Functional analysis of the Zn finger domain encoded by the ATRX gene whose mutations result in X-linked alpha thalassemia mental retardation(ATR-X) syndrome

X连锁阿尔法地中海贫血(ATR-X)综合征是由ATRX基因突变引起的。突变热点之一是其氨基末端与未知功能的锌指结构域(锌指结构域)对应的区域(锌F1、锌氟2和锌氟3)。为了揭示这种遗传性疾病的原因，我们探索了该结构域作为泛素连接酶功能的可能性，以及通过该结构域对色素相关因子的泛素化调节基因表达的可能性。(1)我们发现锌指结构域在体内和体外都是泛素化的。(2)泛素连接酶的活性不仅需要与称为泛素连接酶的环/指同源的ZnF2-ZnF3，而且还需要其N-末端的ZnF1。(3)ATR-X综合征中发现的锌指结构域突变降低了连接酶活性。这些结果表明，ATRX的锌指结构域构成了一个新的泛素连接酶家族，连接酶活性降低导致ATR-X综合征的发生。对野生型锌F(1，2，3)及其突变体的远紫外镉测量表明，单是锌F1结构的破坏就会导致锌指结构域整体结构的扭曲，这可能导致其酶活性的丧失。我们鉴定了三个编码含有ATRX样锌指结构域的蛋白质的基因(ATRXLα、ATRXβ和ATRXLγ)，并从这些蛋白质中分离出具有泛素连接酶活性的ATRX样锌指结构域。对果蝇ATRX基因进行了鉴定。有趣的是，果蝇ATRX是由两个基因编码的，即dATRXN和dATRXC，它们分别含有锌指域和解旋酶结构域。然而，在分离的dATRXN的锌指结构域中没有检测到泛素连接酶活性。已知与ATRX相互作用的HP1和HDAC1不被ATRX泛素化。

X-linked alpha thalassemia mental retardation (ATR-X) syndrome is caused by mutations in the ATRX gene. One of mutational hot spots is the region corresponding to the Zn finger domain of unknown function (ZnF1, ZnF2, and ZnF3) in its amino terminus. To reveal the cause of this genetic disorder, we explored the possibility that this domain functions as a ubiquitin ligase and that ubiquitinylation of chromatine related factors by this domain regulates gene expression.1. (1) We have shown the Zn finger domain is ubiquitinylated both in vivo and in vitro. (2) Ubiquitin ligase activity requires not only ZnF2-ZnF3 homologous to RING/PHD fingers known as ubiquitin ligases but also ZnF1 in their N-terminus. (3) Mutations of the Zn finger domain found in the ATR-X syndrome diminish the ligase activity. These results suggest that the Zn finger domain of ATRX constitutes a new family of ubiquitin ligase and that reduction of ligase activity gives rise to the ATR-X syndrome.2. Far UV CD measurements of wild type ZnF (1, 2, 3) and its mutant in ZnF1 have indicated disruption of the ZnF1 structure alone results in distortion of the overall structure of the Zn finger domain, which might lead to loss of its enzymatic activity.3. Three human genes (ATRXLα, ATRXβ, and ATRXLγ) encoding proteins with ATRX-like Zn finger domains were identified and isolated ATRX-like Zn finger domains from these proteins were also shown to have ubiquitin ligase activity in vitro.4. Drosophila ATRX genes were identified. Interestingly, Drosophila ATRX is encoded by two genes ; namely, dATRXN and dATRXC which contain the Zn finger domain and the helicase domain, respectively. The ubiquitin ligase activity was not, however, detected in the isolated Zn finger domain of dATRXN. HP1 and HDAC1, known to interact with ATRX, were not ubiquitinylated by ATRX.