STRUCTURE-FUNCTION ANALYSIS OF DAX-1 USING Molecular analysis

DAX-1提供了已知的哺乳动物转录抑制因子的罕见例子之一，其功能丧失与先天性疾病肾上腺发育不全(AHC)有关。然而，关于DAX-1的结构-功能或其在分子水平上的功能的信息是有限的。因此，我们首先对来自16个家系的20例疑似X连锁AHC患者进行了DAX-1基因突变分析。我们鉴定了三个移码突变(151 delAG，935delC，1376delTinsG)，三个无义突变(Y91X。Y271X、Q395X)，5个错义突变(V269D、L278R。W291C、L466R)和两个缺失突变。我们发现，这些错义突变降低了它们沉默STAR启动子活性的能力，这表明它们没有功能。所有已报道的错义突变仅位于C末端假定的配体结合区。因此，推测DAX-1蛋白的C-末端可能比N-末端的…更重要更多的L一半用于DAX-1的功能。我们构建的配体结合域的三维结构也支持这些发现。临床分析表明，发病年龄和突变类型之间没有相关性。此外，我们确定了5名青少年和成人患者患有低促性腺激素低促性腺功能低下症。尽管如此，它们保留了正常的间质细胞功能，但它们损害了支持细胞的功能。这表明DAX-1在促性腺激素分泌和精子发生中起一定作用。我们可以证明野生型DAX-1抑制促黄体生成素β启动子的活性，而突变型DAX-1表现出促性腺激素分泌功能的丧失。本项目还提示DAX-1抑制人STAR基因启动子的功能，部分通过与顺式元件结合SF-1发挥作用，并与调节DAX-1作用的辅活化子RIP140结合。然而，这些分析表明，AHC患者广泛的表型变异的全貌可能不能仅仅用DAX-1本身的抑制功能丧失来解释。较少

The DAX-1 provides one of the rare known examples of mammalian transcriptional repressors whose the loss of function is associated with a congenital disorder, Adrenal hypoplasia congenita (AHC). However, the information about the structure-function of the DAX-1 or its function at the molecular level is so limited. Therefore, we performed first in this project the mutation analysis of the DAX-1 gene in the 20 suspected patients with X-linked AHC from 16 families. We identified three frameshift mutation (151 delAG, 935delC, 1376delTinsG), three nonsense mutations (Y91X. Y271X, Q395X), five missense mutations (V269D, L278R. W291C, L466R) and two deletion mutations. We showed that these missense mutations reduced their ability to silence StAR promotor activity, suggesting nonfunctional. All missense mutations reported are located only in the C-terminal presumptive ligand-binding domain. Thus, it was suggested that the C-terminal half of the DAX-1 protein may be important than the N-termina … More l half for DAX-1 function. The 3-D structure of the ligand binding domain we constructed also supported these findings. Clinical analysis showed that there was no correlation between the age at presentation and the type of the mutation. Furthermore, we identified five adolescent and adult patients with hypogonadotropic hypogonadism. Nevertheless, they retained normal Leydig cell functions, but they had impaired Sertoli cell function. This suggests that DAX-1 plays some role in gonadotropin secretion and also spermatogenesis. We could demonstrate that the wild type DAX-1 suppresses the LH β promoter activity, but the mutant DAX-1 displayed the loss of function for gonadotropin secretion.We also analyzed the mechanism of action of DAX-1. This project suggested also DAX-1 suppress the human StAR gene promoter function, that it can act in part through cis element binding SF-1, and associates with the coactivaor RIP140, which modulates DAX-1 action. However, these analysis indicated that the full picture of the wide phenotypic variability in the patients with AHC may not be explained solely by the loss of the repressor function of the DAX-1 itself. Less