Research ArticleMOLECULAR BIOLOGY

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair

See allHide authors and affiliations

Science Advances  24 May 2017:
Vol. 3, no. 5, e1601898
DOI: 10.1126/sciadv.1601898
  • Fig. 1 CTCF localizes to DSBs via its zinc finger domain.

    (A) BrdU presensitized MCF7 cells were subjected to laser micro-irradiation using a 405-nm UV laser. Cells were fixed and stained with the indicated antibodies. (B) The U2OS-LacI-FokI-mCherry DSB reporter cell line was transfected with wild-type (WT) mCherry-FokI or an enzymatically inactivate mutant. After 48 hours, cells were fixed and stained with the indicated antibodies. (C) U2OS-LacI cells were cotransfected with HA-tagged full-length CTCF, or various deletions, along with mCherry-FokI. At 48 hours after transfection, cells were fixed and stained with an anti-HA antibody. (D) Schematic of CTCF fragments used to analyze recruitment to FokI cut sites. (E) Bar graph representing the percentage of cells positive for HA at mCherry-FokI foci. Data are means ± SD. (F) The U2OS-LacI-FokI-mCherry DSB reporter cell line was treated with 1 μM of the PARP inhibitor olaparib or MK4827 for 24 hours. Whole-cell extracts were prepared, and Western blotting was carried out using anti-PAR and β-actin antibodies. (G) Staining for BRCA2 and HA-CTCF in the U2OS-LacI-FokI-mCherry DSB reporter cell line before and after exposure to 1 μM PARP-1 inhibitors for 24 hours. (H) Bar graph representing the percentage of cells duo-positive for HA and mCherry-FokI foci or BRCA2 with mCherry-FokI foci. Data are means ± SD.

  • Fig. 2 CTCF knockdown leads to altered DNA damage repair kinetics.

    (A) Western blotting analyses for CTCF using lysates from three MCF10A clones with heterozygous deletion of CTCF (CTCF+/−) were subjected to immunoblotting with the indicated antibodies. Bar graph represents the quantification of CTCF signal. (B) MCF10A WT and three CTCF heterozygote (CTCF+/−) clone cell lines were fixed at the indicated times after irradiation (2 Gy) and stained with an anti-γH2AX antibody. (C) Quantification of the percent of cells with more than 10 γH2AX foci. Error bars correspond to means ± SEM (n = 3; ***P ≤ 0.005, ** P ≤ 0.01, χ2 test). (D) MCF7 cells were infected with Ctl shRNA or two constructs directed toward CTCF followed by irradiation (2 Gy). Cells were fixed at the indicated time point and stained with an anti-γH2AX antibody. (E) Quantification of percent of cells with more than eight γH2AX foci. Error bars correspond to means ± SEM (n = 3; **P ≤ 0.01, χ2 test). (F) MCF7 cells infected with scrambled control shRNA, or shRNAs directed toward CTCF, were subjected to immunoblotting with the indicated antibodies. (G) Comet assay was performed on MCF7 Ctl or CTCF-depleted cells following irradiation (5 Gy) at the indicated time points. (H) Quantification of the comet assay in percent of head DNA. Error bars correspond to means ± SEM [n = 3, ***P ≤ 0.005, one-way analysis of variance (ANOVA)]. Scale bars, 5 μm.

  • Fig. 3 Acute sensitivity to DNA-damaging agents and PARP inhibitors after loss of CTCF.

    (A to D) Clonogenic survival assay of MCF7 cells comparing CTCF knockdown cells with control cells after treatment with the indicated agents. Cells were grown for 14 days after treatment followed by colony counting and calculation of the survival fraction. Data are means ± SD. (E) Western blot shows CTCF expression in knockdown and control cells. (F) Flow cytometric analysis of phospho-Ser10 histone 3–positive MCF7 cells after irradiation in Ctl and CTCF knockdown cells. Numbers in boxes represent the percentage of cells in mitosis. A representation of three independent experiments is shown.

  • Fig. 4 CTCF regulates HR at I–Sce I– and CRISPR/Cas9-directed double-strand breaks.

