Fig 1.
H2ac associates with telomeres.
(A) Distribution of perfect TTAGGGn /CCCTAAn repeats in the ChIP-seq data performed on cells overexpressing HA or HA-H2ac. Control HA ChIP is shown in black and HA-H2ac in yellow. (B) Dot-blot hybridization to analyze association of telomeric DNA repeat with HA-H2ac. The indicated cells were fixed by formaldehyde and were subjected to ChIP using anti-HA antibodies followed by dot blot analysis using a (CCCTAA)3 probe, Alu sequence and α-satellite. (C) The signals of telomeric DNA in ChIP were quantified and normalized to the corresponding total telomeric signal (Input). The P value was calculated using a Student's two-tailed t-test. (D-E) Dot-blot hybridization to analyze the association of telomeric DNA repeats with endogenous H2ac. The association of telomeric DNA repeat with endogenous histone H3.3, TRF1 and TRF2 served as the positive controls. The signals of telomeric DNA in ChIP were quantified and normalized to the corresponding total telomeric signal (Input). The P value was calculated using a Student's two-tailed t-test. (F-G) Analysis of specificity of interaction between H2ac and telomeric sequence using HA tagged GST, H2ac mutant, and H2al and H2am overexpressed in MCF-7 cells. The signals of telomeric DNA in ChIP were quantified and normalized to the corresponding total telomeric signal (Input). The P value was calculated using a Student's two-tailed t-test.
Fig 2.
Analysis of the interaction of H2ac with TRF2 and POT1 by co-immunoprecipitation.
(A) Endogenous H2ac interacts with endogenous TRF2 and POT1 in MCF-7 cells. Nuclear extracts were immunoprecipitated with mouse anti-H2ac and were blotted with antibodies against TPP1, -POT1, -TRF2, -TIN2, -TRF1 and -H2ac antibodies. Mouse IgG was used as a negative control. (B) Co-immunoprecipitation assay with anti-TRF2 (top) or anti-POT1 (bottom) and western blot with anti-H2ac in MCF-7 nuclear extracts. (C) The wild-type or mutant HA-H2ac was transiently expressed in MCF-7 cells. MCF-7 nuclear lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-TRF1, anti-TRF2 and anti-POT1 antibodies, respectively. (D) Association of H2ac-TRF2 and H2ac-POT1, respectively in MCF-7 cells were not affected after treatment with DNase I, EtBr or RNase A. (E) Purified recombinant HA-H2ac interacted directly with purified recombinant FLAG-TRF2. HA-H2acS16A was used as a negative control. (F) Purified recombinant HA-H2ac interacted directly with purified recombinant MYC-POT1. HA-H2acS16A was used as a negative control.
Fig 3.
The amino-terminal basic domain of TRF2 is required for H2ac recruitment to telomeres.
(A) Co-expression of HA-H2ac and truncated mutants of TRF2 were fused to FLAG and blotted with anti-FLAG in MCF-7 nuclear extracts. (B) Co-expression of truncated mutants of POT1 were fused to FLAG and blotted with anti-FLAG in MCF-7 nuclear extracts. (C) Western blotting of cell extracts prepared from control KD, POT1 KD and TRF2 KD cells using antibodies labeled on the right. (D) siControl or siTRF2-transfected MCF-7cells were analyzed by ChIP assay with anti-TRF2, anti-H2ac, or IgG control and were probed by hybridization with (CCCTAA)3 probe or Alu sequences. Quantification of telomeric-repeat DNA recovered in each ChIP is shown. (E) TRF2△B (TRF246-500) transfected MCF-7 cells or control cells were assayed by ChIP with antibodies to TRF2, H2ac, or control IgG and probed with (CCCTAA)3 probe and Alu sequences. Quantification of telomeric-repeat DNA recovered in each ChIP is shown. (F) POT1(1–250) transfected MCF-7 cells or control cells were assayed by ChIP with antibodies to TRF2, H2ac, or control IgG and were probed with (CCCTAA)3 probe and Alu sequences. Quantification of telomeric-repeat DNA recovered in each ChIP is shown.
