Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T
Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T
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DOI：
10.1073/pnas.1615133114
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发表时间：
2017-02-21
影响因子：
11.1
通讯作者：
Funabiki, Hironori
中科院分区：
文献类型：
--
作者：
Giunta, Simona;Funabiki, Hironori
着丝粒是高度特化的染色质结构域，使染色体分离和协调忠实的细胞分裂。人类着丝粒由a-卫星DNA的串联阵列组成，其跨度可达数兆碱基。关于维持a-卫星DNA重复序列长阵列完整性的机制知之甚少。在这里，我们使用染色体定向荧光原位杂交（CO-FISH）监测人类细胞中着丝粒重复序列的稳定性。该实验在原代组织培养细胞中检测到与姐妹染色单体交换频率为5%的异常着丝粒CO-FISH模式，而在一些癌细胞系和复制衰老过程中观察到更高的水平。为了了解维持着丝粒完整性的机制，我们研究了着丝粒特异性组蛋白变体CENP-A和组成着丝粒相关网络（CCAN）、CENP-C、CENP-T和CENP-W的作用。耗尽CENP-A和CCAN蛋白导致着丝粒畸变增加，而通过其他方法增强染色体错分离则不会，这表明CENP-A和CCAN蛋白有助于维持着丝粒的完整性，而不依赖于它们在染色体分离中的作用。此外，使用结构照明显微镜对着丝体CO-FISH进行超分辨率成像表明，CENP-A可以保护a-卫星重复序列免受大量重排。我们的研究指出，在人类细胞增殖过程中，有一种着丝粒特异性机制积极地维持a-卫星重复完整性。
Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of a-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of a-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects a-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specificmechanism that actively maintains a-satellite repeat integrity during human cell proliferation.