DNA sequence-dependent regulation of SF-1-mediated transcription.
DNA sequence-dependent regulation of SF-1-mediated transcription.
复制标题
SF-1 介导的转录的 DNA 序列依赖性调节。
DOI：
10.1089/dna.2005.24.148
复制
发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Mellon,SynthiaH
中科院分区：
文献类型：
--
作者：
Brake,PaulB;Bair,SusannaR;Mellon,SynthiaH
大鼠P450 c17基因转录受多种核因子调控，包括类固醇生成因子-1（SF-1）、神经生长因子诱导蛋白B（NGF-IB，Nurr 77）、COUP-TF、SET和Ku自身免疫抗原。该基因的一个区域，-447/-419，介导基础和cAMP刺激的转录，含有两个孤儿核受体的结合位点。虽然SF-1通过单个结合位点激活转录，但我们表明，SF-1和cAMP的转录激活需要-447/-419处的两个结合位点。SF-1和一种新的转录因子类固醇生成因子诱导因子2（StF-IT-2）都与该区域结合，这表明StF-IT-2和SF-1之间的DNA依赖性相互作用可能是完全转录活性所必需的。两个孤儿核受体位点-429/-424和-444/-439中的每一个都足以与SF-1结合，但不足以与SF-1介导的转录结合。增加这两个位点之间的距离或改变这两个位点的方向不会影响基础或SF-1刺激的活性。测量蛋白质结合引起的DNA弯曲程度的圆形排列分析表明，SF-1与-447/-419结合诱导的DNA弯曲程度与在另一个SF-1反应位点诱导的不同。然而，SF-1蛋白的类似结构域是其在这两个区域发挥作用所必需的。Southwestern印迹表明StF-IT-2是一种分子量约为33 kDa的蛋白质，凝胶迁移分析表明它主要在啮齿动物发育早期的性腺和大脑中表达。这些数据表明，SF-1刺激转录的机制是DNA序列依赖性的，可能需要额外的蛋白质，如StF-IT-2，在DNA的特定区域激活。
Rat P450c17 gene transcription is regulated by several nuclear factors, including steroidogenic factor-1 (SF-1), nerve growth factor-inducible protein B (NGF-IB, Nurr77), COUP-TF, SET, and Ku autoimmune antigen. A region of this gene, –447/–419, that mediates both basal and cAMP-stimulated transcription, contains two binding sites for orphan nuclear receptors. While SF-1 activates transcription through a single binding site, we show that both binding sites at –447/–419 are required for transcriptional activation by SF-1 and cAMP. Both SF-1 and a novel factor, Steroidogenic Factor-Inducer of Transcription-2 (StF-IT-2) bind to this region, suggesting that a DNA-dependent interaction between StF-IT-2 and SF-1 may be required for full transcriptional activity. Each of the two orphan nuclear receptor sites –429/–424 and at –444/–439 are sufficient for SF-1 binding but are insufficient for SF-1–mediated transcription. Increasing the distance between or changing the orientation of these two sites does not affect basal or SF-1–stimulated activity. Circular permutation analysis, which measures the degree of DNA bending caused by protein binding, indicates that SF-1 binding to –447/–419 induces a different degree of DNA bending than it does at another SF-1–responsive site. However, similar domains of the SF-1 protein are required for its actions at these two regions. Southwestern blots suggest that StF-IT-2 is a ∼33 kDa protein, and gel shift assays suggest it is expressed primarily in the gonad and brain early in rodent development. These data suggest that the mechanism by which SF-1 stimulates transcription is DNA sequence dependent, and may require additional proteins, such as StF-IT-2, for activation at specific regions of DNA.