JMJD6 modulates DNA damage response through downregulating H4K16ac independently of its enzymatic activity

Abstract

The initiation and transduction of DNA damage response (DDR) occur in the context of chromatin, and modifications as well as the structure of chromatin are crucial for DDR signaling. How the profound chromatin alterations are confined to DNA lesions by epigenetic factors remains largely unclear. Here, we discover that JMJD6, a Jumonji C domain-containing protein, is recruited to DNA double-strand breaks (DSBs) after microirradiation. JMJD6 controls the spreading of histone ubiquitination, as well as the subsequent accumulation of repair proteins and transcriptional silencing around DSBs, but does not regulate the initial DNA damage sensing. Furthermore, JMJD6 deficiency results in promotion of the efficiency of nonhomologous end joining (NHEJ) and homologous recombination (HR), rapid cell-cycle checkpoint recovery, and enhanced survival after irradiation. Regarding the mechanism involved, we demonstrate that JMJD6, independently of its catalytic activity, interacts with SIRT1 and recruits it to chromatin to downregulate H4K16ac around DSBs. Our study reveals JMJD6 as a modulator of the epigenome around DNA lesions, and adds to the understanding of the role of epigenetic factors in DNA damage response.

Fig. 1

JMJD6 is recruited to DSBs, and limits the spreading of histone ubiquitination around DSBs independently of its enzymatic activity. a EGFP-JMJD6 is recruited to DNA damage sites. EGFP-JMJD6 expression constructs were transfected into U2OS cells, and the localization of EGFP-JMJD6 was observed under a fluorescence microscope following laser microirradiation. Scale bar, 20 μm. b The endogenous JMJD6 is recruited to laser irradiated regions. Cell treated with microirradiation were subjected to immunofluorescent staining using anti-JMJD6 together with anti-γH2A.X. Scale bar, 20 μm. c JMJD6 overexpression does not affect the initial γH2A.X foci formation, but prevents the vanishment of those foci. U2OS cells transfected with FLAG-JMJD6 expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-γH2A.X at 1 and 8 h after irradiation, respectively. Scale bar, 20 μm. d JMJD6 overexpression does not alter MDC1 foci formation, but prevents the vanishment of those foci. Cells were transfected with FLAG-JMJD6 expression constructs, exposed to 10 Gy of IR, and immunostained for FLAG and MDC1 at the indicated time. Scale bar, 20 μm. e JMJD6 overexpression inhibits the spreading of histone ubiquitination in response to IR. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with FK2 antibodies. Scale bar, 20 μm. f The spreading of histone ubiquitination in response to IR is increased upon depletion of endogenous JMJD6. U2OS cells transfected with JMJD6 siRNAs or control siRNAs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using FK2 antibodies. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis using anti-JMJD6 and anti-GAPDH. g JMJD6 overexpression inhibits the spreading of RNF168 around DSBs. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated with 10 Gy of IR, and immunofluorescence assays were performed using anti-FLAG together with anti-RNF168. Scale bar, 20 μm. h JMJD6 knockdown increases the spreading of RNF168 around DSBs. Scale bar, 20 μm. The knockdown effect induced by JMJD6 specific siRNAs was examined by western blot analysis. For Fig. 1c–h, at least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p-value was determined by Student’s t-test. ****p < 0.0001, **p < 0.01, NS = not significant

