PARP1-dependent recruitment of KDM4D histone demethylase to DNA damage sites promotes double-strand break repair

Abstract

Members of the lysine (K)-specific demethylase 4 (KDM4) A-D family of histone demethylases are dysregulated in several types of cancer. Here, we reveal a previously unrecognized role of KDM4D in the DNA damage response (DDR). We show that the C-terminal region of KDM4D mediates its rapid recruitment to DNA damage sites. Interestingly, this recruitment is independent of the DDR sensor ataxia telangiectasia mutated (ATM), but dependent on poly (ADP-ribose) polymerase 1 (PARP1), which ADP ribosylates KDM4D after damage. We demonstrate that KDM4D is required for efficient phosphorylation of a subset of ATM substrates. We note that KDM4D depletion impairs the DNA damage-induced association of ATM with chromatin, explaining its effect on ATM substrate phosphorylation. Consistent with an upstream role in DDR, KDM4D knockdown disrupts the damage-induced recombinase Rad51 and tumor protein P53 binding protein foci formation. Consequently, the integrity of homology-directed repair and nonhomologous end joining of DNA breaks is impaired in KDM4D-deficient cells. Altogether, our findings implicate KDM4D in DDR, furthering the links between the cancer-relevant networks of epigenetic regulation and genome stability.

Keywords: PARylation; chromosome instability; histone demethylation.

Fig. 1.

KDM4D Protein is rapidly recruited to sites of DNA damage. (A–C) Time-lapse images show the localization of EGFP-KDM4D fusion at the indicated time points after the induction of laser DNA damage to a single region, marked by a red arrow. Graphs (Right) show fold increase in the relative fluorescence intensity of EGFP-KDM4D at laser-microirradiated sites. Each measurement is representative of at least 10 cells. Error bars indicate SD. (A) U2OS-TetON cells expressing EGFP-KDM4D fusion following the addition of doxycycline. Movie S1 shows the accumulation of full-length EGFP-KDM4D at the laser-microirradiated site. (B and C) WI-38 cells and MEFs transfected with expression vector encoding EGFP-KDM4D fusion. Results shown in A–C are typical of three independent experiments and represent at least 15 different cells each. (D) Recruitment of the endogenous KDM4D to laser-microirradiation sites. U2OS cells (Upper) and A172 cells (Lower) were fixed within 5 min after laser microirradiation and subjected to immunofluorescence analysis using antibodies against γH2AX (red) and KDM4D (green). (E) DNA damage-dependent increase in the levels of KDM4D protein in the chromatin-bound fraction. U2OS cells were exposed to 40 μM etoposide for 40 min and subjected to cellular fractionation as described in Materials and Methods. Chromatin-bound fractions from undamaged and DNA damaged cells were immunoblotted with KDM4D antibody. H3 is used as a loading control. (F) Representative pictures showing the recruitment of the endogenous KDM4D to a single DSB. A plasmid encoding for I-Sce-I endonuclease was transfected into HCA-2 fibroblast cells that contained a single recognition site for I-Sce-I endonuclease. Cells were fixed at 24 h (Upper) and 48 h (Lower) posttransfection and immunostained with γH2AX (red) and KDM4D (gray) antibodies. Results shown are typical of three independent experiments and represent at least 15 different cells. (G) ChIP analysis shows the binding of 6xmyc-KDM4D at DNA near DSBs induced by I-Sec-I endonuclease. U2OS-HR-ind cells were transfected with an expression vector encoding 6xmyc-KDM4D or with an empty vector that served as a negative control. Bars show the enrichment of KDM4D around the I-Sce-I recognition site. The cells were then treated for 30 min with Dex to allow the migration of I-Sce-I to the nucleus and the generation of DSBs at its recognition site. Cell lysates were then prepared and subjected to anti-myc ChIP followed by real-time PCR with primers adjacent to the I-Sce-I recognition site. The occupancy of 6xmyc-KDM4D adjacent to I-SceI–induced DSB was determined by quantitative real-time PCR analysis and normalized to GAPDH input DNA. Error bars represent SD from two independent experiments. Dex, dexamethasone; RF, relative fluorescence.

Fig. 2.

