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.

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Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells

The Journal of Cell Biology ◽

10.1083/jcb.143.3.563 ◽

1998 ◽

Vol 143 (3) ◽

pp. 563-575 ◽

Cited By ~ 120

Author(s):

Emmanuelle Martini ◽

Danièle M.J. Roche ◽

Kathrin Marheineke ◽

Alain Verreault ◽

Geneviève Almouzni

Keyword(s):

Uv Irradiation ◽

Physiological Role ◽

Human Cells ◽

Chromatin Assembly ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor ◽

G2 Cells

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

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In Vivo Study of the Nucleosome Assembly Functions of ASF1 Histone Chaperones in Human Cells

Molecular and Cellular Biology ◽

10.1128/mcb.00510-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3672-3685 ◽

Cited By ~ 28

Author(s):

Angélique Galvani ◽

Régis Courbeyrette ◽

Morgane Agez ◽

Françoise Ochsenbein ◽

Carl Mann ◽

...

Keyword(s):

Dna Synthesis ◽

Histone H3 ◽

Direct Determination ◽

Human Cells ◽

Chromatin Assembly ◽

Histone Chaperones ◽

Nucleosome Assembly ◽

Assembly Factor

ABSTRACT Histone chaperones have been implicated in nucleosome assembly and disassembly as well as histone modification. ASF1 is a highly conserved histone H3/H4 chaperone that synergizes in vitro with two other histone chaperones, chromatin assembly factor 1 (CAF-1) and histone repression A factor (HIRA), in DNA synthesis-coupled and DNA synthesis-independent nucleosome assembly. Here, we identify mutants of histones H3.1 and H3.3 that are unable to interact with human ASF1A and ASF1B isoforms but that are still competent to bind CAF-1 and HIRA, respectively. We show that these mutant histones are inefficiently deposited into chromatin in vivo. Furthermore, we found that both ASF1A and ASF1B participate in the DNA synthesis-independent deposition of H3.3 in HeLa cells, thus highlighting an unexpected role for ASF1B in this pathway. This pathway does not require interaction of ASF1 with HIRA. We provide the first direct determination that ASF1A and ASF1B play a role in the efficiency of nucleosome assembly in vivo in human cells.

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Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0426 ◽

2007 ◽

Vol 18 (1) ◽

pp. 129-141 ◽

Cited By ~ 53

Author(s):

Yasunari Takami ◽

Tatsuya Ono ◽

Tatsuo Fukagawa ◽

Kei-ichi Shibahara ◽

Tatsuo Nakayama

Keyword(s):

Dna Replication ◽

Essential Role ◽

Chromatin Assembly ◽

Nuclear Antigen ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-γ are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

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Interaction between the DrosophilaCAF-1 and ASF1 Chromatin Assembly Factors

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6574-6584.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6574-6584 ◽

Cited By ~ 153

Author(s):

Jessica K. Tyler ◽

Kimberly A. Collins ◽

Jayashree Prasad-Sinha ◽

Elizabeth Amiott ◽

Michael Bulger ◽

...

Keyword(s):

Dna Synthesis ◽

Polytene Chromosomes ◽

Normal Growth ◽

Chromatin Assembly ◽

Truncated Form ◽

Assembly Factor ◽

Development And Differentiation ◽

Growth Development

ABSTRACT The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and theDrosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo inDrosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

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