CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.
CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.
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DOI：
10.1083/jcb.200207112
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发表时间：
2002-12-09
影响因子：
7.8
通讯作者：
Masumoto, Hiroshi
中科院分区：
文献类型：
--
作者：
Ohzeki, Jun-ichirou;Nakano, Megumi;Okada, Teruaki;Masumoto, Hiroshi
着丝粒蛋白（CENP）B盒是CENP-B的识别序列，在人着丝粒α-卫星DNA（α-DNA）中以一定的间隔出现。本研究中，为了确定α DNA一级序列所携带的信息是否参与了人类着丝粒的组装，我们合成了四种重复序列：在所有CENP-B盒中点突变的修饰α DNA，导致所有CENP-B结合活性丧失的α DNA;含有功能性CENP-B盒的未修饰α DNA;和具有或不具有功能性CENP-B盒的非α重复DNA序列。将这四种合成的重复DNA引入培养的人细胞（HT 1080）中，并使用哺乳动物人工染色体（MAC）形成测定来评估从头着丝粒组装。我们发现CENP-B盒和α DNA序列都是从头MAC形成和功能性着丝粒组分如CENP-A、CENP-C和CENP-E组装所必需的。使用染色质免疫沉淀分析，我们发现，CENP-A和CENP-B在细胞中与合成的α DNA的直接组装需要功能性CENP-B盒。据我们所知，这是第一个报道的证据，在人类着丝粒特异性DNA序列和着丝粒染色质组装之间的功能分子联系。
Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric α-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.