Ito M, Yu R N, Jameson J L
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Mol Endocrinol. 1998 Feb;12(2):290-301. doi: 10.1210/mend.12.2.0059.
The orphan nuclear receptor, steroidogenic factor-1 (SF-1), plays an important role in the development of the adrenal gland and in sexual differentiation. SF-1 regulates the transcription of variety of genes, including several steroidogenic enzymes, Müllerian inhibiting substance, and gonadotropin genes. In this report, we sought to identify domains in SF-1 that are required for transactivation and to determine whether SF-1 interacts with a subset of known coactivators. Natural variants of the FTZ-F1 locus include embryonal long terminal repeat-binding protein (ELP)-1, ELP-2, and SF-1, which share the DNA-binding domain. Analyses of the transcriptional activity of these variants revealed that the activity of ELP-2 and SF-1 was much greater than ELP-1, which contains a distinct carboxy terminus. Further studies were performed using GAL4-SF-1 fusion proteins that were constructed by replacement of the zinc finger region and FTZ-F1 box of SF-1 with the DNA-binding domain of GAL4. Elimination of the putative AF-2 domain at the carboxy terminus of GAL4-SF-1 proteins resulted in a complete loss of transactivation. Several lines of evidence demonstrated that SF-1 interacts with steroid receptor coactivator-1 (SRC-1). Full-length SRC-1 enhanced GAL4-SF-1-mediated transactivation, whereas a dominant negative form of SRC-1, consisting of its interaction domain alone, inhibited the activity of GAL4-SF-1. In mammalian two-hybrid assays, fusion of the VP16 activation domain to the interaction domain of SRC-1 confirmed the interaction between SRC-1 and GAL4-SF-1 and demonstrated that the AF-2 domain is required for interaction with SRC-1. Furthermore, SRC-1, together with the cAMP responsive element binding protein (CBP) or a closely related factor, p300, synergistically enhanced transcriptional activity of GAL4-SF-1. We conclude that the carboxy-terminal AF-2 region of SF-1 functions as an activation domain and that SRC-1 and CBP/p300 are components of the coactivator complex with SF-1.
孤儿核受体类固醇生成因子-1（SF-1）在肾上腺发育和性别分化中起重要作用。SF-1调节多种基因的转录，包括几种类固醇生成酶、苗勒管抑制物质和促性腺激素基因。在本报告中，我们试图确定SF-1中反式激活所需的结构域，并确定SF-1是否与已知共激活因子的一个子集相互作用。FTZ-F1基因座的天然变体包括胚胎长末端重复序列结合蛋白（ELP）-1、ELP-2和SF-1，它们共享DNA结合结构域。对这些变体转录活性的分析表明，ELP-2和SF-1的活性远高于含有独特羧基末端的ELP-1。使用GAL4-SF-1融合蛋白进行了进一步研究，该融合蛋白是通过用GAL4的DNA结合结构域替换SF-1的锌指区域和FTZ-F1框构建的。去除GAL4-SF-1蛋白羧基末端的假定AF-2结构域导致反式激活完全丧失。几条证据表明SF-1与类固醇受体共激活因子-1（SRC-1）相互作用。全长SRC-1增强了GAL4-SF-1介导的反式激活，而仅由其相互作用结构域组成的显性负性形式的SRC-1抑制了GAL4-SF-1的活性。在哺乳动物双杂交试验中，将VP16激活结构域与SRC-1的相互作用结构域融合证实了SRC-1与GAL4-SF-1之间的相互作用，并表明AF-2结构域是与SRC-1相互作用所必需的。此外，SRC-1与环磷酸腺苷反应元件结合蛋白（CBP）或密切相关因子p300协同增强了GAL4-SF-1的转录活性。我们得出结论，SF-1的羧基末端AF-2区域作为激活结构域发挥作用，并且SRC-1和CBP/p300是与SF-1共激活复合物的组成部分。
