Journal: Nucleic Acids Research

Article Title: CTCF primes repressive chromatin for rapid BRCA1/BARD1 recruitment to drive homologous recombination repair of DNA double-strand breaks

doi: 10.1093/nar/gkag715

Figure Lengend Snippet: CTCF is essential for recruitment of BRCA1 and BARD1 at DNA lesions. Recruitment of BRCA1 ( A ) and BARD1 ( B ) to mCherry-LacI-FokI-induced DSBs in the endogenous CTCF- (siCTCF), BARD1- (siBARD1), and BRCA1-depleted (siBRCA1) FokI-U2OS reporter cells, compared to the control cells (siCTL). The knockdown of CTCF by siRNA was confirmed by a Western blotting. Immunofluorescence was performed 4 h after induction of DSBs. The scale bar represents 10 μm. The plot represents the percentage of cells with BRCA1 (A) or BARD1 (B) focus located at the FokI-induced DSB site. Data are the means ± SD of at least three independent experiments. More than 100 cells were counted in each experiment. *** P ≤ .001 (See A for results using additional siRNAs #1–#3 targeting different regions of CTCF mRNA.). ( C, D ) CTCF- (siCTCF), BARD1- (siBARD1), and BRCA1-depleted (siBRCA1) U2OS cells, along with the control (siCTL), were treated with phleomycin and fixed after 1 h. Cells were stained with antibodies against BRCA1 and γH2AX (C) or BARD1 and γH2AX (D). The plot represents the number of BRCA1 (C) or BARD1 (D) foci per cell. Data are the means ± SD of at least three independent experiments. More than 100 cells were counted in each experiment. ** P ≤ .01; *** P ≤ .001 (See A for results using additional siRNAs #1–#3 targeting different regions of CTCF mRNA.). ( E ) ChIP-qPCR was performed with an antibody against BRCA1 (left) or BARD1 (right) in the CTCF-depleted (siCTCF) or control (siCTL) AsiSI-ER-U2OS cells, with (+) or without (−) induction of DSBs. PCR primers were designed to target two DSBs in transcribed intragenic (CCBL2 gene located at chromosome 1:89 458 296) and non-transcribed intergenic (at chromosome 1:110 319 090) regions. The fold enrichment values were relative to those of cells without induction of DSBs. Data are means ± SD of at least three independent experiments, and all qPCR reactions were performed in triplicate. ** P ≤ .01; *** P ≤ .001. ( F, G ) CTCF-depleted (siCTCF) or control (siCTL) U2OS cells were subjected to laser micro-irradiation. Cells were fixed at indicated times and stained with the indicated antibodies. The scale bar represents 10 μm. Bar graph represents the intensity of BRCA1 (F) or BARD1 (G) on the micro-irradiated strips relative to that in the control U2OS cells. Data are the means ± SD of at least three independent experiments. More than 30 cells were counted in each experiment. ** P ≤ .01; *** P ≤ .001.

Article Snippet: The other antibodies used for IB, IF, IP, and ChIP analyses were as follows: FLAG (F3165 from Sigma–Aldrich, 1:3000 for IB), HA (ab9110 from Abcam, 1:2000 for IB and IP), GFP (ab290 from Abcam, 1:2000), H2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 1 μg for each ChIP sample), Tubulin (05-829 from Millipore, 1:3000 for IB), BRCA1 (sc-6954 from Santa Cruz, 1:500 for IB and IF; A300-000A from Bethyl laboratories, 1:2000 for IF), BARD1 (GTX132094 from Genetex, 1:2000 for IF and IB; A300-263A from Bethyl laboratories, 1:1000 for IP), HP1α (05-689 from Millipore, 1:2000 for IB), HP1β (MAB3448 from Millipore, 1:2000 for IB), HP1γ (05-690 from Millipore, 1:1000 for IP and 1 μg for each ChIP sample; ab217999 from Abcam, 1:2000 for IF and IB), KAP1 (ab22553 from Abcam, 1:2000 for IF, IB, and IP; 1 μg for each ChIP sample), SUV39H1 (#39785 from Active motif, 1:1000 for IF and 1 μg for each ChIP sample), SETDB1 (ab107225 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), KDM5A (ab70892 from Abcam, 1:2000 for IF and IP, and 1 μg for each ChIP sample), KDM5B (H00010765-MO2 from Abnova, 1:2000 for IB and IF), KDM5C (ab34718 from Abcam, 1:2000 for IB and IF), H3K9me2 (07-441 from Millipore, 1 μg for each ChIP sample), H3K9me3 (ab8898 from Abcam, 1:2000 for IB and 1 μg for each ChIP sample; 05-1250 from Millipore, 1:2000 for IF), H3K4me0 (05-1341 from Millipore, 1 μg for each ChIP sample) H3K4me1 (ab8895 from Abcam, 1 μg for each ChIP sample), H3K4me2 (07-303 from Millipore, 1 μg for each ChIP sample), H3K4me3 (ab8580 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), pan-H3 (07-690 from Millipore, 1 μg for each ChIP sample), CtIP (61 141 from Active Motif, 1:1000 for IF), RNF168 (ab58063 from Abcam, 1:2000 for IF), SMARCAD1 (A301-593A from Bethyl laboratories, 1:2000 for IF), EXO1 (ab95012 from Abcam, 1:2000 for IF), DNA2 (ab96488 from Abcam, 1:2000 for IF), RAD51 (ABE257 from Millipore, 1:2000 for IF), and BRCA2 (05-666 from Millipore, 1:2000 for IF).

