Agata Y, Matsuda E, Shimizu A
Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
J Biol Chem. 1999 Jun 4;274(23):16412-22. doi: 10.1074/jbc.274.23.16412.
We have isolated two novel Krüppel-like zinc finger proteins containing the evolutionarily conserved Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated that they repress transcription when heterologously targeted to DNA. Their repression activity appeared to be mediated by the putative corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with KAP-1 lack repression activity, and KAP-1 has intrinsic repressor activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for repression. Using a mammalian two-hybrid assay, we further demonstrated that KAP-1 deletions lacking repression activity fused to the VP16 transactivation domain strongly activated transcription when coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the first evidence that KAP-1 functionally interacts with KRAB in mammalian cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1 complex, and they further support the hypothesis that KAP-1 functions as a corepressor for the large class of KRAB-containing zinc finger proteins.
我们分离出了两种含有进化上保守的Krüppel相关框（KRAB）的新型Krüppel样锌指蛋白，即KRAZ1和KRAZ2，并证明当它们异源靶向DNA时可抑制转录。它们的抑制活性似乎是由假定的共抑制因子KAP-1（KRAB相关蛋白-1）介导的，因为KRAZ1/2与KAP-1结合，但不能与KAP-1相互作用的KRAZ1/2的KRAB突变体缺乏抑制活性，并且KAP-1具有内在的抑制活性并增强KRAZ1/2介导的抑制作用。我们将KAP-1蛋白分解为一个KRAB相互作用结构域和一个抑制所必需的区域。使用哺乳动物双杂交试验，我们进一步证明，与VP16反式激活结构域融合的缺乏抑制活性的KAP-1缺失在与KRAZ1共表达时强烈激活转录。相反，保留抑制活性的VP16-KAP-1融合导致抑制作用。这些结果提供了首个证据，表明KAP-1在哺乳动物细胞中与KRAB发生功能相互作用，并且似乎在与DNA结合的KRAB-KAP-1复合物中发挥抑制活性，它们进一步支持了KAP-1作为一大类含KRAB锌指蛋白的共抑制因子发挥作用的假说。
