Yu R N, Ito M, Jameson J L
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Mol Endocrinol. 1998 Jul;12(7):1010-22. doi: 10.1210/mend.12.7.0131.
The Dax-1 gene encodes a protein that is structurally related to members of the orphan nuclear receptor superfamily. Dax-1 is coexpressed with another orphan nuclear receptor, steroidogenic factor-1 (SF-1), in the adrenal, gonads, hypothalamus, and pituitary gland. Mutations in Dax-1 cause adrenal hypoplasia congenita, a disorder that is characterized by adrenal insufficiency and hypogonadotropic hypogonadism. These developmental and endocrine abnormalities are similar to those caused by disruption of the murine Ftz-F1 gene (which encodes SF-1), suggesting that these nuclear receptors act along the same developmental cascade. Cloning of the murine Dax-1 gene revealed a candidate SF-1-binding site in the Dax-1 promoter. In transient expression assays in SF-1-deficient JEG-3 cells, SF-1 stimulated expression of the Dax-1 promoter. However, deletion or mutation of the consensus SF-1-binding site did not eliminate SF-1 stimulation. Further analyses revealed the presence of a cryptic SF-1 site that creates an imperfect direct repeat of the SF-1 element. When linked to the minimal thymidine kinase promoter, each of the isolated SF-1 sites was sufficient to mediate transcriptional regulation by SF-1. Mutation of both SF-1 sites eliminated SF-1 binding and stimulation of the Dax-1 promoter. Unexpectedly, mutation of either half of the composite SF-1 sites increased basal activity in JEG-3 cells, suggesting interaction of a repressor protein. Gel shift analyses of the composite response element revealed an additional complex that was not supershifted by SF-1 antibodies. This complex was eliminated by mutation of either half-site, and it was supershifted by antibodies against chicken ovalbumin upstream promoter-transcription factor (COUP-TF). We propose that Dax-1 is stimulated by SF-1, and that SF-1 and COUP-TF provide antagonistic pathways that converge upon a common regulatory site.
Dax-1基因编码一种蛋白质，其结构与孤儿核受体超家族的成员相关。Dax-1与另一种孤儿核受体——类固醇生成因子-1（SF-1）在肾上腺、性腺、下丘脑和垂体中共同表达。Dax-1的突变会导致先天性肾上腺发育不全，该疾病的特征是肾上腺功能不全和促性腺激素缺乏性性腺功能减退。这些发育和内分泌异常与小鼠Ftz-F1基因（编码SF-1）破坏所导致的异常相似，这表明这些核受体在同一发育级联中起作用。小鼠Dax-1基因的克隆揭示了Dax-1启动子中的一个候选SF-1结合位点。在SF-1缺陷的JEG-3细胞的瞬时表达试验中，SF-1刺激了Dax-1启动子的表达。然而，共有SF-1结合位点的缺失或突变并未消除SF-1的刺激作用。进一步分析发现存在一个隐蔽的SF-1位点，它形成了SF-1元件的不完美直接重复序列。当与最小胸苷激酶启动子相连时，每个分离出的SF-1位点都足以介导SF-1的转录调控。两个SF-1位点的突变消除了SF-1与Dax-1启动子的结合及刺激作用。出乎意料的是，复合SF-1位点任一半的突变都会增加JEG-3细胞中的基础活性，提示存在一种阻遏蛋白的相互作用。对复合反应元件的凝胶迁移分析揭示了一种额外的复合物，该复合物不会被SF-1抗体超迁移。该复合物可通过任一半位点的突变而消除，并可被抗鸡卵清蛋白上游启动子转录因子（COUP-TF）的抗体超迁移。我们提出Dax-1受SF-1刺激，并且SF-1和COUP-TF提供了在一个共同调控位点汇聚的拮抗途径。