Techniques: Control, Knockdown, Western Blot, Immunofluorescence, Staining, ChIP-qPCR, Irradiation

Journal: Nucleic Acids Research

Article Title: CTCF primes repressive chromatin for rapid BRCA1/BARD1 recruitment to drive homologous recombination repair of DNA double-strand breaks

doi: 10.1093/nar/gkag715

Figure Lengend Snippet: CTCF-BARD1 interaction is required for CTCF-dependent rapid BRCA1/BARD1 recruitment to DNA lesions. ( A ) In the absence of DNA damage (−), CTCF interacts with BARD1 but has little interaction with BRCA1. Treatment with 50 μM etoposide for 1 h (+) increases CTCF interaction with BRCA1/BARD1. Co-IP assays were performed using the indicated antibodies against CTCF, BRCA1, and BARD1, and the indicated proteins were probed in the immunoprecipitates by IB. ( B ) Co-IP assays were performed with control IgG and anti-CTCF antibody using WCEs from 293Tcells treated with or without (−) 10 Gy of γ-irradiation and lysed at the indicated times (5, 20, 40, and 60 min) after radiation. IB analyses were performed with the indicated antibodies. The CTCF-BARD1 interaction strength in non-irradiated cells was set to 1, and the interaction strengths at each time point were normalized to the level of immunoprecipitated CTCF. ( C ) (Upper) U2OS cells were co-transfected with EGFP-tagged BARD1 and mRFP-tagged CTCF constructs and were subjected to laser micro-irradiation. The spatiotemporal dynamics of BARD1 and CTCF in live cells were monitored for up to 180 s using time-lapse microscopy. (Lower) U2OS cells were transfected with EGFP-tagged CTCF construct and processed as described in (Upper). Enlarged images for the areas marked with rectangles are shown in separate panels. Black arrow heads indicate micro-irradiation line. ( D, E ) Schematic representations of truncated mutants of CTCF (D) and BARD1 (E) used in this study. The fragments of CTCF and BARD1 that interact with each other are presented in black—N-terminal and zinc finger (ZF) domains of CTCF in panel (D); RING and Ankyrin domains of BARD1 in panel (E). The 293T cells were transfected with the indicated HA-tagged CTCF truncation constructs (D) or FLAG-tagged BARD1 truncation constructs (E). Cell lysates were immunoprecipitated with anti-BARD1 (D) or anti-CTCF (E) antibody, and IB analyses were performed. In the IB in panel (D), the FL, N-terminal, and ZF of CTCF co-immunoprecipitated with endogenous BARD1 (indicated by arrow). In the IB in panel (F), the FL, 1–424, 1–570, and 424R of BARD1 co-immunoprecipitated with endogenous CTCF (indicated by arrows). ( F ) In vitro interaction between CTCF and BARD1. The three FLAG-BARD1 fragmented proteins (amino acid residues, 1–424, 424–777, and 570–777 indicated as 1–424, 424R, and 570R, respectively) were immobilized on anti-FLAG M2-conjugated agarose beads and incubated with the three GST-CTCF fragmented proteins (N, ZF, and C). GST-CTCF fragment(s) bound to immobilized FLAG-BARD1 proteins were analyzed via IB with an anti-GST antibody (upper). Inputs represent 10% of the used GST-CTCF fragments. Equivalent amounts of FLAG-BARD1 fragments were used (lower). ( G ) Control (siCTL) or endogenous CTCF-depleted U2OS cells complemented by the indicated HA-tagged CTCF fragment constructs were subjected to laser micro-irradiation. Cells were fixed at 5 min after micro-irradiation and stained with the indicated antibodies. The scale bar represents 10 μm. Box plot represents the intensity of BARD1 on the micro-irradiated strips relative to that in the control U2OS cells. Data are the means ± SD of at least three independent experiments. More than 30 cells were counted in each experiment; * P ≤ .05; ** P ≤ .01; *** P ≤ .001 (See C for the recovery of recruitment of BRCA1 onto the micro-irradiated strips by introducing the BARD1-interactable CTCF fragments into CTCF-depleted cells.). ( H ) Control (siCTL) or endogenous CTCF-depleted U2OS cells complemented by the indicated EGFP fusion CTCF proteins were treated with phleomycin and processed for BARD1 immunofluorescence. The dot plot represents the number of BARD1 foci per cells. Data are the means ± SD of at least three independent experiments. More than 100 cells were counted in each experiment; * P ≤ .05; ** P ≤ .01; *** P ≤ .001 (See D for the recovery of BRCA1 foci by introducing the BARD1-interactable CTCF fragments into CTCF-depleted cells).