The C-terminal, but not the demethylase activity, of KDM4D is essential and sufficient for its recruitment to DNA breakage sites. (A) A representative picture showing transient recruitment of the demethylase-dead mutant, KDM4D-S200M, to laser-microirradiated regions (marked with red arrowhead). Shown are time-lapse images of U2OS cells transfected with EGFP-KDM4D-S200M and subjected to laser-microirradiation. The graph shows the increase in the relative fluorescence intensity of EGFP-KDM4D-WT and EGFP-KDM4D-S200M at laser-microirradiated sites. Error bars represent SD of 10 different cells. (B) Deletion-mapping analysis showing that the C-terminal region of KDM4D is essential and sufficient for KDM4D recruitment to sites of DNA damage. U2OS cells were transfected with expression vector encoding EGFP fused to full-length or to the indicated fragments of KDM4D. Laser microirradiation was then applied, and the ability of the deletion mutants to accumulate at DNA breakage sites was assessed. (C) A representative picture showing the rapid recruitment of the C-terminal region of KDM4D (KDM4D^313-523) to laser-microirradiated regions. Results shown are typical of at least five independent experiments and represent more than 50 different cells. The graph shows the increase in the relative fluorescence intensity of EGFP-KDM4D-WT and EGFP- KDM4D^313-523 at laser-microirradiated sites. Error bars represent SD of 10 different cells for each fusion. Movie S2 shows the accumulation of EGFP-KDM4D^313-523 at the laser-microirradiated site. RF, relative fluorescence.

Fig. 3.

PARP1-dependent recruitment of KDM4D to laser-microirradiated sites. (A) The effect of PARP1 siRNA knockdown on the recruitment of KDM4D to DNA damage sites. Representative time-lapse images show the recruitment of EGFP-KDM4D after laser microirradiation of mock and PARP1-depleted cells (marked by red arrow) (Left). The intensity of EGFP-KDM4D signal at the damaged area was measured using Carl Zeiss Zen software. The graph displays the percentage of PARP1-depleted cells that show accumulation of EGFP-KDM4D to laser-microirradiated regions, compared with mock transfected cells at 72 h after transfection (Right). Error bars represent the SEM from two independent experiments. (B and C) Chemical inhibition of PARP1/2 suppresses the recruitment of EGFP-KDM4D (B) and EGFP-KDM4D^313-523 (C) fusions to laser-microirradiated sites. U2OS-TetON cells expressing EGFP-KDM4D or EGFP-KDM4D^313-523 fusions were treated for 1 h with 5 μM PARP1/2 inhibitor, Ku-0059436. Representative time-lapse images show the localization of fusion proteins at the indicated times after laser microirradiation of a single region (marked by arrow). Results shown are typical of three independent experiments and represent at least 30 different cells. Graphs (Right) show fold increase in the relative fluorescence intensity of EGFP-KDM4D at laser-microirradiated sites. (D) PARP-dependent recruitment of the endogenous KDM4D to single-DSBs induced by I-Sce-I endonuclease, as described in Fig. 1F. Mock and PARPi-treated HR-ind cells were immunostained with γH2AX (red) and KDM4D (gray) antibodies. Results shown represent 15 different cells.

Fig. 4.

KDM4D is PARylated in response to DNA damage. (A–C) In vitro PARylation of KDM4D using recombinant PARP1 enzyme in the presence or absence of either PARP1 or NAD⁺. The reaction mixtures were immunoblotted using PAR antibody and stained with Coomassie (A and B) or Ponceau S (C) for loading control. (A) The full-length 6xHis-tagged KDM4D is PARylated in vitro. (B) In vitro PARylation of 6xHis-tagged KDM4D^1-350aa protein only following long exposure time. (C) PARylation of the GST-tagged KDM4D^348-523aa protein. GST tag was used as a negative control. (D) DNA damage-dependent PARylation of EGFP-KDM4D in cells. U2OS-TetON-EGFP-NLS and U2OS-TetON-EGFP-KDM4D cell lines were treated with etoposide or CPT genotoxic agents, subjected to GFP-TARP pull down, and immunoblotted using PAR antibody (Upper). Next, the membrane was stripped and immunostained with GFP antibody (Lower). (E) PARP inhibition suppresses the DNA damage-dependent PARylation of EGFP-KDM4D in cells. As in D, except that the cells were grown in the presence of 20 µM PARPi for 3 h before etoposide treatment.

Fig. 5.

KDM4D PARylation is essential for its recruitment to sites of DNA damage. (A and B) Alanine substitution of R450, R451, R455, and E357 (KDM4D-4M) abrogates the recruitment of EGFP-KDM4D^313-523aa and full-length EGFP-KDM4D to laser-microirradiated sites. U2OS cells were transfected with expression vectors encoding either EGFP-KDM4D^313-523aa-4M (A, Upper), EGFP-KDM4D^313-523aa-WT (A, Lower), full-length EGFP-KDM4D-4M (B, Upper), or full-length EGFP-KDM4D-WT (B, Lower) and subjected to laser microirradiation (red arrow). Results shown are typical of three independent experiments and represent at least 30 different cells. (C) In vitro PARylation shows that PARylation of KDM4D-4M lost its ability to undergo PARylation, compared with the wild-type KDM4D protein. The reaction mixtures were either immunoblotted using PAR antibody (Upper) or stained with Coomassie (Lower).

Fig. 6.

