ING3 is required for ATM signaling and DNA repair in response to DNA double strand breaks

Abstract

Inhibitor of Growth 3 (ING3) is a candidate tumor suppressor gene whose expression is lost in tumors such as hepatocellular carcinoma, head and neck squamous cell carcinoma and melanoma. In the present study, we show that ING3-depleted human cells and yeast cells deleted for its ortholog YNG2 are sensitive to DNA damage suggesting a conserved role in response to such stress. In human cells, ING3 is recruited to DNA double strand breaks and is required for ATM activation. Remarkably, in response to doxorubicin, ATM activation is dependent on ING3 but not on TIP60, whose recruitment to DNA breaks also depends on ING3. These events lead to ATM-mediated phosphorylation of NBS1 and the subsequent recruitment of RNF8, RNF168, 53BP1, and BRCA1, which are major mediators of the DNA damage response. Accordingly, upon genotoxic stress, DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR) were impaired in absence of ING3. Finally, immunoglobulin class switch recombination (CSR), a physiological mechanism requiring NHEJ repair, was impaired in the absence of ING3. Since deregulation of DNA double strand break repair is associated with genomic instability, we propose a novel function of ING3 as a caretaker tumor suppressor involved in the DNA damage signaling and repair.

Fig. 1

ING3 mediates resistance to DNA damage and cancer cell lines are dependent on ING3 for survival. (a) Wild type (WT) and mutant strains deleted for RAD9 (rad9Δ), YNG1 (yng1Δ), YNG2 (yng2Δ), or PHO23 (pho23Δ) were treated with various types of DNA damaging agents: 5 or 100 mM of hydroxyurea (HU), 0.5 μg/mL of bleocin (Bleo), or 10 Gy of ionizing radiation (IR). Plates were analyzed after 48 h. Summary table of the DNA damage sensitivity assay. (−) resistant, (+) to (+++) increased sensitivity, (++++) no survival. (b) Colony formation assay performed on U2OS cells transfected with siCT, siING3#1, siING3#2, or si53BP1 (53BP1 siRNA was used as a positive control) and treated with various type of DNA damaging agents: Cisplatin (CSP) (50 μM), CPT (10 nM), HU (2.5 mM), Mitomycin C (MMC) (100 ng/mL), MMS (0.5 μM), or Doxorubicin (Dox, 0.125 μM). Graph shows the number of colony formation; three independent experiment were performed. Bar graphs represent mean ± SEM (^NSP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001). c Distribution of ING genes CERES score in cancer cell lines (v.2018Q1 of Avana 1.0 library). d Mean CERES score of ING genes in cancer cell lines (***P < 0.0001; n = 391 per group)

Fig. 2

ING3 is recruited at DNA damage sites and is required for 53BP1 and BRCA1 recruitment. a Immunofluorescence analysis of cells transfected with Halo-ING3 or GFP-ING3 and damaged by laser micro-irradiation. U2OS or A549 cells were probed, respectively, for NBS1 or 53BP1. Scale bars represent 9 µM. b Immunofluorescence analysis of U2OS cells transfected with Halo-ING3 or not and subjected to laser micro-irradiation. Cells were fixed 5 min after laser micro-irradiation and probed for γH2AX and for 53BP1 or NBS1. Scale bars represent 8 µM. c Immunofluorescence analysis of U2OS cells transfected with Halo-ING3 and subjected to laser micro-irradiation. Fluorescence intensity was assessed on 30 nuclei for each time and represented on a graph. Scale bars represent 11 µM. d Immunofluorescence analysis of 53BP1 or BRCA1 in U2OS cells damaged by laser micro-irradiation. Cells were probed for 53BP1 or HA-BRCA1. Graph shows the intensity of 53BP1 or HA-BRCA1 recruitment at DNA DSBs, at least 30 cells were analyzed. Bar graphs represent mean ± SEM (****P < 0.0001). Scale bars represent 9 µM. Western blot analysis of U2OS cells transfected with siCT or siING3#1 or siING3#2, treated with Dox for 3 h and probed with indicated antibody. All bar graphs represent mean ± SEM (***P < 0.001)

