Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex
Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex
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DOI：
10.1093/nar/gkz962
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发表时间：
2019-12-16
影响因子：
14.9
通讯作者：
Erhardt, Sylvia
中科院分区：
文献类型：
--
作者：
Demirdizen, Engin;Spiller-Becker, Matthias;Erhardt, Sylvia
在大多数真核生物中，着丝粒的功能需要组蛋白H3变体CENP-A的存在。CENP-A的精确定位和蛋白量对正确的染色体分离至关重要，调控不当可导致非整倍体。为了描述CENP-A装载到非着丝粒染色质上的特征，我们在黑腹果蝇培养细胞中使用了不同的截断和定位缺陷的CENP-A突变体构建体，并表明黑腹果蝇CENP-A的n端是核定位和蛋白质稳定性所必需的，并且决定其定位的是CENP-A相关蛋白，而不是CENP-A本身。突变体CENP-A与其装载因子CAL1共表达导致CENP-A的排他性着丝粒装载，而与组蛋白结合蛋白RbAp48共表达导致CENP-A的排他性非着丝粒掺入。非着丝性CENP-A相互作用伙伴的质谱分析鉴定出含有rbap48的NuRD染色质重塑复合体。进一步分析证实，异位的CENP-A结合需要NuRD，并且NuRD的RbAp48和mta1样亚基与CENP-A的n端尾部一起介导相互作用。总之，我们的数据表明，果蝇的CENP-A对着丝粒染色质没有内在的特异性，并且利用不同的装载机制将其纳入着丝粒和异位位点。这表明，CENP-A相互作用因子的特异性关联和可用性是CENP-A装载特异性的主要决定因素。
Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.