Zhu Lirong, Zhou Guisheng, Poole Suzanne, Belmont John W
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
Hum Mutat. 2008 Jan;29(1):99-105. doi: 10.1002/humu.20606.
ZIC3, a GLI superfamily transcription factor, is involved in establishing normal embryonic left-right patterning. Multiple abnormalities in the central nervous system (CNS) and axial skeleton have also been observed in mice bearing a Zic3 null allele, mice with a Zic3 overexpression allele, and the majority of patients carrying ZIC3 mutations. Previous studies indicate that ZIC3 protein can bind to the GLI consensus binding site (GLIBS) and physically interact with GLI3, a transcription factor involved in multiple aspects of neural and skeletal development. We investigated in vitro interactions of ZIC3 with GLI3 and the effect of ZIC3 mutations identified in patients with either heterotaxy or isolated cardiovascular malformations. Electrophoresis mobility shift assay (EMSA) revealed that all five intact zinc finger (ZF) domains were necessary for binding of ZIC3 to GLIBS. Inclusion of GLIBS upstream of a basal TK promoter had no effect on the activation of the promoter by ZIC3 alone, but it enhanced the synergistic activation of ZIC3 and GLI3. Wild-type (WT) ZIC3 showed specific binding to GLI3 in GST-pull-down assays. Nonsense and frameshift ZIC3 mutants lacking one or more of the zinc finger domains did not physically interact with GST-GLI3; however, two missense mutants c.1213A>G (p.K405E, fifth ZF domain), and c.649C>G (p.P217A, conserved N-terminal domain) retained binding. Luciferase reporter assays indicated that both p.P217A and p.K405E mutants also retained coactivation with GLI3 of reporter gene expression activity, while all the GLI3-nonbinding ZIC3 mutants lacked this activity. Interestingly, no CNS or skeletal abnormalities were observed in patients bearing the p.P217A or p.K405E mutations.
ZIC3是一种GLI超家族转录因子，参与建立正常的胚胎左右模式。在携带Zic3无效等位基因的小鼠、具有Zic3过表达等位基因的小鼠以及大多数携带ZIC3突变的患者中，也观察到中枢神经系统（CNS）和轴向骨骼的多种异常。先前的研究表明，ZIC3蛋白可以结合GLI共有结合位点（GLIBS），并与GLI3发生物理相互作用，GLI3是一种参与神经和骨骼发育多个方面的转录因子。我们研究了ZIC3与GLI3的体外相互作用，以及在患有内脏反位或孤立性心血管畸形的患者中鉴定出的ZIC3突变的影响。电泳迁移率变动分析（EMSA）显示，所有五个完整的锌指（ZF）结构域对于ZIC3与GLIBS的结合都是必需的。在基础TK启动子上游包含GLIBS对单独的ZIC3激活启动子没有影响，但它增强了ZIC3和GLI3的协同激活。在谷胱甘肽S-转移酶（GST）下拉实验中，野生型（WT）ZIC3显示出与GLI3的特异性结合。缺少一个或多个锌指结构域的无义突变和移码ZIC3突变体不与GST-GLI3发生物理相互作用；然而，两个错义突变体c.1213A>G（p.K405E，第五个ZF结构域）和c.649C>G（p.P217A，保守的N端结构域）保留了结合能力。荧光素酶报告基因实验表明p.P217A和p.K405E突变体也保留了与GLI3共同激活报告基因表达活性的能力，而所有不与GLI3结合的ZIC3突变体都缺乏这种活性。有趣的是，携带p.P217A或p.K405E突变的患者未观察到CNS或骨骼异常。
