Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection

Abstract

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

Fig. 1.

Reduced repair of DNA breaks in INO80-depleted cells. (A) Seventy-two hours after ES cells were transfected with esiRNA against luciferase (mock) or Ino80 (siIno80), mRNA levels were assessed by RT-PCR. (B) Western blot of total extracts from ES cells transfected with esiRNAs against luciferase (mock) or Ino80 (siIno80) with an antibody against Ino80 3 days after transfection. (C) ES cells were transfected with esiRNA against Ino80 (siIno80) or luciferase (mock). Three days after transfection cells were irradiated with 20 Gy of ionizing radiation, left to recover for the indicated times, and subjected to alkaline comet assay. Representative images of comets are given for each time point. NI, not irradiated. (D) Tail moment of at least 100 comets was measured in each of two independent experiments; bars represent distribution of mean tail moments and error bars show standard deviation from the mean (SDM). (E) Cell cycle profiles of control and Ino80-depleted cells irradiated with 20 Gy of IR after different times of recovery.

Fig. 2.

Reduced HR repair rate in INO80-depleted cells. (A) Mock- and Ino80-silenced ES cells (72 h after transfection with esiRNA) were transfected with I-SceI-expressing plasmid (+I-SceI) or empty vector (−I-SceI) and analyzed by fluorescence-activated cell sorting (FACS) 48 h later. The GFP-positive population is surrounded by ellipses; data are means of 3 independent experiments, and error bars show SDM. (B) Repair efficiency of Ino80-depleted cells, relative to the control. (C) ES cells were mock silenced or Ino80 silenced and 72 h after esiRNA transfection were transfected with pEGFP-N3 plasmid. Forty-eight hours after plasmid transfection, the percentage of GFP-positive cells was assessed by flow cytometry and is given as the expression efficiency of the CMV promoter.

Fig. 3.

Association of human Ino80 with DSB. (A) Mouse 3T3 cells were irradiated with 20 Gy of IR and fractionated into Triton-soluble (soluble) and chromatin-bound (chr-bound) fractions. Fractions from control and irradiated cells were analyzed by Western blotting. (B) After densitometry, the relative amount of Ino80 was estimated. Data are means of two experiments, and error bars show SDM. (C) U20S-AsiSI-ER cells were treated with different concentrations of 4-OHT. Genomic DNA was extracted after 3 h of treatment, and the efficiency of AsiSI cutting was assessed by real-time PCR with primers spanning the recognition site on chromosome 1. NT, no treatment. (D) U20S-AsiSI-ER cells were treated with 1 μM 4-OHT for the times indicated, lysed, and subjected to ChIP with an antibody against Ino80. PCR was carried out with primers amplifying a region 500 bp away from the AsiSI recognition site. (E) ChIP was carried out as in D, and the amounts of precipitated DNA from the site 0.5 kb from the AsiSI site (white bars), from 10 kb away from that site (light gray bars), and from a region in the β-globin locus (dark bars) were analyzed by real-time PCR. The data from 2 experiments are shown as fold enrichment of Ino80 at the respective sites.

Fig. 4.

Repair focus formation in Ino80-silenced cells. Mock- or Ino80-silenced ES cells were irradiated with 8 Gy and 0.5, 1, or 2 h later were fixed and stained with an antibody against 53BP1. Representative images (A) and quantification of cells with foci (B) are shown (black bars, mock-silenced cells; gray bars, Ino80-silenced cells) of two independent experiments are shown ± SDM. (C) Number of foci per cell containing foci; at least 100 cells were counted for each point, and error bars represent SDM. (D) Control and Ino80-silenced cells were irradiated with 8 Gy, left to recover for the times indicated, and stained with an antibody against Rad51. (E) Cells with more than 5 foci were counted. The data are from 3 independent experiments. (F and G) Protein levels of 53BP1 and Rad51 in mock- and Ino80-silenced cells.

Fig. 5.

ssDNA formation in Ino80-silenced cells. (A) 3T3 cells were silenced for 72 h and labeled with 30 μg/ml BrdU during the last 36 h. Cells were irradiated with 20 Gy of IR, fixed, and stained at the indicated times after irradiation. (B) The percentage of BrdU-positive cells was estimated (black bars, control mock-silenced cells; gray bars, Ino80-silenced cells). Data are from 2 independent experiments, and error bars show SDM. (C) Western blot of total extracts from 3T3 cells transfected with esiRNAs against GFP (mock) or Ino80 (siIno80) with an antibody against Ino80 3 days after transfection. DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 6.

RPA focus formation in Ino80-silenced cells. (A) PC3 cells were silenced for 72 h with esiRNA against Ino80 or GFP as the control. Following irradiation with 8 Gy of IR, cells were fixed at indicated times and stained with an anti-RPA32 antibody. (B) The number of cells containing foci were counted. Results are from 3 experiments ± SDM. (C) Number of foci per cell containing foci. (D) Western blot of total extracts from PC3 cells transfected with esiRNAs against GFP (mock) or Ino80 (siIno80) with an antibody against Ino80 3 days after transfection.

Fig. 7.

H2AX phosphorylation in Ino80-silenced cells. PC3 cells were silenced for 72 h with esiRNA against Ino80 or GFP as the control. Following irradiation with 8 Gy of IR, cells were fixed at indicated times and stained with a γ-H2AX antibody.

Fig. 8.

RPA focus formation in Arp8-silenced cells. (A) PC3 cells were silenced for 72 h with esiRNA against Arp8 or GFP as the control. Following irradiation with 8 Gy of IR, cells were fixed at indicated times and stained with an anti-RPA32 antibody. (B) The number of cells containing foci were counted. Results are from 2 experiments. (C) Number of foci per cell containing foci. (D) RT-PCR to assess Arp8 mRNA levels in PC3 cells transfected with esiRNAs against GFP (mock) or Arp8 (siArp8) 3 days after transfection.