Article Snippet: The other antibodies used for IB, IF, IP, and ChIP analyses were as follows: FLAG (F3165 from Sigma–Aldrich, 1:3000 for IB), HA (ab9110 from Abcam, 1:2000 for IB and IP), GFP (ab290 from Abcam, 1:2000), H2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 1 μg for each ChIP sample), Tubulin (05-829 from Millipore, 1:3000 for IB), BRCA1 (sc-6954 from Santa Cruz, 1:500 for IB and IF; A300-000A from Bethyl laboratories, 1:2000 for IF), BARD1 (GTX132094 from Genetex, 1:2000 for IF and IB; A300-263A from Bethyl laboratories, 1:1000 for IP), HP1α (05-689 from Millipore, 1:2000 for IB), HP1β (MAB3448 from Millipore, 1:2000 for IB), HP1γ (05-690 from Millipore, 1:1000 for IP and 1 μg for each ChIP sample; ab217999 from Abcam, 1:2000 for IF and IB), KAP1 (ab22553 from Abcam, 1:2000 for IF, IB, and IP; 1 μg for each ChIP sample), SUV39H1 (#39785 from Active motif, 1:1000 for IF and 1 μg for each ChIP sample), SETDB1 (ab107225 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), KDM5A (ab70892 from Abcam, 1:2000 for IF and IP, and 1 μg for each ChIP sample), KDM5B (H00010765-MO2 from Abnova, 1:2000 for IB and IF), KDM5C (ab34718 from Abcam, 1:2000 for IB and IF), H3K9me2 (07-441 from Millipore, 1 μg for each ChIP sample), H3K9me3 (ab8898 from Abcam, 1:2000 for IB and 1 μg for each ChIP sample; 05-1250 from Millipore, 1:2000 for IF), H3K4me0 (05-1341 from Millipore, 1 μg for each ChIP sample) H3K4me1 (ab8895 from Abcam, 1 μg for each ChIP sample), H3K4me2 (07-303 from Millipore, 1 μg for each ChIP sample), H3K4me3 (ab8580 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), pan-H3 (07-690 from Millipore, 1 μg for each ChIP sample), CtIP (61 141 from Active Motif, 1:1000 for IF), RNF168 (ab58063 from Abcam, 1:2000 for IF), SMARCAD1 (A301-593A from Bethyl laboratories, 1:2000 for IF), EXO1 (ab95012 from Abcam, 1:2000 for IF), DNA2 (ab96488 from Abcam, 1:2000 for IF), RAD51 (ABE257 from Millipore, 1:2000 for IF), and BRCA2 (05-666 from Millipore, 1:2000 for IF).

