Anti-Rad51 antibody - ChIP Grade (ab176458)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Add protease inhibitors to all lysis solutions before use.	-Use at an assay dependent concentration. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Upstream tips
-Add protease inhibitors to all lysis solutions before use.
Protocol tips
-Use at an assay dependent concentration. -Keep cells on ice between the rounds of homogenisations. - Increase or decrease the homogenization step to maximize the yield of nuclei depending on cell line.

Publication protocol

Nucleic Acids Research
Oxford University Press
CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks
Soon Young Hwang, Mi Ae Kang, [...], and Jong-Soo Lee

Additional article information

Associated Data
Supplementary Materials
ABSTRACT
The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

INTRODUCTION
DNA double-strand breaks (DSBs) represent the most damaging DNA injuries that can compromise genomic integrity and viability. DSBs can result from exogenous (UV, ionizing radiation and cytotoxic chemicals) and endogenous (cellular metabolites, reactive oxygen species and replication errors) insults. DSBs, if left unrepaired, can lead to fatal diseases, including cancer, growth and mental retardation, immune deficiency and developmental defects. To repair DSBs, eukaryotic cells employ mutually exclusive error-prone non-homologous end joining (NHEJ) or error-free homologous recombination (HR) repair. NHEJ involves ligation of the broken DNA ends and may create mutations, given that a homologous template is not available for repair. HR repair employs mostly homologous DNA in the sister chromatid as a template, which restores the correct DNA sequence. HR repair occurs predominantly during the S and G2 phases of the cell cycle, while NHEJ occurs throughout G1, S and G2.

The first control point for the DSB repair pathway occurs at the processing of the 5′ DNA end resection, which is catalyzed by MRE11 and CtIP (1,2). DNA end resection inversely influences the selection of the two major DSB repair pathways. Accordingly, extensive end resection suppresses NHEJ and permits HR repair (3). HR repair commences with the formation of extensive 3′-overhang single-stranded DNA (ssDNA), which requires the recruitment of MRE11 and CtIP at the DSB sites, facilitation of the nuclease activity of MRE11 controlled by CtIP, and involvement of the nucleases EXO1 and BLM/DNA2. Replication protein A (RPA) loads rapidly onto the resulting ssDNA and is simultaneously phosphorylated (3). Subsequently, the recombinase RAD51 displaces RPA in concert with BRCA1–BARD1, PALB2 and BRCA2 to form a helical nucleoprotein filament, thereby allowing homology search, strand invasion and sister chromatid exchange (4,5). Therefore, DNA end resection is a key step that controls the choice of the DSB repair pathway.

Although extensive studies have uncovered much about these critical steps in the regulation of DNA end resection and HR pathway choice between different DSB repair mechanisms, the process is complex and involves many additional proteins. Hence, how DSB repair proteins play a role in selecting the HR repair pathway within this exquisite network and how this process is controlled are largely underexplored.

Recently, a novel role of the multifunctional nuclear protein CCCTC-binding factor (CTCF) in HR-mediated DSB repair has been unveiled (6). CTCF is a transcription factor with 11 zinc finger (ZF) domains that function in many nuclear processes, including genomic organization, transcriptional regulation, insulator activity, VDJ recombination and HR-mediated repair. CTCF mutations in humans are linked to microcephaly and intellectual disability (7). In vivo evidence from CTCF knockout mice implicate CTCF as a haploinsufficient tumor suppressor (8), since heterozygous CTCF+/− mutations display greater susceptibility to irradiation-induced carcinogenesis, while homozygous CTCF−/− mutations result in embryonic lethality (8). Along with these mutation phenotypes, recent findings show that CTCF is recruited to damaged DNA sites and facilitates HR repair (9–11). Additionally, CTCF interacts with BRCA2 (10) and RAD51 (11), which are implicated in DSB repair by HR. Nevertheless, very little is known about the precise role of CTCF in HR and the key mechanism by which CTCF promotes HR.

In this study, we investigated the role of CTCF in HR-mediated DSB repair and its underlying mechanism. Via a proteomic approach, we identified MRE11 and CtIP as novel CTCF-interacting partners with a functional link to HR repair. We further corroborated that CTCF is recruited to DNA lesions in an MRE11-dependent manner, followed by CtIP recruitment, leading to DNA end resection at DSB sites and initiating HR. Consistent with these observations, CTCF depletion or truncation mutants are incapable of binding to CtIP sensitized cells to DNA damage. These data provide insights into a previously undescribed role of CTCF in promoting HR-mediated DNA repair by facilitating the formation of an initial HR complex for the onset of DNA end resection.

