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

Abstract

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.

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

“…This is consistent with an earlier observation using electron microscopy by Kierszenbaum and Huang [66] which clearly indicates a loosen host chromatin containing less nucleosomes in HCMV-infected fibroblasts. Our result of chromatin decondensation also resembles the previously published effect caused by depleting p150 or p60 or overexpressing the dominant-negative form of p150 [8,9,51]. Given that p150 and IE2 colocalize with UL44 and BrdU ( Figure 4) and that ectopically expressing p150 (Δ506-563), a p150 mutant defective in IE2 interaction, partially restores the host-chromatin integrity during HCMV infection ( Figure 9B), we propose that the HCMV-induced chromatin decondensation is likely due to viral IE2-mediated CAF1 mislocation to viral replication compartments.…”

“…p150 utilizes its carboxylterminal third to bind p60 [6], and knocking down either subunit disrupts the chromatin assembly activity, delays cell cycle progression and leads to cell death [7][8][9]. p48, an Rb-interacting partner [10], is thought to escort various histone-metabolizing enzymes to their target sites [11].…”

Chromatin disassembly and reassembly during DNA repair

“…In addition to functioning together in vitro, Asf1 and CAF1 are likely to assemble chromatin onto newly-replicated DNA in cells. For example, Asf1 and CAF-1 localize to DNA replication forks [21,22] and their inactivation results in major defects in chromatin assembly of the newly-replicated DNA in cells [23,24]. H2A/H2B deposition following DNA replication is presumably also mediated by histone chaperones, where possible candidates include the NAP1/SET/TAFI [25] and FACT/SPN proteins [26].…”

Nucleosome assembly and epigenetic inheritance

“…In vivo data from different model organisms supported the conclusion of above in vitro experiments. Both RNAi mediated knockdown and overexpression of a dominant negative CAF-1 in cultured human cells result in defective assembly of newly replicated chromatin (Hoek and Stillman, 2003;Ye et al, 2003;Nabatiyan and Krude, 2004). Conditional depletion of CAF-1 p150 or p60 subunit in chicken DT40 cells leads to delayed S phase progression concomitant with slower DNA synthesis (Takami et al, 2007).…”