Techniques: Co-Immunoprecipitation Assay, Control, Irradiation, Immunoprecipitation, Transfection, Construct, Time-lapse Microscopy, In Vitro, Incubation, Staining, Immunofluorescence

Journal: Nucleic Acids Research

Article Title: CTCF primes repressive chromatin for rapid BRCA1/BARD1 recruitment to drive homologous recombination repair of DNA double-strand breaks

doi: 10.1093/nar/gkag715

Figure Lengend Snippet: CTCF alone and in conjunction with HP1γ acts in rapid BARD1 recruitment onto DNA lesions. ( A ) HP1γ-depleted (siHP1γ), CTCF/HP1γ-codepleted (siCTCF + siHP1γ), or control (siCTL) U2OS cells were subjected to laser micro-irradiation. Cells were fixed at 5 min post-micro-irradiation and stained with the indicated antibodies. The scale bar represents 10 μm. (See also A and B for late BARD1 recruitment and rapid and late BRCA1 recruitment on micro-irradiated strips, respectively). ( B ) HP1γ-depleted (siHP1γ) or control (siCTL) U2OS cells were subjected to laser micro-irradiation without or with ectopic expression of the indicated HA-tagged FL or fragmented HP1γ. Cells were fixed and stained as described in panel (A). The scale bar represents 10 μm. The bar graph represents the intensity of BARD1 on the micro-irradiated strips relative to that in control U2OS cells. Data are the means ± SD of at least three independent experiments. The intensity of BARD1 on micro-irradiated strips in >30 cells were evaluated in each experiment. *** P ≤ .001, ns, not significant. ( C ) CTCF- and HP1γ-codepleted (siCTCF/siHP1γ) or control (siCTL) U2OS cells without or with ectopic expression of the indicated HA-tagged FL or fragmented CTCF were subjected to laser micro-irradiation. Cells were fixed and stained as described in panel (A). The scale bar represents 10 μm. Bar graph was plotted as described in panel (B). ( D ) CTCF-depleted (siCTCF) or control (siCTL) U2OS cells with endogenous BARD1 knockdown and ectopically expressing FLAG-tagged wild type (WT) or HP1-binding motif mutant of BARD1 (PEELI), together with the HA-tagged FL or fragmented CTCF (N, ZF, or C fragments), were subjected to laser micro-irradiation. Cells were fixed and stained as in panel (A). Key: The residues required for HP1 binding, PxVxI, were changed to PEELI, and thus the PEELI BARD1 mutant largely lacks the ability to bind the CSD of all HP1 subtypes (see Fig. ). ( E ) Bar graph was plotted as described in panel (B) by assessing the intensity of BARD1 on the micro-irradiated strips in >30 cells from panel (D). * P ≤ .05, ** P ≤ .01, *** P ≤ .001. ( F ) WCEs of endogenous BARD1-deficient 293T cells expressing FLAG-tagged WT or mutant (PEELI) BARD1 were immunoprecipitated with antibodies against CTCF and FLAG. The inputs and immunoprecipitates were analyzed via IB with the indicated antibodies. Note that the PEELI BARD1 mutant (D), which is unable to bind to HP1γ, marginally interacts with CTCF. ( G ) WCEs of HP1γ-depleted (siHP1γ) or control (siCTL) 293T cells, treated with or without etoposide, were immunoprecipitated with antibodies against CTCF and BARD1. Inputs and immunoprecipitates were analyzed via IB as described in panel (F). Note that HP1γ deprivation reduces BARD1 interaction with CTCF independent of the DNA damaging agent etoposide.

