCAAGGPyCPu基序结合以调节其表达。在本研究中，我们通过研究SF-1在调节大鼠性腺组织中P450芳香化酶基因中的作用来扩展这些发现。通过基于聚合酶链反应的方法，使用与已发表的芳香酶序列的5 '和3'末端相对应的引物，分离大鼠芳香酶基因的5 '侧翼区。DNA序列分析显示，我们的序列和以前发表的序列之间有三个差异，包括一个44碱基对（bp）的插入。此外，转录起始位点，引物延伸分析确定，不同于以前提出的。新的转录起始位点位于TATA盒3'端23 bp处。当通过BEST-FIT分析将修订的大鼠序列与人芳香化酶PII启动子的序列进行比较时，鉴定出在两个启动子之间80%保守的约300 bp的区域。一个潜在的SF-1位点，CCAAGGTCA，被确定在该区域内的位置-82。在凝胶迁移率变动测定中使用含有该推定SF-1位点的寡核苷酸探针。与以前的研究一致，一个特定的复杂的观察与性腺类固醇生成组织的核提取物，但不存在与非类固醇生成组织的核提取物。SF-1在这种类固醇生成细胞特异性复合物中的作用接下来更直接地解决。含有SF-1-谷胱甘肽S-转移酶融合蛋白的细菌提取物与假定的SF-1位点特异性相互作用，并且针对SF-1-谷胱甘肽S-转移酶的多克隆抗血清特异性地废除了与大鼠卵巢或R2 C大鼠Leydig肿瘤细胞的核提取物形成的复合物。最后，芳香化酶SF-1元件在瞬时转染实验中以类固醇生成细胞选择性方式增加了SV 40启动子/荧光素酶构建体的表达。总的来说，这些研究暗示SF-1在调节类固醇羟化酶基因的表达在非肾上腺组织，显着扩展以前的研究在肾上腺皮质细胞。
In a concerted analysis of the genes encoding three mouse steroid hydroxylases, we identified and characterized a transcriptional regulatory protein, designated steroidogenic factor 1 (SF-1), that contributes to the coordinate expression in adrenocortical cells. SF-1, an orphan member of the nuclear receptor family, binds to PyCAAGGPyCPu motifs upstream of the steroid hydroxylases to regulate their expression. In the present study, we extend these findings by examining the role of SF-1 in regulation of the rat P450 aromatase gene in gonadal tissues. The 5'-flanking region of the rat aromatase gene was isolated by a polymerase chain reaction-based approach, using primers corresponding to the 5'- and 3'-ends of a published aromatase sequence. DNA sequence analysis revealed three differences between our sequence and the previously published sequence, including a 44-base pair (bp) insertion. Moreover, the transcription initiation site, as determined by primer extension analysis, differed from that previously proposed. The new transcription initiation site is located 23 bp 3' of a putative TATA box. When a revised rat sequence was compared to that of the human aromatase PII promoter by BEST-FIT analysis, a region of about 300 bp was identified that was 80% conserved between the two promoters. A potential SF-1 site, CCAAGGTCA, was identified at position -82 within this region. An oligonucleotide probe containing this putative SF-1 site was used in gel mobility shift assays. Consistent with previous studies, a specific complex was observed with nuclear extracts from gonadal steroidogenic tissues but was absent with nuclear extracts from nonsteroidogenic tissues. The role of SF-1 in this steroidogenic cell-specific complex was next addressed more directly. Bacterial extracts containing an SF-1-glutathione S-transferase fusion protein interacted specifically with the putative SF-1 site, and polyclonal antisera against SF-1-glutathione S-transferase specifically abolished the complex formed with nuclear extracts from rat ovaries or R2C rat Leydig tumor cells. Finally, the aromatase SF-1 element increased expression of an SV40 promoter/luciferase construct in transient transfection experiments in a steroidogenic cell-selective manner. Collectively, these studies implicate SF-1 in the regulation of steroid hydroxylase gene expression in nonadrenal tissues, significantly extending previous studies in adrenocortical cells.