Wu L, Wu H, Ma L, Sangiorgi F, Wu N, Bell J R, Lyons G E, Maxson R
Department of Biochemistry and Molecular Biology, USC Norris Cancer Hospital and Research Institute, University of Southern California School of Medicine, Los Angeles 90033, USA.
Mech Dev. 1997 Jul;65(1-2):3-17. doi: 10.1016/s0925-4773(97)00032-4.
Msx2 is a homeobox gene with a regulatory role in inductive tissue interactions, including those that pattern the skull. We demonstrated previously that individuals affected with an autosomal dominant disorder of skull morphogenesis (craniosynostosis, Boston type) bear a mutated form of Msx2 in which a histidine is substituted for a highly conserved proline in position 7 of the N-terminal arm of the homeodomain (p148h). The mutation behaves as a dominant positive in transgenic mice. The location of the mutation in the N-terminal arm of the homeodomain, a region which in other homeodomain proteins plays a key part in protein-protein interactions, prompted us to undertake a yeast two hybrid screen for Msx2-interacting proteins. Here we present a functional analysis of one such protein, designated Miz1 (Msx-interacting-zinc finger). Miz1 is a zinc finger-containing protein whose amino acid sequence closely resembles that of the yeast protein, Nfi-1. Together these proteins define a new, highly conserved protein family. Analysis of Miz1 expression by Northern blot and in situ hybridization revealed a spatiotemporal pattern that overlaps that of Msx2. Further, Miz1 is a sequence specific DNA binding protein, and it can function as a positive-acting transcription factor. Miz1 interacts directly with Msx2 in vitro and enhances the DNA binding affinity of Msx2 for a functionally important element in the rat osteocalcin promoter. The p148h mutation in Msx2 augments the Miz1 effect on Msx2 DNA binding, suggesting a reason why this mutation behaves in vivo as a dominant positive, and providing a potential explanation of the craniosynostosis phenotype.
Msx2是一种同源框基因，在诱导组织相互作用（包括形成颅骨的相互作用）中起调节作用。我们先前证明，患有常染色体显性遗传性颅骨形态发生障碍（颅缝早闭，波士顿型）的个体携带Msx2的一种突变形式，其中在同源结构域N端臂的第7位（p148h），一个组氨酸取代了高度保守的脯氨酸。该突变在转基因小鼠中表现为显性阳性。由于该突变位于同源结构域的N端臂，而在其他同源结构域蛋白中该区域在蛋白质-蛋白质相互作用中起关键作用，这促使我们进行酵母双杂交筛选以寻找与Msx2相互作用的蛋白。在此，我们对一种这样的蛋白进行功能分析，该蛋白命名为Miz1（Msx相互作用锌指蛋白）。Miz1是一种含锌指的蛋白，其氨基酸序列与酵母蛋白Nfi-1的序列非常相似。这些蛋白共同定义了一个新的、高度保守的蛋白家族。通过Northern印迹和原位杂交分析Miz1的表达，揭示了一种与Msx2重叠的时空模式。此外，Miz1是一种序列特异性DNA结合蛋白，并且它可以作为一种正向作用的转录因子发挥功能。Miz1在体外直接与Msx2相互作用，并增强Msx2对大鼠骨钙素启动子中一个功能重要元件的DNA结合亲和力。Msx2中的p148h突变增强了Miz1对Msx2 DNA结合的作用，这表明该突变在体内表现为显性阳性的原因，并为颅缝早闭表型提供了一种潜在的解释。
