Régnier Vinciane, Vagnarelli Paola, Fukagawa Tatsuo, Zerjal Tatiana, Burns Elizabeth, Trouche Didier, Earnshaw William, Brown William
Department of Biochemistry, Oxford University, South Parks Road, OX1 3QU Oxford, United Kingdom.
Mol Cell Biol. 2005 May;25(10):3967-81. doi: 10.1128/MCB.25.10.3967-3981.2005.
CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.
着丝粒蛋白A（CENP - A）是组蛋白H3的一种在进化上保守的、着丝粒特异性变体，被认为在指导动粒组装和着丝粒功能中起核心作用。在此，我们分析了在鸡DT40细胞系中破坏CENP - A基因的后果。在缺乏CENP - A的细胞中，动粒蛋白组装受损，这表现为动粒内部蛋白CENP - I、CENP - H和CENP - C以及外部成分Nuf2/Hec1、Mad2和CENP - E的定位错误。然而，BubR1和着丝粒内部蛋白INCENP能有效地募集到动粒上。在缺乏CENP - A后，染色体在有丝分裂纺锤体上不能正常排列，并且在前中期有短暂延迟。缺乏CENP - A的细胞进一步进入后期和胞质分裂，染色体分离不均等，这表明在CENP - A大量缺失后仍保留一些动粒功能。我们还证明，在检查点激活的条件下，缺乏CENP - A的细胞在维持检查点蛋白BubR1的动粒定位方面表现出特定缺陷。因此，我们的数据表明CENP - A在组装能够维持有丝分裂检查点信号的动粒中具有特定作用。
