1 Nature Communications 2013 Vol: 3():676-. DOI: 10.1038/ncomms1676

Regulation of histone modification and chromatin structure by the p53–PADI4 pathway

Histone proteins are modified in response to various external signals; however, their mechanisms are still not fully understood. Citrullination is a post-transcriptional modification that converts arginine in proteins into citrulline. Here we show in vivo and in vitro citrullination of the arginine 3 residue of histone H4 (cit-H4R3) in response to DNA damage through the p53–PADI4 pathway. We also show DNA damage-induced citrullination of Lamin C. Cit-H4R3 and citrullinated Lamin C localize around fragmented nuclei in apoptotic cells. Ectopic expression of PADI4 leads to chromatin decondensation and promotes DNA cleavage, whereas Padi4−/− mice exhibit resistance to radiation-induced apoptosis in the thymus. Furthermore, the level of cit-H4R3 is negatively correlated with p53 protein expression and with tumour size in non-small cell lung cancer tissues. Our findings reveal that cit-H4R3 may be an 'apoptotic histone code' to detect damaged cells and induce nuclear fragmentation, which has a crucial role in carcinogenesis.

Mentions
Figures
Figure 1: Citrullination of core histones in the damaged cells.(a) Histones extracted from U-2 OS cells 48 h after ADR treatment (lane 2) were immunoblotted with anti-modified citrulline (anti-MC) antibody (left panel). U-2 OS cells without ADR treatment were used as a negative control (lane 1). CBB stain is shown in the right panel to ensure equal loading. Whole-cell extracts were subjected to immunoblotting with anti-PADI4, anti-p53 or anti-β-actin antibody. (b) siRNA against PADI4 was trasfected 10 h before DNA damage treatment. siEGFP was used as a control. Purified histones were immunoblotted with anti-MC antibody (upper left panel). Whole-cell extracts were subjected to immunoblotting with anti-PADI4, anti-p53 or anti-β-actin antibody (lower left panel). Quantitative PCR analysis of PADI4 or PADI3 expression (right panel). Error bars represent range (n=2). GAPDH was used for normalization of expression levels. (c) Histones were extracted from PADI4-introduced or mock-transfected HEK293T cells, and immunoblotted with anti-MC antibody. Whole-cell extracts were subjected to immunoblotting with anti-PADI4 or anti-β-actin antibody. (d) In vitro citrullination followed by western blotting with anti-MC, anti-citrullinated histone H4R3 (cit-H4R3), anti-citrullinated histone H3R2,8,17 (cit-H3R2,8,17), or anti-His-tag (6×His) antibody. Each recombinant histone was incubated with wild-type or mutant PADI4 protein in the presence of 10−3 M of calcium at 37 °C for 1 h. (e) Alignment of amino-acid sequences of the N-terminal regions of histone H2A and H4. Figure 2: Citrullination of histone H4R3 in the damaged cells.(a) Western blotting for histones extracted from ADR-treated U-2 OS cells (lane 2) using antibodies against citrullinated histone H4R3 (cit-H4R3) or citrullinated histone H3R2,8,17 (cit-H3R2,8,17). Whole-cell extracts were subjected to immunoblotting with anti-PADI4, anti-p53 or anti-β-actin antibody. U-2 OS cells without ADR treatment were used as a control (lane 1). (b) siRNA against PADI4 or p53 was transfected 10 h before ADR treatment. siEGFP was used as a control. Extracted histones were immunoblotted with anti-cit-H4R3 antibody. Whole-cell extracts were subjected to immunoblotting with anti-PADI4, anti-p53 or anti-β-actin antibody. CBB staining was shown to ensure equal loading. (c) Histones were extracted from HEK293T cells transfected with plasmid-expressing HA tagged-wild-type PADI4, mutant PADI4 (D350A or D473A), or mock and immunoblotted with anti-cit-H4R3 antibody. Whole-cell extracts were subjected to immunoblotting with anti-HA or anti-β-actin antibody. Figure 3: Localization of cit-H4R3 around the fragmented nuclei during apoptosis.