Zhang P, Compagnone N A, Fiore C, Vigne J L, Culp P, Musci T J, Mellon S H
Department of Obstetrics & Gynecology & Reproductive Sciences, Center for Reproductive Sciences, and The Metabolic Research Unit, University of California, San Francisco 94143-0556, USA.
DNA Cell Biol. 2001 Oct;20(10):613-24. doi: 10.1089/104454901753340604.
Cytochrome P450c17 catalyzes the 17alpha-hydroxylase/17,20 lyase activity needed for sex steroid synthesis. We recently characterized the nuclear phosphoprotein SET as a novel transcriptional regulator that binds to the -447/-399 region of the rat P450c17 gene, along with the transcription factors COUP-TF II, NGF-IB, and SF-1. Gel shift studies localized SET binding to nucleotides -410/-402. We have shown that SET activates transcription of the rat P450c17 gene in neuronal precursor cells and now show that it also activates transcription from the -418/-399 region of the rat P450c17 gene in mouse Leydig MA-10 cells. Studying the ontogenic expression of SET and P450c17 in the rodent gonad, we found that SET expression preceded P450c17 expression in the embryonic genital ridge, suggesting that SET may be important for initiating P450c17 expression in this region. Expression of SET also preceded P450c17 expression in the testis and ovary, and its expression was much greater during embryogenesis than in the adult gonad. In the adult rat testis, P450c17 was expressed only in Leydig cells, while SET was expressed in Leydig cells and in spermatocytes. In the adult rat ovary, P450c17 was expressed only in theca cells, while SET was expressed in theca cells and also in oocytes. Because SET is expressed early in development in the genital ridge and in the testis and ovary, and because SET has many functions in addition to its activity as a transcription factor, we determined whether SET acts a transcription factor in oocytes. The SET protein was detected by Western blots in Xenopus oocytes from stages II through VI and in mature oocytes. Using extracts of Xenopus oocytes in gel shift assays, we detected a protein that bound to the -418/-399 region of the rat P450c17 gene, to which SET binds. Nuclear injection of either a -418/-399TK32LUC wildtype reporter construct or a construct containing a mutant SET site into Xenopus oocytes from stages III through VI resulted in activation of luciferase activity with the wildtype but not the mutant construct in all stages. These data suggest that Xenopus SET is able to bind to specific DNA sequences to activate transcription at all stages of Xenopus oogenesis. These data indicate that SET is an evolutionarily conserved transcription factor that participates in the early ontogenesis of the gonadal system, regulates P450c17 gene transcription in Leydig cells, and may also activate other genes expressed in immature oocytes, thus playing a role in oocyte development.
细胞色素P450c17催化性类固醇合成所需的17α-羟化酶/17,20裂解酶活性。我们最近将核磷蛋白SET鉴定为一种新型转录调节因子，它与转录因子COUP-TF II、NGF-IB和SF-1一起结合到大鼠P450c17基因的-447 / -399区域。凝胶迁移研究将SET结合定位到核苷酸-410 / -402。我们已经表明SET在神经元前体细胞中激活大鼠P450c17基因的转录，现在表明它也在小鼠睾丸间质MA-10细胞中激活大鼠P450c17基因-418 / -399区域的转录。研究SET和P450c17在啮齿动物性腺中的个体发生表达，我们发现在胚胎生殖嵴中SET表达先于P450c17表达，这表明SET可能对于在该区域启动P450c17表达很重要。SET的表达在睾丸和卵巢中也先于P450c17表达，并且其在胚胎发生期间的表达比在成年性腺中高得多。在成年大鼠睾丸中，P450c17仅在睾丸间质细胞中表达，而SET在睾丸间质细胞和精母细胞中表达。在成年大鼠卵巢中，P450c17仅在卵泡膜细胞中表达，而SET在卵泡膜细胞以及卵母细胞中表达。因为SET在生殖嵴以及睾丸和卵巢的发育早期表达，并且因为SET除了作为转录因子的活性外还有许多功能，我们确定SET在卵母细胞中是否作为转录因子起作用。通过蛋白质免疫印迹在II至VI期的非洲爪蟾卵母细胞和成熟卵母细胞中检测到SET蛋白。使用非洲爪蟾卵母细胞提取物进行凝胶迁移分析，我们检测到一种与大鼠P450c17基因的-418 / -399区域结合的蛋白质，SET也结合到该区域。将-418 / -399TK32LUC野生型报告基因构建体或包含突变SET位点的构建体核注射到III至VI期的非洲爪蟾卵母细胞中，导致野生型构建体而非突变构建体在所有阶段均激活荧光素酶活性。这些数据表明非洲爪蟾SET能够结合特定的DNA序列以在非洲爪蟾卵子发生的所有阶段激活转录。这些数据表明SET是一种进化保守的转录因子，它参与性腺系统的早期个体发生，调节睾丸间质细胞中P450c17基因的转录，并且还可能激活未成熟卵母细胞中表达的其他基因，从而在卵母细胞发育中发挥作用。
