Molecular mechanism of sex determination and its disease

在人类中，与其他哺乳动物一样，性别决定是由一个显性开关控制的，该开关被称为睾丸决定因子（Testis Determining Factor）。SRY基因被认为是TDF，其编码具有一个HMG盒作为DNA结合结构域的转录因子。在人类15%的XY性反转病例中，已经确定了SRY HMG盒中的突变。将小鼠SRY基因（Sry）导入XX雌性小鼠，诱导睾丸分化和随后的雄性发育。然而，关于SRY的转录调控机制还知之甚少。WT 1基因突变在伴有泌尿生殖系统畸形的Denys-Drash综合征（DDS）患者中较为常见，我们在分析WT 1基因相关蛋白时发现，WT 1基因与Sox 30结合，后者编码一种新的转录因子，具有一个HMG盒。进一步分析表明WT 1与其HMG盒结合。这一观察结果促使我们分析WT 1和SRY之间的相互作用，这两者都在早期性腺的体细胞中表达。结果表明：① WT 1在体外和培养细胞中与SRY结合; ② WT 1和SRY协同激活SRY结合序列启动子和SRY启动子的转录; ③在DDS中发现的WT 1突变体没有这种活性; ④ WT 1以SRY依赖的方式被募集到SRY结合序列上，而DDS突变体的募集显著减少。最近，我们建立了同时表达WT 1和SRY的细胞系。我们目前正在分析该细胞系的表达谱，这将揭示受WT 1和SRY调控的靶基因。

In humans, as in other mammals, sex determination is controlled by a dominant switch termed TDF for Testis Determining Factor. The SRY gene is thought to be the TDF, which encodes a transcription factor with one HMG box as a DNA binding domain. Mutations in the SRY HMG box have been identified in 15% cases of XY sex reversal in humans. Introduction of mouse SRY gene (Sry) into XX female mice induced testis differentiation and subsequent male development. However, little is known about mechanism of transcriptional regulation by SRY. WT1 mutations have frequently been observed in Denys-Drash syndrome (DDS) patients with urogenital malformation.During analysis of WT1-associated proteins, we found that WT1 bound to Sox30, which encodes a novel transcription factor with one HMG box. Further analysis showed that WT1 bound to its HMG box. This observation prompted us to analyze interaction between WT1 and SRY, both of which are expressed in the somatic cells of early gonads. We showed the following results:i) WT1 binds to SRY in vitro and in cultured cells.ii) WT1 and SRY synergistically activate transcription from a promoter that contains SRY binding sequence and also from the SRY promoter.iii) WT1 mutants found in DDS, however, did not show this activity.iv) WT1 is recruited on a SRY-binding sequence in a SRY-dependent manner, while recruitment of DDS mutants is significantly reduced.These observations suggest that WT1 and SRY interaction plays an important role in early gonadal development and its disease. Recently we have established cell lines which express both WT1 and SRY. We are currently analyzing the expression profile of this cell line, which will reveal target genes regulated by WT1 and SRY.