Article Snippet: The other antibodies used for IB, IF, IP, and ChIP analyses were as follows: FLAG (F3165 from Sigma–Aldrich, 1:3000 for IB), HA (ab9110 from Abcam, 1:2000 for IB and IP), GFP (ab290 from Abcam, 1:2000), H2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 1 μg for each ChIP sample), Tubulin (05-829 from Millipore, 1:3000 for IB), BRCA1 (sc-6954 from Santa Cruz, 1:500 for IB and IF; A300-000A from Bethyl laboratories, 1:2000 for IF), BARD1 (GTX132094 from Genetex, 1:2000 for IF and IB; A300-263A from Bethyl laboratories, 1:1000 for IP), HP1α (05-689 from Millipore, 1:2000 for IB), HP1β (MAB3448 from Millipore, 1:2000 for IB), HP1γ (05-690 from Millipore, 1:1000 for IP and 1 μg for each ChIP sample; ab217999 from Abcam, 1:2000 for IF and IB), KAP1 (ab22553 from Abcam, 1:2000 for IF, IB, and IP; 1 μg for each ChIP sample), SUV39H1 (#39785 from Active motif, 1:1000 for IF and 1 μg for each ChIP sample), SETDB1 (ab107225 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), KDM5A (ab70892 from Abcam, 1:2000 for IF and IP, and 1 μg for each ChIP sample), KDM5B (H00010765-MO2 from Abnova, 1:2000 for IB and IF), KDM5C (ab34718 from Abcam, 1:2000 for IB and IF), H3K9me2 (07-441 from Millipore, 1 μg for each ChIP sample), H3K9me3 (ab8898 from Abcam, 1:2000 for IB and 1 μg for each ChIP sample; 05-1250 from Millipore, 1:2000 for IF), H3K4me0 (05-1341 from Millipore, 1 μg for each ChIP sample) H3K4me1 (ab8895 from Abcam, 1 μg for each ChIP sample), H3K4me2 (07-303 from Millipore, 1 μg for each ChIP sample), H3K4me3 (ab8580 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), pan-H3 (07-690 from Millipore, 1 μg for each ChIP sample), CtIP (61 141 from Active Motif, 1:1000 for IF), RNF168 (ab58063 from Abcam, 1:2000 for IF), SMARCAD1 (A301-593A from Bethyl laboratories, 1:2000 for IF), EXO1 (ab95012 from Abcam, 1:2000 for IF), DNA2 (ab96488 from Abcam, 1:2000 for IF), RAD51 (ABE257 from Millipore, 1:2000 for IF), and BRCA2 (05-666 from Millipore, 1:2000 for IF).

Techniques: Control, Irradiation, Staining, Expressing, Knockdown, Binding Assay, Mutagenesis, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: CTCF primes repressive chromatin for rapid BRCA1/BARD1 recruitment to drive homologous recombination repair of DNA double-strand breaks

doi: 10.1093/nar/gkag715

Figure Lengend Snippet: CTCF facilitates transient H3K9 methylation to quickly accumulate BRCA1/BARD1 at DSBs. ( A ) Immunofluorescence microscopy of H3K9me3 was performed 4 h after induction of DSBs by ER-mCherry-lacI-FokI-DD in CTCF-depleted (siCTCF) and control (siCTL) FokI-U2OS cells. Scale bar represents 10 μm. Boxed areas are shown in higher magnification. The plot represents the relative foci intensity of cells positive for H3K9me3 colocalized at the mCherry-FokI focus. *** P ≤ .001. ( B ) CTCF-depleted (siCTCF) or control (siCTL) U2OS cells were subjected to laser micro-irradiation. Cells were fixed at the indicated time points and stained with the indicated antibodies. The scale bar represents 10 μm. The bar graph represents the intensity of H3K9me3 on the micro-irradiated strips relative to that in the control U2OS cells. Data are the means ± SD of at least three independent experiments. More than 30 cells were counted in each experiment. *** P ≤ .001. ( C ) ChIP-qPCR was performed with the indicated antibodies against γH2AX or H3K9me3 in AsiSI-ER-U2OS cells transfected with the control (siCTL) or CTCF siRNA (siCTCF), with (+) or without (−) induction of DSBs by AsiSI (AsiSI-ER). The fold-enrichment values were relative to those of cells without DSB induction on two chromosome 1 positions 89 458 296 (intragenic region; INTRA) and 110 319 090 (intergenic region; INTER). Data are presented as means ± SD of three independent experiments, and all qPCR reactions were performed in triplicate; *** P ≤ .001. ( D ) Similar to panel (B), with indicated antibodies against SETDB1, SUV39H1, and KAP1. The scale bar represents 10 μm. The bar graph represents the intensity of SETDB1, SUV39H1, or KAP1 on the micro-irradiated strips relative to that in control U2OS cells. Data are the means ± SD of at least three independent experiments. More than 30 cells were counted in each experiment. *** P ≤ .001. HP1γ-, SETDB1-, SUV39H1-, and KAP1 are essential for H3K9me3 accumulation ( E ) and rapid BRCA1/BARD1 recruitment ( F ) at DNA DSBs. HP1γ-, SETDB1-, SUV39H1-, and KAP1-depleted (siHP1γ, siSETDB1, siSUV39H1, and siKAP1, respectively) or control (siCTL) U2OS cells were fixed at 5 min post micro-irradiation and stained with the indicated antibodies. The scale bar represents 10 μm. Bar graph in panel (F) represents the intensity of BRCA1/BARD1 on the micro-irradiated strips relative to that in the control U2OS cells. Data are the means ± SD of at least three independent experiments. More than 30 cells were counted in each experiment. *** P ≤ .001.