    (A) Schematic representing the CRISPR-mClover assay for quantification of gene targeting by HR. (B) Quantification of mClover-positive MCF10A control or CTCF+/− cells by flow cytometry, representing the gene targeting efficiency at the LMNA locus. mClover expression was also quantified in CTCF add-back cells (+CTCF). Data are means ± SEM (n = 3; *P ≤ 0.05, one-way ANOVA). (C) Western blotting of CTCF for cells used in mClover experiments described in (B). (D) Schematic representing the DR-GFP assay for measuring gene targeting at I–Sce I cut sites by HR. (E) U2OS DR-GFP HR reporter cells were infected with control, CTCF, or RAD51 shRNAs and transfected with I–Sce I or an empty vector for 48 or 72 hours. The resultant cells were subjected to flow cytometric analysis for GFP-positive cells. (F) Quantification of data from experiment described in (A) depicted by bar graphs. Error bars correspond to means ± SEM (n = 3; *P ≤ 0.05, two-tailed Student’s t test). (G) Western blot for CTCF and Rad51 expression in cells used for experiments described in (E).

  • Fig. 5 CTCF modulates the recruitment of BRCA2 to FokI-induced DSB.

    (A) U2OS-LacI-FokI-mCherry DSB reporter cells infected with control or CTCF shRNA were treated with Shield-1 and 4-hydroxytamoxifen for 6 hours to induce FokI expression as per Materials and Methods. Cells were next fixed and immunostained with the indicated antibodies and DAPI. (B) Quantification of colocalization between mCherry-FokI and 53BP1, LIGIV, BRCA2, γH2A.X, and Rad51. Error bars correspond to means ± SEM (n = 3; *P ≤ 0.05, two-tailed Student’s t test). (C) U2OS Ctl or CTCF knockdown cells were treated with NCS (150 ng/ml) for 3 hours followed by fixation and staining with the indicated antibodies. Representative images are shown. (D) Quantification of Ctl or CTCF knockdown cells harboring five or more 53BP1, BRCA1, or RAD51 foci. Error bars correspond to means ± SEM (n = 3; *P ≤ 0.05, paired Student’s t test).

  • Fig. 6 CTCF association with BRCA2 is PARylation-dependent.

    (A) U2OS-LacI-FokI-mCherry DSB reporter cells with CTCF knockdown and reconstituted, full-length CTCF, CTCF deletions, or CTCFPARMUT (defective for PARylation) were probed for BRCA2 colocalization with FokI foci. (B) Histogram representing quantification of colocalization events between mCherry-FokI and BRCA2 for experiments depicted in (A). Error bars correspond to means ± SEM (n = 3; *P ≤ 0.05, two-tailed Student’s t test). (C) Western blot showing reconstitution of full-length CTCF after knockdown with an shRNA targeting the 3′ untranslated region of CTCF. (D) Co-IP showing HA-CTCF interaction with endogenous BRCA2 ± 24 hours of exposure to 5 μM of the PARP-1 inhibitor MK4827 followed by 5-Gy γ-irradiation or light treatment (100 J/m2). Lysates were collected 1 hour after damage. (E) Co-IP showing HA-CTCF or HA-CTCFPARMUT, with endogenous BRCA2 subjected to treatment with UV (100 J/m2).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1601898/DC1

    fig. S1. Live-cell imaging of CTCF at laser micro-irradiation tracks.

    fig. S2. CTCF association with PARylation increases as a response to DNA-damaging agents.

    fig. S3. Impact of CTCF loss on γH2AX and 53BP1 foci resolution.

    fig. S4. Loss of CTCF increases sensitivity to PARP inhibitors.

    fig. S5. Loss of CTCF impairs Rad51 foci formation following infrared.

    fig. S6. DNA damage increases the association between CTCF and BRCA2.

    table S1. sgRNA sequences targeting Cas9 to CTCF.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Live-cell imaging of CTCF at laser micro-irradiation tracks.
    • fig. S2. CTCF association with PARylation increases as a response to DNAdamaging
      agents.
    • fig. S3. Impact of CTCF loss on γH2AX and 53BP1 foci resolution.
    • fig. S4. Loss of CTCF increases sensitivity to PARP inhibitors.
    • fig. S5. Loss of CTCF impairs Rad51 foci formation following infrared.
    • fig. S6. DNA damage increases the association between CTCF and BRCA2.
    • table S1. sgRNA sequences targeting Cas9 to CTCF.

    Download PDF

    Files in this Data Supplement:

Navigate This Article