Shono Nobuaki, Ohzeki Jun-ichirou, Otake Koichiro, Martins Nuno M C, Nagase Takahiro, Kimura Hiroshi, Larionov Vladimir, Earnshaw William C, Masumoto Hiroshi
Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.
Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
J Cell Sci. 2015 Dec 15;128(24):4572-87. doi: 10.1242/jcs.180786. Epub 2015 Nov 2.
Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.
虽然人们普遍认为，含有组蛋白H3变体CENP-A的染色质是维持着丝粒身份的一种表观遗传标记，但导致着丝粒染色质形成和维持的途径仍不清楚。我们之前构建了着丝粒含有包含四环素操纵子的合成α-卫星（α-卫星）DNA阵列（α-卫星（tetO））的人类人工染色体（HAC）。我们还获得了在染色体臂上的异位整合位点携带α-卫星（tetO）阵列的细胞系。在这里，我们通过连接大量着丝粒因子和染色质修饰剂的四环素阻遏物（tetR）融合体，研究了着丝粒处CENP-A组装以及异位阵列上从头组装的调控。该分析揭示了影响CENP-A组装的四类因子。有趣的是，许多动粒结构成分在异位位点诱导了CENP-A的从头组装。我们表明，这些成分通过招募CENP-C并随后招募M18BP1发挥作用。此外，我们发现CENP-I也可以招募M18BP1，因此增强了M18BP1在CENP-C下游的着丝粒上的组装。因此，我们认为CENP-C和CENP-I是连接动粒与CENP-A组装的关键因子。