Fig. 2

JMJD6 controls the accumulation of BRCA1 and 53BP1 as well as the transcriptional silencing around DSBs. a U2OS cells were synchronized by double thymidine treatment, and treated with IR at S phase. The cell cycle of the synchronized cells was detected by flow cytometry. b JMJD6 overexpression inhibits the accumulation of BRCA1 at DSBs in response to IR treatment. U2OS cells transfected with FLAG-JMJD6 or FLAG-mutant expression constructs were treated as shown in a, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with anti-BRCA1. Scale bar, 20 μm. c JMJD6 knockdown leads to increased formation of BRCA1 foci after IR treatment. Synchronized U2OS cells were infected by lentiviruses containing JMJD6 shRNA sequences and a GFP cassette, treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using antibodies against BRCA1. Scale bar, 20 μm. d JMJD6 or its enzymatic mutant overexpression inhibits the accumulation of 53BP1 at DSBs in response to IR treatment. Scale bar, 20 μm. e JMJD6 knockdown leads to increased formation of 53BP1 foci after IR treatment. Scale bar, 20 μm. For Fig. 2b-e, at least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p-value was determined by Student’s t-test. ****p < 0.0001, **p < 0.01. f JMJD6 depletion results in increased association of RNF168 and downstream repair factors with chromatin. U2OS cells transfected with control or JMJD6 specific siRNAs were treated with 10 Gy of IR. Then the nuclear soluble fraction (N) and chromatin-bound proteins (P) were extracted and subjected to western blot analysis using antibodies against the indicated proteins. Total cell lysates were also subjected to western blot analysis. g Depletion of JMJD6 allows 53BP1 to spread beyond its physiological boundaries. U2OS cells were transfected with indicated siRNAs for 72 h and immunostained with the indicated antibodies 1 h after 0.25 Gy of IR treatment. Scale bar, 20 μm. h JMJD6 knockdown leads to increased accumulation of 53BP1 but no change of γH2A.X level near the DSB. U2OS-DR-GFP cells stably expressing control or JMJD6 shRNAs were transfected with empty vector or HA-I-SceI expression constructs. ChIP assays were performed using IgG, anti-γH2A.X or anti-53BP1, and the final DNA exactions were amplified by quantitative real-time PCR using primers that cover the DNA sequences near the I-SceI site. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t test. *p < 0.05. i JMJD6 affects the accumulation of BRCA1 near the DSB. U2OS-DR-GFP cells transfected as indicated were treated with nocodazole; ChIP assays were performed using IgG or anti-BRCA1. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. ***p < 0.001, **p < 0.01. j Depletion of JMJD6 leads to unscheduled transcriptional silencing. U2OS cells transfected with the indicated siRNAs were treated with 0.25 Gy of IR, incubated in the presence of 5-ethinly uridin (5-EU) for the last 1 h, and then immunostained with anti-53BP1. The 5-EU incorporation to nascent mRNA was developed with Click-iT chemistry. Scale bar, 20 μm

Fig. 3

JMJD6 affects the efficiency of NHEJ and HR, as well as the cellular response to IR treatment. a Sketch map of NHEJ in U2OS-EJ5-GFP cells. b Depletion of JMJD6 leads to increased NHEJ efficiency. U2OS-EJ5-GFP cells were transfected with control or JMJD6 siRNAs, and 24 h later, HA-I-SceI expression constructs were transfected into these cells. The percentage of GFP+ cell was examined by FACS analysis 48 h after I-SceI transfection. The expression of JMJD6 and HA-I-SceI was determined by western blot analysis. c Overexpression of JMJD6 or its enzymatic mutant results in decreased NHEJ efficiency. U2OS-EJ5-GFP cells were transfected with empty vectors, FLAG-JMJD6 or FLAG-mutant expression constructs together with HA-I-SceI expression constructs, and 48 h later, the percentage of GFP+ cell was examined by FACS analysis. The expression of FLAG-JMJD6 and HA-I-SceI was determined by western blot analysis. d Sketch map of HR in U2OS-DR-GFP cells. e Depletion of JMJD6 leads to increased HR efficiency. f Overexpression of JMJD6 or its enzymatic mutant results in decreased HR efficiency. g JMJD6 depletion leads to a more rapid and efficient recovery from cell-cycle arrest after irradiation. U2OS cells stably expressing JMJD6 or control shRNAs were collected at indicated times after 2 Gy of IR treatment, and then subjected to propidium iodide staining and flow cytometry. h Cylindrical graphs presenting the change of percentage of G₂/M cells detected by flow cytometry in figure G. i JMJD6 knockdown allows increased cell survival after IR treatment. Cell survival after irradiation in control or JMJD6 knockdown cells was measured by colony formation. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05

Fig. 4

JMJD6 is required for the association of SIRT1 with chromatin. a Immunoaffinity purification of JMJD6-containing protein complexes. Cellular extracts from 293T cells expressing FLAG (vector) or FLAG-JMJD6 were immunopurified with anti-FLAG affinity columns and eluted with FLAG peptides. The eluates were resolved by SDS-PAGE and silver-stained. The protein bands were retrieved and analyzed by mass spectrometry. b JMJD6 interacts with SIRT1 in U2OS cells. Immunoprecipitation assays were performed with antibodies against the indicated proteins, followed by immunoblot analysis in U2OS cells. c JMJD6 interacts with SIRT1 in A549 cells. d The molecular detail of the interaction between JMJD6 and SIRT1. Whole-cell lysates from U2OS cells transfected with empty vector, FLAG-JMJD6, FLAG-mutant, or FLAG-JMJD6-N expression constructs were prepared, and immunoprecipitation was performed with anti-FLAG, followed by immunoblot analysis with indicated antibodies. e JMJD6 is required for the recruitment of SIRT1 to chromatin. The nuclear-soluble fraction (N) and chromatin-bound proteins (P) of U2OS cells, transfected with control or JMJD6-specific siRNAs, were extracted, and western blot analysis was performed using antibodies against the indicated proteins

