Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites.
Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites.
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DOI：
10.1083/jcb.136.3.501
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发表时间：
1997-02-10
期刊：
影响因子：
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通讯作者：
Sullivan KF
中科院分区：
文献类型：
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作者：
Shelby RD;Vafa O;Sullivan KF
我们通过将表位标记的CENP-A衍生物转染到HeLa细胞中，研究了靶向着丝粒组蛋白H3同源物CENP-A在人类细胞中着丝粒处组装的要求。着丝粒靶向仅由CENP-A的保守组蛋白折叠结构域驱动。使用组蛋白H3的晶体结构作为指导，构建了一系列CENPA/组蛋白H3嵌合体，以测试组蛋白折叠结构域的离散结构元件的作用。确定了有效靶向着丝粒所必需的三个元件。两个对应于组蛋白H3和核小体DNA之间的接触位点。第三个映射到一个同型H3-H3相互作用的网站重要的（H3/H4）2异源四聚体的组装。免疫沉淀证实CENP-A在体内自缔合。此外，靶向需要CENP-A表达在S期期间与组蛋白H3合成解偶联。CENP-A mRNA在细胞周期中的积累晚于组蛋白H3，在G2达到峰值。人CENP-A基因的分离揭示了启动子区域中的调控基序，其指导其他细胞周期依赖性转录物如cdc 2、cdc 25 C和细胞周期蛋白A的晚期S/G2表达。我们的数据表明，在核小体水平介导的一系列合作的分化CENP-A-DNA接触点排列在一个CENP-A核小体的表面和一个独特的组装途径发生在细胞周期后期的着丝粒DNA的分子识别机制。
We investigated the requirements for targeting the centromeric histone H3 homologue CENP-A for assembly at centromeres in human cells by transfection of epitope-tagged CENP-A derivatives into HeLa cells. Centromeric targeting is driven solely by the conserved histone fold domain of CENP-A. Using the crystal structure of histone H3 as a guide, a series of CENPA/histone H3 chimeras was constructed to test the role of discrete structural elements of the histone fold domain. Three elements were identified that are necessary for efficient targeting to centromeres. Two correspond to contact sites between histone H3 and nucleosomal DNA. The third maps to a homotypic H3–H3 interaction site important for assembly of the (H3/H4)2 heterotetramer. Immunoprecipitation confirms that CENP-A self-associates in vivo. In addition, targeting requires that CENP-A expression is uncoupled from histone H3 synthesis during S phase. CENP-A mRNA accumulates later in the cell cycle than histone H3, peaking in G2. Isolation of the gene for human CENP-A revealed a regulatory motif in the promoter region that directs the late S/G2 expression of other cell cycle–dependent transcripts such as cdc2, cdc25C, and cyclin A. Our data suggest a mechanism for molecular recognition of centromeric DNA at the nucleosomal level mediated by a cooperative series of differentiated CENP-A–DNA contact sites arrayed across the surface of a CENP-A nucleosome and a distinctive assembly pathway occurring late in the cell cycle.