Nabatiyan Arman, Krude Torsten
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom.
Mol Cell Biol. 2004 Apr;24(7):2853-62. doi: 10.1128/MCB.24.7.2853-2862.2004.
In eukaryotic cells, chromatin serves as the physiological template for gene transcription, DNA replication, and repair. Chromatin assembly factor 1 (CAF-1) is the prime candidate protein to mediate assembly of newly replicated DNA into chromatin. To investigate the physiological role of CAF-1 in vivo, we used RNA interference (RNAi) to silence the 60-kDa subunit of CAF-1 (p60) in human cells. Transfection of a small interfering RNA (siRNA) directed against p60 resulted in efficient silencing of p60 expression within 24 h. This silencing led to an induction of programmed cell death in proliferating but not in quiescent human cells. Concomitantly, proliferating cells lacking p60 accumulated DNA double-strand breaks and increased levels of the phosphorylated histone H2A.X. Nuclear extracts from cells lacking p60 exhibited a 10-fold reduction of nucleosome assembly activity during DNA synthesis, which was restored upon addition of recombinant p60 protein. Nascent chromatin in cell nuclei lacking p60 showed significantly increased nuclease sensitivity, indicating chromatin assembly defects during DNA synthesis in vivo. Collectively, these data identify CAF-1 as an essential factor not only for S-phase-specific chromatin assembly but also for proliferating cell viability.
在真核细胞中，染色质是基因转录、DNA复制和修复的生理模板。染色质组装因子1（CAF-1）是介导新复制的DNA组装成染色质的主要候选蛋白。为了研究CAF-1在体内的生理作用，我们使用RNA干扰（RNAi）在人类细胞中沉默CAF-1的60-kDa亚基（p60）。转染针对p60的小干扰RNA（siRNA）导致p60表达在24小时内有效沉默。这种沉默导致增殖的人类细胞而非静止细胞中程序性细胞死亡的诱导。同时，缺乏p60的增殖细胞积累了DNA双链断裂并增加了磷酸化组蛋白H2A.X的水平。缺乏p60的细胞的核提取物在DNA合成过程中核小体组装活性降低了10倍，加入重组p60蛋白后恢复。缺乏p60的细胞核中的新生染色质显示出核酸酶敏感性显著增加，表明体内DNA合成过程中染色质组装存在缺陷。总体而言，这些数据表明CAF-1不仅是S期特异性染色质组装的必需因子，也是增殖细胞活力的必需因子。