MATERIALS AND METHODS
Cells and reagents
HeLa, 293T and U2OS cell lines were purchased from ATCC, and these lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Kyung-san, Republic of Korea) with 10% FBS (Hyclone, GE Healthcare, Chicago, IL, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). U2OS-based DSB reporter cell lines, which stably express inducible AsiSI-ER (AsiSI-ER-U2OS; a generous gift from Dr. Gaëlle Legube, Universitéde Toulouse) (12) or inducible ER-mCherry-LacI-FokI-DD (FokI-U2OS; a generous gift from Dr. Roger Greenberg, University of Pennsylvania) (13) fusion protein, were grown in DMEM-containing puromycin. U2OS-based DR-GFP, EJ2-GFP, EJ5-GFP and SA-GFP cells (14) were kindly provided by Dr. Jeremy Stark (Beckman Research Institute of the City of Hope); they were maintained in DMEM without sodium pyruvate (Hyclone) containing 10% FBS and 1 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA). H1299.GC and H1299.EJ cell lines (15) were kindly provided by Dr. Hans Will and were cultured in DMEM-containing puromycin and G418, respectively. All cells were incubated at 37°C in a 5% CO2 incubator (BB15, Thermo Fisher Scientific).

Plasmid construction and transfection
GFP- and HA-tagged full-length and truncation mutants of CTCF and MRE11 were constructed by a classical PCR method using pEGFP-N1 (Addgene #6085-1) and pcDNA3-HA, respectively. The oligonucletoide sequences of the primers used for cloning are shown in Supplementary Table S1. All junction regions of CTCF and MRE11 constructs and the coding sequences of CTCF fragments were verified by DNA sequencing. Double-stranded short hairpin RNAs (shRNAs) targeting CTCF, MRE11 and CtIP were generated using pSUPER.retro.puro, an H1 promoter-driven RNA interference retroviral vector (Oligoengine, Seattle, WA). The sequences of the shRNAs are shown in Supplementary Table S2. Transfections were performed using Effectene Transfection Reagent (Qiagen, Carlsbad, CA) for expression in mammalian cells.

Antibodies
The CTCF antibodies were obtained from Abcam (ab128873, 1:2000 dilution for immunoblotting (IB) and 1:2000 for immunofluorescence (IF); ab70303, 2 μg for each chromatin immunoprecipitation (ChIP) sample), Cell Signaling Technology (#2899S, 1:1000 for immunoprecipitation (IP)) and Millipore (07-729, 2 μg for each ChIP sample). The other antibodies used for IB, IF, IP and ChIP analyses were as follows: HA (ab9110 from Abcam, 1:2000 for IB, ChIP, and IP), GFP (ab290 from Abcam, 1:2000 for IB and 1:1000 for IP), γH2AX (05-636 from Millipore, 1:2000 for IF and IB; ab2893 from Abcam, 2 μg for each ChIP sample), CtIP (61141 from Active Motif, 1:1000 for IF, IB, IP and ChIP), RPA (ab2175 from Abcam, 1:1000 for IF and 2 μg for ChIP), phospho-RPA (S4/S8) (A300-245A from Bethyl, 1:2000 for IB and IF), MRE11 (ab214 from Abcam, 1:2000 for IF, ChIP, IP and IB and 2 μg for ChIP; #4895S from Cell Signaling Technology for IP), NBS1 (ab32074 from Abcam, 1:2000 for IB), RAD50 (ab89 from Abcam, 1:2000 for IB and IP), Tubulin (05-829 from Millipore, 1:3000 for IB), RAD51 (ab176458 from Abcam, 2 μg for each ChIP sample), p53 (sc-126 from Santa Cruz, 1:2000 for IB), p21 (05-345 from Millipore, 1:2000 for IB), phospho-(serine/threonine) ATM/ATR substrate multi-monoclonal antibody (#6966S from Cell Signaling Technology, IB), BRCA1 (sc-642 from Santa Cruz, 1:1000 for IB), 53BP1 (NB100-305 from Novus, 1:2000 for IB) and β-actin (sc-47778 from Santa Cruz, 1:3000 for IB).

Tandem affinity purification (TAP) and mass spectrometry
The 293T cells transiently expressing S protein-Flag-Streptavidin binding peptide (SFB)-tagged CTCF (SFB-CTCF) were treated with or without γ-irradiation. The cells were lysed with TAP-NETN buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, pH 8.0). After removal of cell debris by centrifugation, the crude lysates were incubated with Streptavidin-sepharose beads (GE Healthcare, Chicago, IL, USA). The bead-bound proteins were washed and eluted with biotin (Sigma Aldrich) in TAP-NETN buffer. The eluates were then incubated with S-protein agarose beads (Millipore, Burlington, MA, USA). The S-protein bead-bound proteins were washed, separated by SDS-PAGE and then visualized by staining with Instant Blue (Expedeon, Heidelberg, Germany). For LC-MS/MS analyses, the gel lanes were sliced into different bands and processed as follows. Briefly, the acetylated protein bands were divided into 10-mm sections and digested in the gel with trypsin. The tryptic digests were separated by online reversed-phase chromatography using a Thermo Scientific EASY-nLC 1200 UHPLC equipped with an autosampler using a reversed-phase peptide trap Acclaim PepMap™ 100 (75-μm inner diameter, 2-cm length) and a reversed-phase analytical column PepMap™ RSLC C18 (75-μm inner diameter, 15-cm length, 3-μm particle size), both from Thermo Scientific. This was followed by electrospray ionization at a flow rate of 300 nl·min−1. The chromatography system was coupled in line with an Orbitrap Fusion Lumos Mass Spectrometer. The obtained spectra were screened against the UniProt human database using Proteome Discoverer Sorcerer 2.1 software with a SEQUEST-based search algorithm. The comparative analysis of proteins identified in this study was performed using Scaffold 4 Q+S. All raw mass spectrometry proteomics data obtained in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD014441.

