Regulation of p53 target gene expression by peptidylarginine deiminase 4

Abstract

Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.

FIG. 1.

PAD4 inhibitor Cl-amidine inhibits the activity of PAD4. (A and B) The structure of Cl-amidine (A) is similar to that of the PAD4 substrate peptidylarginine (B). (C) The activity of GST-PAD4 in histone citrullination was inhibited by preincubating GST-PAD4 with Cl-amidine. (D) The amount of histone citrullination signal detected in panel C was quantified using the NIH Image J program and graphed, with the concentration of the inhibitor indicated.

FIG. 2.

Cl-amidine treatment increases p21 expression. (A) The number of U2OS cells treated with 200 μM of Cl-amidine was 48.3% ± 3.6% (n = 3) of the number of mock-treated cells, demonstrating that Cl-amidine has a negative effect on cell growth and proliferation. (B) Western blot analyses of the levels of PAD4, H3R17Me, H3Cit, p21, p53, and p53 Ser15 phosphorylation in U2OS cells after treatment with Cl-amidine for 24 h. The level of PAD4 was unaltered (note the doublet of PAD4 detected on the Western blot). Tubulin and histone H3 were monitored to ensure equal protein loading. (C) The effects of Cl-amidine on the expression of p21 in U2OS cells without or with the depletion of p53 using a pHTP/p53-shRNA plasmid. (D) Lanes 1 and 2, the changes of PAD4, p53, and p21 in the p53^+/+ HCT116 cells after the Cl-amidine treatment for 24 h detected by Western blotting. The p21 expression was increased by 7.7- ± 1.3-fold (n = 3). Lanes 3 and 4, changes in PAD4, p53, and p21 in the p53^−/− HCT116 cells after the Cl-amidine treatment for 24 h. The increase in p21 expression was not observed.

FIG. 3.

Effect of PAD4 depletion by siRNAs on the expression of p53 target genes and apoptosis in U2OS cells. (A) PAD4 protein was decreased in U2OS cells after the PAD4 siRNA treatment compared to that after the GFP siRNA treatment. Note the doublet of PAD4 in U2OS cells. PAD4 siRNA caused the PAD4 protein level to decrease ∼60%. An approximately sixfold increase in p21 was observed. Tubulin blotting showed equal protein loading. (B) The levels of GADD45, p21, and PUMA mRNAs were increased in U2OS cells after the PAD4 siRNA treatment over that in the cells treated with the GFP siRNA. In contrast, the levels of other p53 target genes, such as CDC25C and MDM2, were unchanged. (C) Effect of PAD4 depletion by siRNAs on the expression of p21 in U2OS cells without or with the depletion of p53 using a pHTP/p53-shRNA plasmid. (D) Cells were treated with the PAD4 siRNAs or the GFP siRNA as a control for 60 h. The number of PAD4 siRNA-treated cells was 52.3% ± 4.3% (n = 3) of that of the GFP siRNA-treated cells, suggesting that PAD4 depletion inhibits cell growth and proliferation. (E) PAD4 staining and TUNEL staining in cells treated with GFP siRNAs (a to c) or PAD4 siRNAs (d to f). Approximately equal amounts of PAD4 were detected in all cells after the GFP siRNA treatment (a). In contrast, a large fraction of cells in subpanel d exhibited little or no PAD4 staining following PAD4 siRNA treatment (denoted by arrows); TUNEL-positive cells were detected in PAD4 siRNA-treated cells (e) but not in the GFP siRNA-treated cells (b); in the merged images (f), cells with decreased PAD4 were found to be TUNEL positive.

FIG. 4.

Depletion of PAD4 increased p21 expression, apoptosis, and cell cycle arrest in a p53-dependent manner in HCT116 cells. (A) Lanes 1 and 2: the changes of PAD4, p21, and p53 proteins after PAD4 depletion by shRNA in the p53^+/+ HCT116 cells were detected by Western blotting. The expression of p21 was increased by 2.3- ± 0.3-fold (n = 3). Lanes 3 and 4: an increase in p21 expression was not detected after PAD4 depletion in the p53^−/− HCT116 cells. (B) The expression of p21 was analyzed by RT-PCR experiments. Depletion of PAD4 by the PAD4 shRNA increased the p21 expression in the p53^+/+ HCT116 cells. (C) The percentages of annexin V-positive cells were analyzed by flow cytometry in the p53^+/+ and p53^−/− HCT116 cells after transfection with the pHTP vector (control) or the PAD4-shRNA and selection with puromycin for 6 days. (D) Flow cytometry analyses of the effects of the PAD4 shRNA treatment on cell cycle progression in the p53^+/+ and p53^−/− HCT116 cells. Ctrl, control.

