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. 2013 Jul 19;288(29):21376-21388.
doi: 10.1074/jbc.M113.491514. Epub 2013 Jun 6.

Kdm4b histone demethylase is a DNA damage response protein and confers a survival advantage following γ-irradiation

Leah C Young  1 , Darin W McDonald  1 , Michael J Hendzel  2
Affiliations

Kdm4b histone demethylase is a DNA damage response protein and confers a survival advantage following γ-irradiation

Leah C Young et al. J Biol Chem. .

Abstract

DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.

Keywords: Chromatin Regulation; Chromatin Remodeling; Chromatin Structure; DNA Damage Response; DNA Repair; Heterochromatin; Histone Methylation.

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Figures

FIGURE 1.
FIGURE 1.
Kdm4b- and KDM4D-EGFP recruit to DNA damage. U2OS osteosarcoma cells were transfected with Kdm4b-EGFP. 24 h after transfection 2-photon laser micro-irradiation was used to measure the recruitment of EGFP-tagged Kdm4 demethylases to DNA damage. A, both Kdm4b-EGFP and KDM4D-EGFP recruited readily to the DNA damage tracks. Graphs represent the mean values for 18, 30, 25, and 16 nuclei ± S.E. (Kdm4a–c, respectively). B, Kdm4a-EGFP also recruited to the DNA damage tracks but at later time points. C, representative images show the accumulation of Kdm4b-EGFP (C, top) and KDM4D-EGFP (C, bottom) on the DNA damage tracks.
FIGURE 2.
FIGURE 2.
The influence of demethylase activity on the recruitment of Kdm4b-EGFP to DNA damage. A, deletion of either the JmjN domain or the JmjC domain abolished Kdm4b-EGFP recruitment to the DNA damage tracks. Mutational inactivation of Kdm4b-EGFP (H189A mutation in the JmjC domain) resulted in at least an 80% reduction in recruitment. Graphs represent the mean values for at least 12 nuclei ± S.E. B and D, Kdm4b-EGFP recruited more readily to the DNA damage tracks in the Suv39H1/H3-double null MEFS (n = 25) than in the isogenic wild type control MEFs (n = 15). P, pre-irradiation. C and E, similarly, reduction of H3K9me2 levels through the inhibition of the G9a methyltransferase (n = 21) resulted in enhanced Kdm4b-EGFP recruitment to the DNA damage tracks, as compared with DMSO control cells (n = 18). F, shown is an example of the decreased H3K9me2 methylation observed after 3 days of treatment with 800 nm G9a inhibitor UNC0638. Mean decreases in H3K9me2 between experiments ranged from 60 to 70%.
FIGURE 3.
FIGURE 3.
Kdm4b-EGFP is dependent on PARP-1 for recruitment to DNA damage. U2OS cells were transiently transfected with Kdm4b-EGFP. 24 h after transfection two-photon micro-irradiation was used to determine the requirement of Kdm4b-EGFP on key DDR proteins for the recruitment to DNA damage. The recruitment of Kdm4b-EGFP to the DNA damage tracks was not affected by ATM inhibition (A) or DNA-PK inhibition (B). C, inhibition of PARP-1 resulted in a 30% decrease in Kdm4b-EGFP recruitment. Cells were treated for 1 h before micro-irradiation. Graphs represent the mean values for at least 18 nuclei ± S.E.
FIGURE 4.
FIGURE 4.
FRAP. FRAP was used to compare the chromatin binding of Kdm4b-EGFP both on and off the DNA damage tracks induced by 2-photon micro-irradiation (n = 10). Kdm4b-EGFP recovered significantly more rapidly on the DNA damage track (p < 0.001, curve-fitting analysis GraphPad Prism).
FIGURE 5.
FIGURE 5.
Kdm4-EGFP does not accumulate at IRIF. U2OS cells were plated on glass coverslips and transfected with EGFP-tagged Kdm4a (A), Kdm4b (B), Kdm4c (C), or KDM4D (D). 24 h after transfection, cells were treated with 2 Gy of γ-irradiation and fixed at 60 min post-irradiation. Cells were stained for γH2AX, and the nuclei were counterstained with DAPI. Line scans show the fluorescence intensity for the EGFP-tagged Kdm4 protein (gray line, curve area filled) and the Cy3-stained γH2AX (black line). The line scans shown were performed on the lines indicated in the Kdm4-EGFP panels (delineated by arrows).
FIGURE 6.
FIGURE 6.
ATM activation is proficient in Kdm4b-overexpressing cells. U2OS cells were plated on glass coverslips and transfected with Kdm4b-EGFP. 24 h after transfection the plated cells were exposed to 2 Gy γ-irradiation and then fixed at 30 and 60 min recovery. Indirect immunofluorescence was used to stain phosphorylated ATM. Nuclei were counterstained with DAPI. Images were obtained and processed to ensure quantitative micrographs. Exposure times were held constant, and the resulting images were scaled identically. B, γH2AX foci formation 5 min after 2 Gy γ-irradiation as a readout for ATM activation. Kdm4b-EGFP cells were proficient at the formation of ATM-dependent γH2AX foci. C, shown is quantification of γ-irradiation-induced pATM foci in Kdm4b-EGFP-positive and -negative cells. Overexpression of Kdm4b-EGFP resulted in no significant change in pATM foci numbers. n.s., not significant.
FIGURE 7.