Laser micro-irradiation
U2OS cells were plated onto glass-bottomed culture dishes (SPL Life Science, Korea) and transfected with the indicated plasmids and siRNA. The cells were presensitized with 10 μM 5-bromo-2′-deoxyuridine (BrdU, Sigma) for 24–30 h and then subjected to laser micro-irradiation using an Eclipse T1 & A1 confocal system (Nikon Instruments Inc., Melville, NY, USA) at 405 nm for 3 s (32 lines/s) in a 37°C chamber containing 5% CO2. After the laser treatment, the cells were subjected to live-cell imaging or were processed for IF.

Immunofluorescence
After the indicated treatments (micro-irradiation, etoposide, 4′-hydroxytamoxifen [4-OHT], or γ-irradiation), the cells were fixed with 3.7% formaldehyde and permeabilized with 0.5% Triton X-100. After blocking with 1% BSA, the cells were incubated with the indicated primary and secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma). Coverslips were mounted using Vectashield mounting medium (Vector Labs), and images were acquired using a confocal (LSM 710, Carl Zeiss, Oberkochen, Germany) or fluorescence microscope (Eclipse Ti-S, Nikon Instruments Inc.).

Immunoprecipitation
The 293T cells that had been pretreated with or without 10 or 20 μM ATM inhibitor (KU55933 from Santa Cruz) for 1 h and then treated with 50 μM etoposide or vehicle (dimethyl sulfoxide (DMSO)) were lysed with NETN buffer (0.1 mM dithiothreitol (DTT), 1% NP-40, 150 mM NaCl, 40 mM Tris-HCl, pH 8.0) containing protease inhibitors on ice for 10 min. The cell lysates were clarified by centrifugation and incubated with nuclease digestion buffer (0.25 M sucrose, 1.5 mM Tris-HCl [pH 7.4], 80 mM NaCl, 3 mM KCl, 7.5 mM NaCl, 1 mM CaCl2, 0.1 mM DTT, 20 U/ml micrococcal nuclease) containing protease inhibitor with or without 150 U/ml Benzonase® (Sigma) or 0.1 μg/μl ethidium bromide (Qbiogene) for 15 min at 37°C. After centrifugation, the chromatin-bound proteins were collected and incubated with antibody against the indicated proteins for 12 h at 4°C, and then with protein A beads for 1 h at 4°C. The beads were then washed with NETN buffer three times and analyzed by IB. For tagged-protein IP, 293T cells treated with etoposide or vehicle were lysed in IP buffer (40 mM Tris-HCl [pH 7.4], 150 mM NaCl, 2 mM MgCl2, 0.2% Nonidet P-40, 0.4% Triton X-100) containing protease inhibitors on ice for 30 min. The cell lysates were clarified by centrifugation and incubated with the indicated antibody for 12 h at 4°C, followed by incubation with protein A beads for 1 h at 4°C. The precipitates were rinsed with wash buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.2% Triton X-100) three times, and the bound proteins were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

ChIP assay
AsiSI-ER-U2OS cells (12) were treated with 300 nM 4-OHT (Sigma) for 4 h to induce AsiSI-ER nuclear localization and DSB generation by the AsiSI nuclease. FokI-U2OS reporter cells (13) expressing inducible ER-mCherry-LacI-FokI-DD were treated with 300 nM 4-OHT plus 1 μM Shield-I (Clontech) for 4 h to induce nuclear expression and stabilization of ER-mCherry-LacI-FokI-DD and DSB generation at the transgene-harboring Lac operator sequences by the FokI nuclease. Subsequently, ChIP was carried out using the EZ-ChIP Kit according to the manufacturer’s protocols (Millipore). The immunoprecipitated and input DNA were analyzed by quantitative PCR (qPCR) using the Rotor-Gene SYBR® Green PCR kit (Qiagen) on a Rotor-Gene Q system (Qiagen). The PCR conditions were an initial preincubation step at 95°C for 5 min followed by 45 cycles of 95°C for 5 s and 60°C for 30 s. The last amplification cycle was followed by a melting curve analysis to confirm the specificity of the PCR amplification. Relative IP values were calculated based on the threshold cycle (Ct) value using the 2−ΔΔCt method (16).

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