ZMBH, DKFZ-ZMBH-Alliance and CellNetworks - Cluster of Excellence, University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Nucleic Acids Res. 2019 Dec 16;47(22):11589-11608. doi: 10.1093/nar/gkz962.
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.
着丝粒功能需要组蛋白 H3 变体 CENP-A 在大多数真核生物中存在。CENP-A 的精确定位和蛋白量对于正确的染色体分离至关重要，而失调可能导致非整倍体。为了研究 CENP-A 在非着丝粒染色质上的加载情况，我们利用不同的截短和定位缺陷的 CENP-A 突变体构建体在黑腹果蝇培养细胞中进行研究，并表明黑腹果蝇 CENP-A 的 N 端对于核定位和蛋白稳定性是必需的，并且决定其定位的是 CENP-A 相关蛋白，而不是 CENP-A 本身。突变型 CENP-A 与加载因子 CAL1 共表达导致 CENP-A 仅在着丝粒上加载，而与组蛋白结合蛋白 RbAp48 共表达则导致 CENP-A 仅在非着丝粒上掺入。对非着丝粒 CENP-A 相互作用伙伴的质谱分析鉴定了含有 RbAp48 的 NuRD 染色质重塑复合物。进一步的分析证实，NuRD 对于异位 CENP-A 掺入是必需的，并且 NuRD 中的 RbAp48 和 MTA1 样亚基以及 CENP-A 的 N 端尾巴介导了相互作用。总之，我们的数据表明，黑腹果蝇 CENP-A 对着丝粒染色质没有固有特异性，并且利用分离的加载机制将其掺入到着丝粒和异位部位。这表明 CENP-A 相互作用因子的特异性结合和可用性是 CENP-A 加载特异性的主要决定因素。
