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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.

Silencing of Chromatin Assembly Factor 1 in Human Cells Leads to Cell Death and Loss of Chromatin Assembly during DNA Synthesis