Cobb B S, Morales-Alcelay S, Kleiger G, Brown K E, Fisher A G, Smale S T
Howard Hughes Medical Institute, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, California 90095-1662, USA.
Genes Dev. 2000 Sep 1;14(17):2146-60. doi: 10.1101/gad.816400.
Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, we have examined the mechanism by which it is targeted to centromeric foci. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, as only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting was dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain was required only for homodimerization, as targeting was restored when this domain was replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain revealed a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats. Models for the function of Ikaros that are consistent with this targeting mechanism are discussed.
Ikaros是一种序列特异性DNA结合蛋白，对淋巴细胞发育至关重要。尽管最近的研究结果提出了一种假说，即Ikaros将注定要进行遗传性失活的基因招募到含有着丝粒周围异染色质的位点，但对其分子功能仍知之甚少。为了进一步深入了解Ikaros的功能，我们研究了它靶向着丝粒位点的机制。在3T3成纤维细胞中异位表达时观察到Ikaros的有效靶向，这表明不需要淋巴细胞特异性蛋白和淋巴样核结构。着丝粒周围的靶向作用并非与Mi-2重塑因子相互作用的结果，因为在3T3细胞中只有一小部分Mi-2定位于着丝粒位点。相反，靶向作用依赖于Ikaros的氨基末端DNA结合锌指结构域和羧基末端二聚化结构域。羧基末端结构域仅对同源二聚化是必需的，因为当该结构域被亮氨酸拉链取代时靶向作用得以恢复。令人惊讶的是，对氨基末端结构域的详细替代突变分析揭示了DNA结合与着丝粒周围靶向之间的密切相关性。这些结果表明，DNA结合对于Ikaros着丝粒周围定位至关重要，这可能与着丝粒DNA重复序列中存在Ikaros结合位点一致。本文讨论了与这种靶向机制一致的Ikaros功能模型。