Article Snippet: The other antibodies used for IB, IF, IP, and ChIP analyses were as follows: FLAG (F3165 from Sigma–Aldrich, 1:3000 for IB), HA (ab9110 from Abcam, 1:2000 for IB and IP), GFP (ab290 from Abcam, 1:2000), H2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 1 μg for each ChIP sample), Tubulin (05-829 from Millipore, 1:3000 for IB), BRCA1 (sc-6954 from Santa Cruz, 1:500 for IB and IF; A300-000A from Bethyl laboratories, 1:2000 for IF), BARD1 (GTX132094 from Genetex, 1:2000 for IF and IB; A300-263A from Bethyl laboratories, 1:1000 for IP), HP1α (05-689 from Millipore, 1:2000 for IB), HP1β (MAB3448 from Millipore, 1:2000 for IB), HP1γ (05-690 from Millipore, 1:1000 for IP and 1 μg for each ChIP sample; ab217999 from Abcam, 1:2000 for IF and IB), KAP1 (ab22553 from Abcam, 1:2000 for IF, IB, and IP; 1 μg for each ChIP sample), SUV39H1 (#39785 from Active motif, 1:1000 for IF and 1 μg for each ChIP sample), SETDB1 (ab107225 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), KDM5A (ab70892 from Abcam, 1:2000 for IF and IP, and 1 μg for each ChIP sample), KDM5B (H00010765-MO2 from Abnova, 1:2000 for IB and IF), KDM5C (ab34718 from Abcam, 1:2000 for IB and IF), H3K9me2 (07-441 from Millipore, 1 μg for each ChIP sample), H3K9me3 (ab8898 from Abcam, 1:2000 for IB and 1 μg for each ChIP sample; 05-1250 from Millipore, 1:2000 for IF), H3K4me0 (05-1341 from Millipore, 1 μg for each ChIP sample) H3K4me1 (ab8895 from Abcam, 1 μg for each ChIP sample), H3K4me2 (07-303 from Millipore, 1 μg for each ChIP sample), H3K4me3 (ab8580 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), pan-H3 (07-690 from Millipore, 1 μg for each ChIP sample), CtIP (61 141 from Active Motif, 1:1000 for IF), RNF168 (ab58063 from Abcam, 1:2000 for IF), SMARCAD1 (A301-593A from Bethyl laboratories, 1:2000 for IF), EXO1 (ab95012 from Abcam, 1:2000 for IF), DNA2 (ab96488 from Abcam, 1:2000 for IF), RAD51 (ABE257 from Millipore, 1:2000 for IF), and BRCA2 (05-666 from Millipore, 1:2000 for IF).

Techniques: Methylation, Immunofluorescence, Microscopy, Control, Irradiation, Staining, ChIP-qPCR, Transfection

Journal: Nucleic Acids Research

Article Title: CTCF primes repressive chromatin for rapid BRCA1/BARD1 recruitment to drive homologous recombination repair of DNA double-strand breaks