KDM4D promotes cell survival and facilitates the DNA damage-induced phosphorylation of a subset of ATM substrates. (A) Nearly 85% KDM4D knockdown as confirmed using TaqMan real-time PCR. (B) Clonogenic assay shows that KDM4D depletion sensitizes cells to IR. Control and KDM4D-depleted cells were seeded at a density of 600 cells per 6-cm plate and exposed to increasing doses of IR. Plates were stained with crystal violet, and the numbers of surviving colonies were evaluated 12 d after irradiation. The averages of total colony numbers were done in triplicate and are representative of two independent experiments. The numbers of colonies were normalized to the percentage of undamaged cells and plotted as a function of IR dosage. Error bars represent the SEM in two independent experiments. (C–E) KDM4D knockdown impairs the phosphorylation of H2AX-S139, KAP1-S824, and Chk2-T68 but has no detectable effect on the phosphorylation of ATM-S1981 protein. Control and KDM4D-depleted U2OS cells were exposed to IR (5 Gy), and protein extracts were prepared at the indicated time points using a hot-lysis procedure and subjected to Western blotting using antibodies against the indicated proteins. Next, the membranes were stripped and reacted with anti-H3, ATM, KAP1, and actin for loading controls. These results are representative of two independent experiments. (F) KDM4D depletion impairs the association of ATM with chromatin. Control and KDM4D siRNA-transfected U2OS cells were exposed to IR (5 Gy) and subjected to cellular fractionation. Chromatin-bound fractions were immunoblotted using the indicated antibodies. (G) As in F, except that the cells were exposed to either DMSO or etoposide before cellular fractionation. Results shown in F and G are representative of at least two independent experiments.

Fig. 7.

KDM4D depletion affects double-strand break repair. (A–C) KDM4D was depleted from U2OS cells using two different siRNA sequences. Control siRNA was used as a negative control. Cells were seeded in 96-well plates and treated with 40 μM etoposide for 20 min. The drug was then removed, and cells were fixed at the indicated time points after DNA damage and immunostained with γH2AX, Rad51, and 53BP1. A high-content screening microscope (In Cell Analyzer 2000; GE Healthcare) was used for automatic acquisition of at least 500 cells at each time point. (A) The nuclear intensity of γH2AX was calculated using the IN Cell Analyzer Workstation 3.7. (B) The percentage of cells with more than three Rad51 foci. Results are typical of two independent experiments. Error bars show SDs from the mean. (C) The percentage of cells with more than three 53BP1 foci. (D) KDM4D depletion impairs HDR of DSBs induced by I-Sce-I endonuclease. A decrease of 29–34% in the GFP-positive cells was observed following the depletion of KDM4D in HR-ind cells. Results shown are typical of three independent experiments. For positive control, ATM was inhibited using 20 μM KU-55933. Two independent experiments show that ATM inhibition leads to 61% reduction in the GFP-positive cells. Error bars represent the SD. (E) Flow-cytometric analysis shows that KDM4D depletion or ATM inhibition has no significant effect on cell-cycle distribution. (F) The defects in HDR in KDM4D-deficient cells were mended by expressing wild-type KDM4D, but not KDM4D-S200M or KDM4D-4M mutants. Control and KDM4D-siRNA transfected HR-ind cells were transfected with constructs expressing the indicated forms of KDM4D and treated with 0.1 μM Dex for 48 h, and the percentage of GFP-positive cells from the total number of red cells was determined by flow cytometry. Results shown are typical of two independent experiments. Error bars represent SD. (G) The 8-hydroxyquinoline (8-HQ) molecule inhibits the demethylase activity of KDM4D. U2OS-TetON cells expressing EGFP-KDM4D cells were treated with 50 μM 8-HQ inhibitor for 12 h, and protein extracts were prepared using a hot-lysis procedure and subjected for Western blotting using GFP, H3K9me3, and H3 antibodies. (H) Chemical inhibition of KDM4 proteins impairs HDR of DSBs in a dose-dependent manner of 8-HQ. U2OS-HR-ind cells were incubated with increasing concentrations of 8-HQ for 2 h and then treated with 0.1 μM Dex for 48 h to induce the formation of I-Sce1 DSBs. To evaluate the integrity of HDR, cells were harvested, and the percentages of GFP-positive cells were determined using flow-cytometric analysis. Results shown are typical of two independent experiments. Error bars represent SD. (I) KDM4D depletion disrupts the integrity of NHEJ in vivo. A decrease of 65% in the GFP-positive cells was observed following the depletion of KDM4D in HeLa cells containing the NHEJ reporter, pEJSSA. Control and KDM4D siRNA-treated cells were cotransfected with constructs expressing I-Sce-I endonuclease and Red-Monomer (MR) tag. To evaluate the integrity of NHEJ, cells were harvested, and the percentage of GFP-positive cells from the total number of red cells was determined by flow cytometry. Results shown are typical of two independent experiments. Error bars represent the SD. The P values were calculated using two-tailed paired tests, compared with control of each time point. *, **, and *** indicate significance at P < 0.05, 0.01, and 0.001, respectively.