Previously, we found that two isoforms of the ZNF268 gene (ZNF268a and ZNF268b2, with and without the KRAB domain, respectively) might play distinct roles in normal epithelia and in cervical cancer. Here we further investigated that KRAB domain defined the function disparity in part by reinforcing nuclear localization of ZNF268a. We found that the A-box of KRAB alone retained major specific nuclear localization activity. In contrast, the B-box alone did not have nuclear localization activity but enhanced it significantly. Consistent with the critical function of the A-box, each mutation of six conserved residues (V9, V11, F13, E16, E17 and W18) in the A-box dramatically impaired nuclear localization activity. Furthermore, the unique nuclear localization activity of KRAB was verified in seven additional KRAB-containing zinc finger proteins (KRAB-ZFPs), suggesting that it is a universal feature of KRAB-ZFPs. Finally, KRAB exerted its unique nuclear localization activity by interacting with the RBCC domain of its corepressor KAP1. Our results have revealed a novel mechanism by which the KRAB domain reinforces nuclear localization of KRAB-ZFPs by interacting with KAP1. Our study also suggests that loss of the KRAB domain in KRAB-ZFPs due to aberrant alternative splicing might contribute to carcinogenesis.

以前，我们发现ZNF268的两个同工型基因（分别具有和不具有KRAB结构域的ZNF268a和ZNF268b2）在正常上皮细胞和宫颈癌中可能发挥不同的作用。在这里，我们进一步研究了KRAB域部分地通过增强ZNF268a的核定位来定义功能差异。我们发现仅KRAB的A盒保留了主要的特定核定位活性。相比之下，仅B-box并不具有核定位活性，而显着增强了核定位活性。与A-box的关键功能一致，A-box中六个保守残基（V9，V11，F13，E16，E17和W18）的每个突变都大大削弱了核定位活性。此外，还通过另外七个含KRAB的锌指蛋白（KRAB-ZFPs）验证了KRAB独特的核定位活性，这表明它是KRAB-ZFP的通用功能。最终，KRAB通过与其核心加压因子KAP1的RBCC结构域相互作用而发挥其独特的核定位活性。我们的结果揭示了一种新颖的机制，通过该机制，KRAB域通过与KAP1相互作用来增强KRAB-ZFP的核定位。我们的研究还表明，由于异常的可变剪接而导致的KRAB-ZFPs中KRAB结构域的丢失可能有助于癌变。