Fig. 3

ING3 regulates ATM activation and signaling in response to DNA DSBs. a Immunofluorescence analysis of U2OS cells transfected with siCT or siING3 and then subjected to laser micro-irradiation, treated with doxorubicin for 3 h or exposed to X-rays. Cells were fixed 15 min after X-rays exposure. Cells were probed for p-ATM. Scale bars represent 8 µM. b Immunofluorescence analysis of U2OS cells transfected with siCT or siING3 and subjected to laser micro-irradiation. Cells were probed for p-ATM. Scale bars represent 10 µM. Graph shows p-ATM signal intensity and fluorescence intensity was assessed on 50 nuclei for each time and represented on a graph. Bar graphs represent mean ± SEM (****P < 0.0001). c Western blot analysis of A549 cells transfected with siCT, siING3, or siATM, treated with Dox for 3 h and probed with indicated antibody. d Immunofluorescence analysis of U2OS cells transfected with siCT or siING3 and subjected to doxorubicin treatment for 3 h. Cells were probed with p-ATM and Cyclin A or PCNA antibodies. Graph shows the number of p-ATM foci per nucleus in PCNA or Cyclin A positive and negative cells, at least 50 cells were analyzed. Scale bars represent 9 µM. Bar graphs represent mean ± SEM (****P < 0.0001). e U2OS cells were transfected with Halo-ING3, treated with an ATM inhibitor (ATMi, 10 μM) for 6 h and submitted to laser micro-irradiation. Cells were then probed for 53BP1. Scale bars represent 9 µM. Graph shows the intensity of Halo-ING3 recruitment at DNA DSBs, at least 30 cells were analyzed. Bar graphs represent mean ± SEM (^NSP > 0.05). Western blot analysis of U2OS cells treated ATMi and probed with indicated antibody

Fig. 4

ING3 is required for TIP60 accumulation at DNA DSBs. a Immunofluorescence analysis of U2OS cells co-transfected with GFP-TIP60 and siCT or siING3#1 or siING3#2, and then submitted to laser micro-irradiations. Graph shows the intensity of GFP-TIP60 recruitment at DNA DSBs, at least 30 cells were analyzed. Bar graphs represent mean ± SEM (****P < 0.0001). Scale bars represent 9 µM. b Immunofluorescence analysis of U2OS cells transfected with Halo-ING3 and siCT or siTIP60 and subjected to laser micro-irradiation. Cells were probed for Halo-ING3 and 53BP1. Scale bars represent 13 µM. Bar graphs represent mean ± SEM (****P < 0.0001). c Western blot analysis of Halo pull down on U2OS cells transfected with an empty Halo or Halo-ING3 and treated or not with Doxorubicin for 3 h. d Western blot analysis of U2OS cells transfected with siCT or siTIP60 or siING3#1, treated with Dox for 3 h and probed with indicated antibody. e Immunofluorescence analysis of U2OS cells transfected with siCT, siTIP60, or siING3 and treated with Dox for 3 h. Cells were probed for p-ATM. Scale bars represent 13 µM. Graph shows the number of p-ATM foci per nucleus, at least 50 cells were analyzed. Bar graphs represent mean ± SEM (****P < 0.0001)

Fig. 5

ING3 is required for DNA repair in response to DNA DSBs. a Immunofluorescence analysis of U2OS cells transfected with siCT or siING3, treated with Dox for 3 h. Cells were probed for γH2AX. Graph shows the number of γH2AX foci per nucleus, at least 50 cells were analyzed. Scale bars represent 12 µM. Bar graphs represent mean ± SEM (***P < 0.001; ****P < 0.0001). b Immunofluorescence analysis of U2OS cells transfected with siCT or siING3, treated with Dox for 3 h. Cells were probed for PCNA and γH2AX. Scale bars represent 12 µM. Graph shows the number of γH2AX foci per nucleus in PCNA positive and negative cells, at least 50 cells were analyzed. Bar graphs represent mean ± SEM (***P < 0.001; ****P < 0.0001)

Fig. 6

ING3 is required for NHEJ, HR, and class switch recombination. a U2OS cells were transfected with siCT, siING3#1, siING3#2, or si53BP1 and assessed for DNA repair by non-homologous end joining (NHEJ). Bar graphs represent mean ± SEM (*P < 0.05; ***P < 0.001). b U2OS cells were transfected with siCT, siING3#1, siING3#2, or with siBRCA1 and assessed for DNA repair by homologous recombination (HR). Bar graphs represent mean ± SEM (^NSP > 0.05; *P < 0.05). c qPCR analysis of CH12F3 cells transfected with GFP-miR-CT or four different GFP-miR-ING3. Bar graphs represent mean ± SEM (*P < 0.05; **P < 0.01). CSR to IgA in CH12F3 cells electroporated with GFP-miR-CT or GFP-miR-ING3#3 or GFP-miR-ING3#4 was measured by flow cytometry. The data are the means of three independent experiments. Bar graphs represent mean ± SEM (*P < 0.05; ***P < 0.001). d CH12F3 cells transfected with GFP-miR-CT or GFP-miR-ING3#4 were labeled with SNARF. Cells were harvested at the indicated time points following SNARF labeling and analyzed by flow cytometry. The data are representative of three independent experiments. e ATM recruitment and activation is a critical step in the DNA damage response and happens in the early stages of the pathway. Its auto-phosphorylation (on Ser 1981) is impaired when the cells are deficient for the ING3 protein in response to DNA damage, thus impacting the following recruitment of 53BP1 and BRCA1 at lesions sites and the DNA repair by NHEJ or HR