(a) Representative image of HA-PADI4-transfected HEK293T cells stained with anti-citrullinated histone H4R3 (cit-H4R3) antibody (Alexa Fluor 488) and anti-HA antibody (Aleza Fluor 594) (left panel).The proportion of cit-H4R3-positive cells stratified by HA-PADI4 expression was indicated (right panel). Error bars represent s.d. (n=4). **P<0.01 by Student's t-test. Scale bars, 20 μm. (b) Chromatin fractionation followed by western blotting. Isolated nuclei from HEK293T cells transfected with PADI4-expressing plasmids were digested with MNase. Digested nuclei were fractionated into three fractions: transcriptionally active (S1), transcriptionally inactive (S2) and insoluble compact chromatin (P). Aliquots from S1, S2 and P fractions were separated by SDS–PAGE and then analysed by western blotting using anti-cit-H4R3 antibody. (c) Representative image of ADR-treated U-2 OS cells stained with anti-cit-H4R3 antibody (Alexa Fluor 488). Cells were treated with 2 μg ml−1 of ADR for 2 h and fixed 48 h later. Scale bars, 20 μm. (d) Representative image of ADR-treated U-2 OS cells stained with anti-citrullinated histone H4R3 antibody (Alexa Fluor 488). Each cell line was treated with 2 μg ml−1 of ADR for 2 h and fixed 48 h later. Each siRNA was transfected 10 h before the ADR treatment. siEGFP was used as a control. Scale bars, 20 μm. (e) Co-staining of U-2 OS cells with TUNEL (FITC) and anti-cit-H4R3 antibody (Alexa Fluor 594) at 48 h after treatment with 2 μg ml−1 of ADR for 2 h (left panel). The proportion of TUNEL positive cells stratified by cit-H4R3 expression was indicated (right panel). Error bars represent s.d. (n=7). **P<0.01 by Student's t-test. Scale bars, 20 μm. Figure 4: Regulation of DNA cleavage by PADI4.(a) Chromatin was isolated from HEK293T cells transfected with PADI4 expressing plasmid. Chromatin extracted from equal numbers of cells was digested with MNase at 37 °C for 2 min. Soluble DNA released from MNase-treated chromatin were subjected to agarose gel electrophoresis. Arrow heads denote mono- and poly-nucleosomal DNAs. (b) Chromatin was isolated from HEK293T cells transfected with PADI4-expressing plasmid. Chromatin extracted from equal numbers of cells was digested with MNase at 37 °C for 2 min. Soluble core histones released from MNase-treated chromatin were subjected to SDS–PAGE. CBB staining for input chromatin is shown as controls. Input chromatin was subjected to immunoblotting with anti-HA antibody. (c) Chromatin was isolated from U-2 OS cells treated with ADR. Each siRNA was transfected 10 h before DNA damage treatment. siEGFP was used as a control. Chromatin extracted from equal numbers of cells was digested with MNase at 37 °C for 2 min. Soluble core histones released from MNase-treated chromatin were subjected to SDS–PAGE. CBB staining for input chromatin is shown as controls. Input chromatin was subjected to immunoblotting with anti-PADI4 antibody. Arrow head indicates MNase. Figure 5: Lamin C is citrullinated during nuclear fragmentation.(a) Representative image of HA-PADI4-transfected HEK293T cells stained with anti-modified citrulline (MC) antibody (Alexa Fluor 488) and anti-HA antibody (Aleza Fluor 594) (left panel). The proportion of MC-positive cells stratified by HA-PADI4 expression was indicated (right panel). Error bars represent s.d. (n=9). **P<0.01 by Student's t-test. Scale bars, 20 μm. Cells were incubated with 5 μmol l−1 of A23187 for 1 h before fixation. (b) Western blotting for nuclear extracts from HEK293T cells transfected with wild-type PADI4, mutant PADI4 (D350A or D473A), or mock plasmid using each anti-Lamin antibody. (c) HEK293T cells