Fig. 5

JMJD6 regulates the level of H4K16ac in cells. a Depletion of JMJD6 leads to increased H4K16ac level. Total proteins from U2OS cells transfected with the control or JMJD6 siRNAs were extracted, and western blot analysis was performed using antibodies against the indicated proteins. b JMJD6 knockdown does not increase the total level of methylation on H4R3 or H3R2. Total proteins from U2OS cells transfected with the control or JMJD6 siRNAs were extracted, and western blot analysis was performed using antibodies against the indicated proteins. c JMJD6 depletion does not change the mRNA levels of TIP60, MOF, or SIRT1. Total mRNA from U2OS cells transfected with indicated siRNAs was extracted, and quantitative real-time RT-PCR assays were performed. Each bar represents the mean ± S.D. for triplicate experiments. d JMJD6 knockdown does not change the protein level of TIP60, MOF, or SIRT1. e ChIP-seq density heatmaps of JMJD6 and H4K16ac around 6292 JMJD6-binding peaks in HeLa cells. f ChIP-seq profiling of JMJD6 and H4K16ac in HeLa cells over a 1112-bp window. g Knockdown of JMJD6 increases digestion by micrococcal nuclease. Nucleosomes from control or JMJD6-depleted U2OS cells were digested by micrococcal nuclease and then subjected to DNA gel electrophoresis

Fig. 6

JMJD6 modulates the H4K16ac level around DSBs. a Depletion of JMJD6 increases H4K16ac in U2OS cells after ionizing radiation. U2OS cells transfected with control or JMJD6 siRNAs were untreated or treated with 10 Gy of IR, and 1 h later, the cell lysates were extracted and subjected to immunoblot analysis using indicated antibodies. b JMJD6 is required for the recruitment of SIRT1 to chromatin after IR treatment. U2OS cells transfected with control or JMJD6 specific siRNAs were treated with 10 Gy of IR, and 1 h later, the nuclear-soluble fraction (N) and chromatin-bound proteins (P) of U2OS cells were extracted and subjected to western blot analysis using antibodies against the indicated proteins. c Knockdown of JMJD6 increases digestion by micrococcal nuclease in response to IR treatment. Control or JMJD6-depleted U2OS cells were treated with IR or not. The nucleosomes were digested by micrococcal nuclease and subjected to DNA gel electrophoresis. d JMJD6 overexpression leads to increased recruitment of SIRT1 and decreased level of H4K16ac around the DSB. U2OS-DR-GFP cells were transfected with indicated expression constructs. ChIP assays were performed using IgG, anti-SIRT1 or anti-H4K16ac, and the final DNA exactions were amplified by quantitative real-time PCR using primers that cover the DNA sequences around the I-SceI site. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. **p < 0.01, *p < 0.05. e JMJD6 knockdown results in decreased recruitment of SIRT1 and increased level of H4K16ac around the DSB. U2OS-DR-GFP cells stably expressing control or JMJD6 shRNAs were transfected with empty vector or HA-I-SceI expression constructs. ChIP assays were performed using indicated antibodies. Each bar represents the mean ± S.D. for triplicate experiments and the p-value was determined by Student’s t-test. *p < 0.05

Fig. 7

The impaired DDR mediated by JMJD6 overexpression is SIRT1- and BRD4 dependent. a SIRT1 knockdown counteracts the impaired 53BP1 foci formation in JMJD6-overexpressed cells. U2OS cells stably expressing shRNAs specific for SIRT1 or control shRNAs were transfected with FLAG-JMJD6 expression constructs, and immunofluorescence experiments were performed using anti-FLAG together with anti-53BP1 1 h after IR treatment. Scale bar, 20 μm. At least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p-value was determined by Student’s t- test. ****p < 0.0001. b Overexpression of FLAG-JMJD6-N inhibits the recruitment of 53BP1 to DSBs. U2OS cells transfected with FLAG-JMJD6-N expression constructs were treated with 10 Gy of IR, and 1 h later, immunofluorescence assays were performed using anti-FLAG together with anti-53BP1. Scale bar, 20 μm. *p < 0.05. c BRD4 is essential for the recruitment of JMJD6 to chromatin. U2OS cells transfected with control or BRD4 specific siRNAs were treated with or without IR. Chromatin-bound proteins or total proteins were extracted and then subjected to western blot analysis using indicated antibodies. d The impaired loading of 53BP1 in JMJD6-overexpressed cells is counteracted by BRD4 inhibition. U2OS cells transfected with FLAG-JMJD6 expression constructs were untreated or treated with JQ1, and 1 h after IR treatment, immunofluorescence experiments were performed using anti-FLAG together with anti-53BP1. Scale bar, 20 μm. At least 50 nuclei of FLAG-JMJD6 expressing cells or control cells (cells without FLAG-JMJD6 expressing) from triplicate experiments were used to quantify the number of foci, and the p-value was determined by Student’s t-test. ****p < 0.0001