Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells - PubMed (original) (raw)

Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells

Arman Nabatiyan et al. Mol Cell Biol. 2006 Mar.

Abstract

Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.

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Figures

FIG. 1.

FIG. 1.

Response of quiescent human cells to induction of DSBs. (A) Phosphorylation of histone variant H2AX. Proliferating and quiescent human EJ30 cells were treated with the indicated concentrations of bleocin for 24 h. Total amounts of γ-H2AX were analyzed by Western blotting of whole-cell extracts. Forty micrograms of protein was loaded per lane. Ponceau staining is shown as a loading and transfer control. (B) Formation of DSBs. Chromosomal DNAs from nuclei isolated from the cells used for panel A were separated by pulsed-field gel electrophoresis. DNA from 3 × 105 cell nuclei was loaded per lane. Asterisks denote the positions of broken high-molecular-weight DNAs that have entered the gel; loading wells containing large amounts of unbroken DNA are not shown. DNA size markers (Sigma) were included in each panel (lane M). (C) Cell viability assay. Quiescent EJ30 cells were treated with the indicated concentrations of bleocin for the indicated times and stained with trypan blue after trypsinization. For each sample, 200 cells were scored, and the proportion of surviving cells was determined for each time point. (D) Cell cycle effects. The DNA contents of nuclei isolated from untreated and bleocin-treated proliferating and quiescent EJ30 cells were determined by flow cytometry.

FIG. 2.

FIG. 2.

Induction of active CAF-1 by DSBs in quiescent cells. (A) Expression levels of CAF-1. Whole-cell extracts of the indicated cells were analyzed by Western blotting using antibodies specific for the p150 and p60 subunits of CAF-1, PCNA, and the 70-kDa and 32-kDa subunits of RPA. Asterisks denote hyperphosphorylated forms of RPA-32. Forty micrograms of protein was loaded per lane. (B) Nucleosome assembly activity of CAF-1. Nuclear extracts were prepared from untreated and bleocin-treated proliferating EJ30 cells and added at the indicated amounts (μg of protein) to nucleosome assembly reaction mixtures in vitro (23). Nucleosome assembly during cDNA strand synthesis is monitored by DNA supercoiling. The positions of supercoiled form I DNA, linear form III DNA, and nicked form II DNA are indicated. (C) CAF-1 was partially purified in parallel in a single step by cation-exchange chromatography from untreated and bleocin-treated quiescent cells and added at the indicated amounts (μg of protein) to assembly reaction mixtures.

FIG. 3.

FIG. 3.

Recruitment of CAF-1, PCNA, and RPA to damaged chromatin in cells containing single- and double-strand DNA breaks. Proliferating and quiescent EJ30 cells were grown on coverslips and treated with 3 μg/ml bleocin for 24 h or with 5 Gy of IR followed by a 2-h recovery incubation at 37°C. (A) Visualization of chromatin-bound p60 subunit of CAF-1 (red signal) and γ-H2AX (green signal) in the same nuclei by confocal immunofluorescence microscopy. Colocalization of both signals in the merged image gives a yellow signal. Soluble proteins were removed by treatment with the nonionic detergent Triton X-100 prior to fixation, as indicated. (B) Visualization of chromatin-bound p60 (red) and PCNA (green). (C) Visualization of chromatin-bound PCNA (green) and RPA (red). (D) Visualization of chromatin-bound PCNA (green) and sites of DNA synthesis (BrdU; red). (E) Induction of CAF-1 and PCNA and their recruitment to the chromatin of damaged cells are reversible. EJ30 cells were treated with bleocin as indicated (0-24 h). The cultures were divided and either treated with bleocin for another 24 h (bleocin 24-48 h) or washed free of bleocin and cultivated in fresh medium for another 24 h (no bleocin 24-48 h). PCNA (green) and p60 (red) were visualized by confocal immunofluorescence microscopy (top panels), and total expression levels of γ-H2AX and p60 were detected by Western blotting (bottom panels).

FIG. 4.

FIG. 4.

DNA damage-induced expression of CAF-1 in quiescent cells depends on DNA-PK. The expression levels of CAF-1 and PCNA and the phosphorylation of H2AX were investigated in the DNA-PK-proficient MO59K and DNA-PK-deficient MO59J sister cell lines (1, 24). Whole-cell extracts (50 μg of protein) from the indicated cells were analyzed by Western blotting, using antibodies specific for the p60 subunit of CAF-1, PCNA, and γ-H2AX. β-Actin was used as a loading control.

FIG. 5.

FIG. 5.

DNA repair by the NHEJ and BER pathways, but not by HR, is activated in quiescent cells upon bleocin treatment. Responses in proliferating and quiescent human EJ30 cell nuclei were investigated by confocal immunofluorescence microscopy of untreated and bleocin-treated EJ30 cells, as indicated. (A) Activation of HR in proliferating but not in quiescent cells. Visualization of the chromatin-bound HR marker protein Rad51 (red) and of γ-H2AX (green) was performed. (B and C) Activation of NHEJ. (B) Visualization of the chromatin-bound NHEJ marker protein XRCC4 (red) and of γ-H2AX (green). (C) Visualization of XRCC4 (red) and the p150 subunit of CAF-1 (green). (D and E) Activation of BER. (D) Visualization of the chromatin-bound BER marker protein XRCC1 (red) and of γ-H2AX (green). (E) Visualization of XRCC1 (red) and the p150 subunit of CAF-1 (green). Colocalization of both signals gives a yellow signal in the merged channels.

FIG. 6.

FIG. 6.

CAF-1 becomes essential for viability of quiescent cells in the presence of DSBs. (A) RNAi against p60 in bleocin-treated quiescent EJ30 cells. Whole-cell extracts were prepared from untreated cells and from bleocin-treated cells that were untransfected (none) or transfected with a nontarget scrambled sequence (nt siRNA) or with siRNAs against the p60 subunit of CAF-1 (p60-1 and p60-2 siRNAs). Samples were analyzed at 24 and 48 h posttransfection by Western blotting. Ponceau staining of the blotted membrane is shown as a loading and transfer control. (B) Cell viability after RNAi. The indicated cells were subjected to a colorimetric cell viability assay (33). Mean values for the total numbers of viable cells per experiment are presented, with the standard deviations of four independent data acquisitions (n = 4) presented as error bars. (C) Direct visualization of cells at 48 h posttransfection by phase-contrast light microscopy. The proportion of apoptotic cells was determined under these experimental conditions by TUNEL assays as described previously (33), and this value is given above each micrograph. (D) DNA fragmentation after RNAi. Nuclei were isolated from untreated (−) and bleocin-treated quiescent EJ30 cells at 24 h posttransfection with nt siRNA or p60-1 siRNA. At this time point, apoptotic DNA fragmentation was not detected by TUNEL assays (data not shown). Chromosomal DNAs from these nuclei were analyzed by pulsed-field gel electrophoresis as detailed in the legend to Fig. 1.

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