FIGURE 7.
Kdm4b overexpression promotes DSB repair. A, U2OS cells were plated on glass coverslips and transfected with Kdm4b-EGFP. 24 h after transfection, the plated cells were treated with 2 Gy of γ-irradiation (γIR) and fixed at 1, 6, and 24 h post-irradiation. Cells were stained by immunofluorescence for γH2AX, and the nuclei were counterstained with DAPI. The numbers of γH2AX foci were counted after 1, 6, and 24 h recovery in both Kdm4b-EGFP-positive cells and neighboring untransfected (control) cells. The mean number of foci is indicated by the horizontal bar. Statistical differences were determined by using a two-tailed t test. n.s., not significant. B, levels of Kdm4b-EGFP were negatively correlated with the number of remaining γH2AX foci after 6 h recovery. Correlation was measured using one-tailed Pearson correlation calculation. The mean EGFP intensity was measured for individual masked nuclei using CellProfiler. C, two examples show the inverse correlation between Kdm4b-EGFP levels and γH2AX foci at 6 h post-irradiation. D, U2OS cells were plated onto glass coverslips and transfected with either wild type Kdm4b-EGFP or inactive Kdm4bH189A-EGFP. At 24 and 48 h post-transfection, coverslips were fixed and stained for γH2AX foci, and the nuclei were counterstained with DAPI. Foci numbers were counted for the transfected cells as well as the EGFP-negative control cells. Kdm4H189A-EGFP/48h had a significantly (p < 0.001) higher number of γH2AX foci than any other experimental condition (one way analysis of variance); all other comparisons did not show significant differences.
FIGURE 8.
FIGURE 8.
DNA damage-induced changes in H3K9 methylation. 24 h after transfection, U2OS cells expressing low level Kdm4b-EGFP demonstrated a complete loss of H3K9me3 (A) but not H3K9me2 (B). C, U2OS cells were plated on glass-bottom dishes and transfected with Kdm4b-EGFP. 24 h after transfection, DNA damage was induced in EGFP-positive cells using two-photon laser micro-irradiation. Ten minutes after micro-irradiation of the first cell, the plates were fixed and stained for H3K9me2 by immunofluorescence. Two examples are shown. D and E, U2OS plated on glass coverslips were treated with two Gy of γ-irradiation (γIR). Plated cells were fixed at denoted time-points after irradiation. Cells were stained by immunofluorescence for H3K9me3 (D) or H3K9me2 (E), and the nuclei were counterstained with DAPI. H3K9me2 levels were quantified for each nuclei (sample numbers are shown on the graph) using CellProfiler and expressed relative to the mean H3K9me3 levels of the non-irradiated control cells. The mean methylation level was compared with the non-irradiated control using an unpaired t test with Welch's correction for unequal variance. n.s., not significant. p > 0.05. ***, p < 0.0001.
FIGURE 9.
FIGURE 9.
Constitutively reduced H3K9 methylation reduced DSB repair. A, U2OS cells were pretreated with 800 nm concentrations of the G9a inhibitor (G9ai in DMSO) UNC0638 or DMSO alone (control) and plated onto glass coverslips. Three days after treatment initiation, the cells were exposed to 2 Gy of γ-irradiation. The cells were fixed at the time points indicated and stained for γH2AX and H3K9me2 by immunofluorescence. γH2AX foci and H3K9me2 levels were quantified by CellProfiler software. The mean foci number is denoted by the horizontal bar. Total numbers of nuclei assays are indicated on the graph. The number γH2AX foci were compared for each time point using analysis of variance with Bonferroni's correction for multiple comparisons. G9a inhibition resulted in a 63% reduction in H3K9me3 levels at the time of γ-irradiation (Fig. 2F). B, Suv39h1/h2 double null MEFs (DN) and isogenic wild type control MEFs (WT) were assayed for γH2AX foci numbers, as above. n.s., not significant.
FIGURE 10.
FIGURE 10.
Low level Kdm4b overexpression confers decreased sensitivity to γ-irradiation. A, stable U2OS/Kdm4b-EGFP cells were sorted for low levels of Kdm4b-EGFP expression. The resulting U2OS/Kdm4b-EGFPlow and parental U2OS cells were plated at low levels for the colony formation assay. 24 h after plating, the cells were irradiated and then were allowed to recover until the resulting colonies reached at least 50 cells. The plates were then stained with crystal violet to allow visualization of the colonies; both cell lines were plated and stained on the same day. The experiment was performed in triplicate. Colonies were counted and expressed relative to untreated controls. B, U2OS cells were treated with G9a inhibitor or DMSO (control). On day 2 of treatment the pretreated cells were plated for the colony formation assay, as above. C, the sensitivity of the Suv39h1/h2-dn and isogenic wild type MEFs was compared using the colony formation assay, as above γIR, γ-irradiation.
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