FIG. 5.

Interaction of PAD4 and p53. (A) p53 was coimmunoprecipitated by the M2 agarose beads from the Flag-PAD4-expressing 293T cells (lane 6) but not from the parental cells lacking Flag-PAD4 (lane 5). Lanes 1 to 4 had 5, 2.5, 1.25, and 0.625% of input, respectively. (B) Coimmunoprecipitation of endogenous PAD4 by a p53 monoclonal antibody (lane 3) but not by control mouse IgG (lane 2). Lane 1 had 2.5% of the input. (C) Schematic drawing of GST-PAD4 and its derivatives used in the pull-down experiments. (D) p53 was pulled down by GST-PAD4 and GST-PAD4^IgL1&2 (lanes 8 and 9), but not GST, GST-PAD4^IgL1, or beads alone. Lanes 1 to 4 had 5, 2.5, 1.25, and 0.625% of input, respectively. (E) Illustration of GST-p53 and its derivatives used in the pull-down experiments. (F) GST-p53 (lane 5) and GST-p53^301-393 (lane 7), but not GST-p53^1-300 or beads alone, were efficient in mediating the p53 and PAD4 interaction. Lanes 1 to 3 had 2, 1, and 0.5% of the input, respectively. (G) GST pull-down experiments suggest that the N-terminal immunoglobulin-like domains of PAD4 interact with the C-terminal regulatory domain of p53. (H) GST-PAD4 bound to the glutathione agarose beads associated with Flag-His6-p53, suggesting a direct interaction of p53 and PAD4. Lanes 1 to 3 had 5, 2.5, and 1.25% of input, respectively.

FIG. 6.

Dynamic p53 and PAD4 association and histone Arg modifications at the p21 promoter after UV irradiation. (A) Illustration of the p21 gene promoter, including the two p53 binding sites (p53BS1 and p53BS2). (B) Representative ChIP results of p53 and PAD4 association as well as histone Arg modifications at the p53BS2 region of the p21 promoter after 50-J/m² UVC irradiation. (C) PAD4 and p53 were not associated with the GAPDH promoter before and after DNA damage treatment. (D) Q-PCR analyses of the H3Cit, H3R17Me, and PAD4 levels on p53 binding site 1 in the U2OS cells at different time points (0, 1, and 6 h) after UVC treatment (n = 6). (E) The H3Cit and H3R17Me ChIP results in panel D were normalized to those of histone H3. (F) Q-PCR analyses of the H3Cit, H3R17Me, and PAD4 levels on p53 binding site 2 in the U2OS cells at different time points (0, 1, and 6 h) after UVC treatment (n = 6). (G) The H3Cit and H3R17Me ChIP results in panel F were normalized to those of histone H3.

FIG. 7.

Association of PAD4 with the p21 promoter is p53 dependent. (A) The expression of p53 in the p53^−/− H1299 cells was restored by transient transfection of a p53-expressing plasmid. (B) ChIP analyses of the p53 and PAD4 association with p53 binding site 2 of the p21 promoter in H1299 cells with or without the transfection of a p53-expressing plasmid. (C and D) ChIP assays of p53 (C) or PAD4 (D) association with p53 binding site 1 using Q-PCR in U2OS cells without or with the depletion of p53 by shRNA. (E and F) ChIP assays of p53 (E) or PAD4 (F) association with p53 binding site 2 using Q-PCR in U2OS cells without or with the depletion of p53 by shRNA.

FIG. 8.

Detection of paused and elongating RNA Pol II at the p21 promoter. (A) The association of RNA Pol II and Ser5 phosphorylated RNA Pol II was monitored at the +182 region of p21 at different time points after UVC treatment. (B) Permanganate footprinting was used to monitor RNA Pol II at the p21 promoter region during induction by UVC irradiation. Lane 1 shows a background pattern of bands that accompanied the LM-PCR procedure when DNA was not treated with permanganate. Lane 2 shows markers produced by partial cleavage of DNA at purines (G/A). Lane 3 shows permanganate reactivity that was intrinsic to naked DNA. Despite a high background, one can readily detect permanganate-hyperreactive positions at T9, T11, T36, T38, and T50 that occur in cells (lane 4) but not in naked DNA (lane 3). These reactivities were indicative of paused Pol II. Within 30 min after a brief pulse of UV light, permanganate reactivities at T91 and T152 increased and provided evidence of Pol II undergoing productive elongation at 0.5 and 1 h after UV treatment. By 2 and 6 h, transcription appeared repressed since the permanganate reactivity marking paused and elongating forms of Pol II had decreased significantly. (C) A “three-state” model for the role of PAD4 and dynamic histone Arg modifications in the expression of the p21 gene after UV irradiation (see text for further details).