were transfected with the indicated plasmids. Cell extracts were immunoprecipitated using anti-Flag antibody, followed by immunoblotting with anti-MC, anti-Flag, or anti-HA antibody. (d) HEK293T cells were transfected with the indicated plasmids. Cell extracts were immunoprecipitated using anti-Flag antibody, followed by immunoblotting with anti-MC, anti-Flag, or anti-HA antibody. (e) Whole-cell extracts from PADI4-transfected HEK293T cells or ADR-treated U-2 OS cells were subjected to western blotting using antibody against citrullinated Lamin C at arginine 571 and arginine 572 residues (cit-Lamin C), Lamin C, PADI4, or β-actin. Each siRNA was transfected 10 h before DNA damage treatment. siEGFP was used as a control. (f) Representative image of ADR-treated U-2 OS cells stained with anti-cit-Lamin C antibody (Alexa Fluor 488). Scale bars, 20 μm. Figure 6: The role of Padi4 in DNA damage induced apoptosis in vivo.(a) Quantitative real-time PCR analysis of Padi4 and p21Waf1 expression in thymuses from γ-ray-irradiated Padi4−/− or Padi4+/+ mice (3 Gy). Error bars represent range (n=2). Gapdh was used for normalization of Padi4 expression levels. Mice were sacrificed 24 h after irradiation with 3 Gy of γ-ray. (b) Representative images of thymus sections from γ-ray-irradiated Padi4−/− or Padi4+/+ mouse 24 h after irradiation with 3 Gy of γ-ray stained for citrullinated histone H4R3 (cit-H4R3) (left panel, X40). The proportion of positive cells in Padi4−/− or Padi4+/+ mouse was indicated (right panel). Error bars represent s.d. (n=4). **P<0.01 by Student's t-test. (c) Representative images of TUNEL staining for thymus sections from γ-ray-irradiated Padi4−/− or Padi4+/+ mouse 24 h after irradiation with 3 Gy of γ-ray (left panel, X40). The proportion of positive cells in Padi4−/− or Padi4+/+ mouse was indicated (right panel). Error bars represent s.d. (n=4). **P<0.01 by Student's t-test. (d) Representative images of cleaved-caspase-3 staining in thymus sections from γ-ray-irradiated Padi4−/− or Padi4+/+ mouse 24 h after irradiation with 3 Gy of γ-ray (left panel, X40). The proportion of positive cells in Padi4−/− or Padi4+/+ mouse was indicated (right panel). Error bars represent s.d. (n=4). *P<0.05 by Student's t-test. (e) Whole-cell extracts of thymus from X-ray-irradiated Padi4−/− or Padi4+/+ mice were subjected to immunoblotting with anti-cleaved caspase-3, anti-caspase-3 or anti-β-actin antibody (left panel). Mice were sacrificed 48 h after irradiation with 3 Gy of X-ray. The ratio between cleaved caspase-3 and β-actin from independent samples (n=4 for Padi4−/− mice and n=2 for Padi4+/+ mice) was indicated (right panel). *P<0.05 by Student's t-test. Figure 7: Involvement of PADI4 in carcinogenesis.(a) Whole-cell extracts from HEK293T cells transfected with wild-type or mutant PADI4 were subjected to western blotting using anti-citrullinated Lamin C (cit-Lamin C), anti-citrullinated nucleophosmin (cit-NPM), anti-citrullinated histone H4R3 (cit-H4R3), anti-HA, or anti-β-actin antibody (upper panel). For quantification, band intensities were normalized to the signal in cells transfected with wild-type PADI4 (lower panel). (b) MDA-MB-231 and MDA-MB-435S cells were infected with adenovirus vector expressing p53 (Ad-p53). Histones were subjected to western blotting using anti-cit-H4R3 (left panel). Representative images of Ad-p53-infected cells stained for cit-H4R3 is shown in right panel. Scale bars, 20 μm. (c) cit-H4R3 or p53 expression in NSCLC tissues (magnification X10). (d) Kaplan–Meier analysis of survival in patients with NSCLC according to the expression of cit-H4R3 (P=0.24, log-rank test).
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References
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