doi: 10.1093/nar/gkag715

Figure Lengend Snippet: CTCF-reliant rapid BRCA1/BARD1 translocation at DSBs facilitates EXO1/DNA2-mediated extensive DNA end resection. ( A ) Recruitment of SMARCAD1 and exonucleases EXO1 and DNA2 at DSBs depend on CTCF and the CTCF downstream factor HP1γ, which are required for rapid BARD1/BRCA1 localization at DSBs after micro-irradiation. U2OS cells lacking CTCF, HP1γ, BARD1, or BRCA1 following transfection with the indicated siRNAs were subjected to micro-irradiation. Cells were fixed at 5 min (SMARCAD1) or 10 min (EXO1 and DNA2) post micro-irradiation and stained with the indicated antibodies. The graph represents the percentage of cells with SMARCAD1, EXO1, or DNA2 assembled at the micro-irradiated DSBs. Data are presented as the means ± SD of three independent experiments. More than 30 cells were counted in each experiment; *** P ≤ .001. Quantification of ssDNA generated by 5′-end resection at two sites, BamHI-1 and BamHI-2, at chromosome position 1:89 458 296 in AsiSI-ER-U2OS cells ( B ) transfected with the indicated siRNAs or ( C ) pretreated with the indicated inhibitors for 2 h (short) or 12 h (long) prior to AsiSI-induced DNA DSBs. Data are the means ± SD of at least three independent experiments, and all qPCR reactions were performed in triplicate; ** P ≤ .01, *** P ≤ .001. ( D ) Recrutiment of RAD51 at DSBs depend on CTCF and the CTCF downdstream factors that are required for HR. U2OS cells lacking CTCF, HP1γ, SUV39H1, SETDB1, KAP1, KDM5A, SMARCAD1, EXO1/DNA2, BRCA1, or BRCA2 following transfection with the indicated siRNAs were subjected to microirradation. Cells were fixed at 20 min post-micro-irradation and stained with the indicated antibodies. The graph represents the percentage of cells with RAD51 assembled at the micro-irradiated DSBs. Data are presented as means ± SD of three independent experiments. More than 30 cells were counted in each experiment; *** P ≤ .001. ( E ) Upon DSB formation, CTCF governs the BRCA1/BARD1 recruitment cascade pathway. CTCF recruits repressive chromatin factors, HP1γ, SUV39H1/SETDB1, KAP1, and KDM5A, at DSB chromatin, allowing subsequent methylation of H3K9 and demethylation of H3K4me3, whereby BRCA1/BARD1 is promptly enriched. BRCA1/BARD1 promotes recruitment of SMARCAD1 at DSBs with a subsequent recruitment of EXO1/DNA2, which in turn facilitates EXO1/DNA2-mediated extensive resection.

Article Snippet: The other antibodies used for IB, IF, IP, and ChIP analyses were as follows: FLAG (F3165 from Sigma–Aldrich, 1:3000 for IB), HA (ab9110 from Abcam, 1:2000 for IB and IP), GFP (ab290 from Abcam, 1:2000), H2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 1 μg for each ChIP sample), Tubulin (05-829 from Millipore, 1:3000 for IB), BRCA1 (sc-6954 from Santa Cruz, 1:500 for IB and IF; A300-000A from Bethyl laboratories, 1:2000 for IF), BARD1 (GTX132094 from Genetex, 1:2000 for IF and IB; A300-263A from Bethyl laboratories, 1:1000 for IP), HP1α (05-689 from Millipore, 1:2000 for IB), HP1β (MAB3448 from Millipore, 1:2000 for IB), HP1γ (05-690 from Millipore, 1:1000 for IP and 1 μg for each ChIP sample; ab217999 from Abcam, 1:2000 for IF and IB), KAP1 (ab22553 from Abcam, 1:2000 for IF, IB, and IP; 1 μg for each ChIP sample), SUV39H1 (#39785 from Active motif, 1:1000 for IF and 1 μg for each ChIP sample), SETDB1 (ab107225 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), KDM5A (ab70892 from Abcam, 1:2000 for IF and IP, and 1 μg for each ChIP sample), KDM5B (H00010765-MO2 from Abnova, 1:2000 for IB and IF), KDM5C (ab34718 from Abcam, 1:2000 for IB and IF), H3K9me2 (07-441 from Millipore, 1 μg for each ChIP sample), H3K9me3 (ab8898 from Abcam, 1:2000 for IB and 1 μg for each ChIP sample; 05-1250 from Millipore, 1:2000 for IF), H3K4me0 (05-1341 from Millipore, 1 μg for each ChIP sample) H3K4me1 (ab8895 from Abcam, 1 μg for each ChIP sample), H3K4me2 (07-303 from Millipore, 1 μg for each ChIP sample), H3K4me3 (ab8580 from Abcam, 1:2000 for IB and IF, and 1 μg for each ChIP sample), pan-H3 (07-690 from Millipore, 1 μg for each ChIP sample), CtIP (61 141 from Active Motif, 1:1000 for IF), RNF168 (ab58063 from Abcam, 1:2000 for IF), SMARCAD1 (A301-593A from Bethyl laboratories, 1:2000 for IF), EXO1 (ab95012 from Abcam, 1:2000 for IF), DNA2 (ab96488 from Abcam, 1:2000 for IF), RAD51 (ABE257 from Millipore, 1:2000 for IF), and BRCA2 (05-666 from Millipore, 1:2000 for IF).

Techniques: Translocation Assay, Irradiation, Transfection, Staining, Generated, Methylation