Fig 4.
H2ac depletion induces telomere-repeat loss and telomere dysfunction.
(A) Western blotting of cell extracts prepared from control KD and H2ac KD cells using antibodies labeled on the right. (B) Telomere length analysis of MCF-7 and IMR-90 cells with control or H2ac siRNAs by telomere restriction fragment (TRF) analysis. MCF-7 and IMR-90 cells were harvested at day 5 after two separate transfections with control and H2ac siRNA. Telomere-repeat length and intensity was measured by restriction digest of genomic DNA with AluI/MboI and Southern hybridization with DIG-labeled (TTAGGG)4 probe (top panel). The GAPDH region was used as a control for DNA loading (bottom panel). The position of molecular weights (MWs; kb) is indicated on the left. (C) Telomere-ChIP assay indicates the effect of H2ac depletion on the occupancy of TRF1, TRF2 and POT1 at telomeres with telomere-specific sequences or Alu sequences using dot blotting. Quantification of TTAGGG repeat DNA in the panel recovered in each ChIP is shown. The average of experiments performed in triplicate is shown. The P value was calculated using a Student's two-tailed t-test. (D) Effect of H2ac depletion on soluble and chromatin-bound TRF1, TRF2 and POT1 proteins. Equal cell equivalents of cytoplasmic proteins (CP), nucleoplasmic proteins (NP), and the chromatin-bound fraction (CB) were analyzed. α-tubulin is cytoplasmic and histone H3 is a chromatin protein.
Fig 5.
H2ac depletion induces chromosomal abnormalities.
Telomeric FISH on metaphases spreads from MCF-7 cells with siH2ac. The telomeric hybridization signal is shown in red and DAPI-counterstained chromosomes in blue. (A) Representative images of normal telomeric DNA signals (in red) and chromosomes (in blue). (B-F) Representative images of multiple chromosome abnormalities. Knockdown of H2ac induced multiple chromosomal aberrations, including chromosome with telomere free ends (B); sister-chromatid telomere losses (C); chromosome fusions with or without TTAGGG repeats at the fusion sites (D); chromatid fusions (E); and chromosome breakages (F). The frequency of cytogenetic aberrations is quantitated under each panel. (G) The induction of anaphase bridges and micronuclei is shown. MCF-7 and IMR-90 cells were harvested at day 6 after two separate transfections with control or H2ac siRNAs. The black-and-white images showed DNA staining by DAPI. Quantification of DAPI-positive anaphase bridges and micronuclei in control or H2ac siRNA-treated in MCF-7 or IMR-90 cells. (n = 100, the P value was calculated using a Student's two-tailed t-test.).
Fig 6.
H2ac depletion induces cell arrest and cellular senescence.
(A) BrdU incorporation assay of MCF-7 and IMR-90 cells, respectively with transfected H2ac-siRNA, H2al-siRNA, H2am-siRNA or control siRNA. 1, 3 and 5 days after H2ac depletion, cells were pulsed with BrdU for 4 hr. Then cells were stained with anti-BrdU antibody and percentage of BrdU the positive cells is indicated. The P value was calculated using a Student's two-tailed t-test. (B) Cell-cycle analysis of MCF-7 and IMR-90 cells, respectively with transfected H2ac-siRNA, H2al-siRNA, H2am-siRNA or control siRNA by using propidium iodide and flow cytometric analysis. (C) SA-β-gal staining was done for the analysis of cellular senescence of MCF-7 and IMR-90 cells transfected with control siRNAs or siH2ac for 5 days. Quantification of SA-β-gal-positive cells relative to total cells was obtained by counting 200 cells in three randomly chosen fields per dish. Three independent experiments were conducted. Data obtained from the representative experiment are shown. Bar represents the mean ± SD of three experiments. The P value was calculated using a Student's two-tailed t-test.
Fig 7.
H2ac knockdown triggers an ATM-dependent DNA damage signal at telomeres.
(A) Representative images of confocal sections of γ-H2AX foci (red) and telomere (green) in nuclei of MCF-7 and IMR-90 cells expressing either control KD or H2ac KD. The magnification of the images is shown as a scale bar (5 μm). (B) Representative images of confocal sections of 53BP1 foci (red) and telomere (green) in nuclei of MCF-7 and IMR-90 cells expressing either control knockdown (KD) or H2ac KD. The magnification of the images is shown as a scale bar (5 μm). (C) Quantitation of γ-H2AX-associated and 53BP1-associated telomeres in TIFs assays in cytogenetic preparations (mean ± SD; n = 3). (D) Using phosphorylated γ-H2AX (pS-139) and ATM (pS-1981) antibodies to perform telomere-ChIP analysis in MCF-7 extracts with transfected with control or H2ac siRNA, respectively, prepared at 0, 1, 2, 4, 6, 8, 10 and 12 hr after release from double-thymidine block. (E) Western blotting of cell extracts prepared from control KD, and H2ac KD cells using antibodies labeled on the right. (F) Western blotting of cell extracts prepared from control KD, H2ac KD, H2ac+ATM double KD and H2ac+ATR double KD cells using antibodies labeled on the right. (G) Percentage of cells containing three or more γ-H2AX-positive TIFs in control KD, H2ac KD, H2ac+ATM KD and H2ac+ATR KD cells (mean ± SD; n = 3; The P value was calculated using a Student's two-tailed t-test).
Fig 8.
XPF is essential for H2ac depletion-mediated telomere deletion and loss of 3’ G-strand overhangs.
(A) Knockdown of XPF rescued the telomere deletion induced by the treatment with H2ac-siRNA, as examined by TRF assay. (B) Telomere-ChIP assay showing the effect of H2ac depletion on the occupancy of XPF in telomeres with telomere-specific sequences or Alu sequences using dot blot. Quantification of telomeric-repeat DNA recovered in each ChIP is shown. Results are average of experiments performed in triplicate. The P value was calculated using a Student's two-tailed t-test. (C) Telomere-ChIP analysis MCF-7 extracts with transfected control siRNA and H2ac-siRNA, respectively, prepared 0, 1, 2, 4, 6, 8, 10 and 12 hr after release from double-thymidine block using XPF antibody. (D) Non-denaturing hybridization analysis of telomeric G-strand overhangs from control KD, H2ac KD and H2ac+XPF KD cells. Undigested genomic DNA was hybridized to DIG-labeled C-rich (CCCTAA)3 probe and then gel fractionated in 0.5× TBE. Relative amounts of overhangs were calculated by normalizing signals from non-denaturing gel (overhang signals) to the EtBr signal (representing total genomic DNA) and plotted. Results shown are representative of three independent experiments. Error bars represent one standard deviation. The P value was calculated using a Student's two-tailed t-test.
Fig 9.
XPF is essential for the H2ac depletion-mediated DNA damage response and genome abnormalities.
(A) Knockdown of XPF reduced the consequences of TIFs induced by the treatment with H2ac specific siRNA, as examined by telomere-FISH and immunofluorescence assay. The bar graph shows the mean number of TIFs per nucleus. The data are represented as mean ± SD. The P value was calculated using a Student's two-tailed t-test. (B) Western blotting of cell extracts prepared from control knockdown (KD), H2ac KD, H2ac+XPF double KD and H2al KD cells using antibodies labeled on the right. (C) The reduction of anaphase bridges and micronuclei in simultaneous knockdown of H2ac and XPF. Quantification of DAPI-positive anaphase bridges and micronuclei of cells treated with control, H2ac siRNA, or H2ac and XPF double siRNAs. 100 anaphase cells or interphase cells were analyzed. The P value was calculated using a Student's two-tailed t-test. (D) Knockdown of XPF reduced the cell arrested induced by the treatment with H2ac specific siRNA. Cell-cycle analysis of the indicated cells was assessed using propidium iodide staining and flow cytometry.