brca1  (Addgene inc)

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: A Domain organisation of the BRCA1-A complex. All components were used full-length except RAP80 265-413 . Rectangles: folded domains; lines: disordered regions; Dashed segments: untraceable in cryo-EM density. RAP80, grey segments: absent from expression construct. BRE, grey numbers: ancestral UEV domain boundaries . Ins-1, Ins-2: JAMM insertions 1 and 2. B BRCA1-A cleaves long polyUb chains more efficiently. Left: time course of K63 polyUb digestion by 5 nM BRCA1-A (Coomassie); lane E: enzyme control. Right: band intensities for each Ub species. Long chains (Ub 15+ ) are processed first; shorter species (Ub 2–6 ) are then digested in order of decreasing length. Log₂ time scale; time 0 (plotted at 2 –4 min) is denoted as (0). A.U., arbitrary units. C Design of a di-ubiquitin probe for metalloproteases. Top: native K63 isopeptide bond. Bottom: K63 diUbATA superposed on the JAMM active site. A His–His–Asp triad coordinates the catalytic zinc, which activates a water molecule for isopeptide bond hydrolysis . DiUb ATA replaces the water with a thiol warhead that chelates the zinc. ATA portions mimicking Gly76 and Lys63 coloured red and blue; carbons labelled with Greek letters. Residue numbering after BRCC36. D diUb ATA binds BRCA1-A. Co-elution by size-exclusion chromatography; SDS-PAGE of peak fractions shown (Coomassie). E diUb ATA inhibits diUb cleavage by BRCA1-A. Time course of K63-diUb cleavage ± diUbATA (anti-Ub western blot). F DiUb ATA can be polymerized using E1/E2/E3 combinations to generate chains alternating ATA warheads and cleavable isopeptide bonds. K63-linked probe chains were assembled using UBA1, UBC13:MMS2 and RNF8. G CryoEM structure of BRCA1-A bound to poly-Ub ATA chains. Sharpened cryo-EM density segmented by subunit (ABRAXAS, blue/dark blue; BRCC36, purple/mauve; BRE, gold; BABAM1, red; RAP80, green; unmodelled density, grey). Visible ubiquitin moieties: UbD (teal), UbP (light blue), Ub elbow (pink), Ub wrist (purple), UbP’ (cyan). The ABRAXAS’-finger (blue) is indicated. Ub elbow and Ub wrist density is weak in the global reconstruction but recovered by focused refinement. Ct and free Lys63 (white dot) indicated for each visible ubiquitin moiety. H Schematic of the structure. Subunits and ubiquitin moieties coloured as in ( G ) for one arm; second arm in grey. Primes denote subunits of the first arm. ABX: ABRAXAS.

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Cryo-EM Sample Prep, Expressing, Construct, Control, Ubiquitin Proteomics, Residue, Co-Elution Assay, Size-exclusion Chromatography, SDS Page, Western Blot

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: A The two active sites of BRCA1-A bind diUb ATA in different conformations. Sharpened cryo-EM density of the two active sites of BRCA1-A:polyUb ATA (State C + P) in equivalent orientations. Top: State C, catalytically competent. Bottom: State P, pre-catalytic; UbP’ is displaced from the active site. Subunits of the State P pentamer and its bound chain are denoted with primes (e.g., BRCC36’, UbP’). ABRAXAS’-finger residues 44–52 are disordered in State P. Colours: BRCC36, purple; BRCC36’, mauve; UbD, teal; UbP, light blue; UbP’, cyan; ABRAXAS, blue; BRE, gold; unmodelled Ins-2 density, grey. B State C and State P differ in the conformation of UbP. Cartoon representation of State C (top) and State P (bottom) in equivalent orientations. UbD and BRCC36 are similar in both states; UbP position differs. Colours as in ( A ). Ct: C-terminus. C Ins-1 positions both Ub moieties of the substrate and seals the reaction chamber. Active site in State C. BRCC36 Ins-1 (residues 80–93, pink) positions both ubiquitin moieties; Arg89 seals the reaction chamber. The ABRAXAS’-finger acts in trans, contacting BRCC36 Arg89 and the UbD basic patch (Arg42, Arg72, Arg74). BRCC36 Lys87, Asp88 and His133 position UbP via UbP Gln2 and Glu64. Key residues shown as sticks. Colours as in ( A ). D The ABRAXAS-finger coordinates with Ins-1 during substrate engagement. Superposition of apo (white) and polyUb ATA -bound BRCC36 (purple, State C). Ins-1 shifts downward to accommodate the C-terminus of UbD (teal), displacing Asp88 from the active site. The ABRAXAS’-finger (apo, light blue; State C, blue) stabilises this transition by engaging Arg89. Nt and Ct: amino and carboxyl termini of the ATA warhead. H₂O: catalytic water in apo structure. E The ABRAXAS-finger and Arg89 of BRCC36 are necessary for efficient catalysis. K63 polyUb chain cleavage assay with 5 nM wild-type BRCA1-A, BRCA1-A ABX3A or BRCA1-A BRCC36-R89A (37 °C, Coomassie). Lane E: enzyme control. O/N: overnight. F Quantification of chain cleavage kinetics in ( E ). Disappearance of Ub 15+ and peak maxima of Ub 2–6 are delayed by ~4 and ~7–8 log₂ time points for BRCA1-A ABX3A and BRCA1-A BRCC36-R89A respectively, corresponding to ~16-fold and ~128–256-fold reductions in catalytic efficiency. Ub 15+ quantified from gel region indicated by red bar. Log₂ time scale; time 0 (plotted at 2 –4 min) is denoted as (0); overnight approximated to 1024 min. A.u., arbitrary units.

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Cryo-EM Sample Prep, Ubiquitin Proteomics, Cleavage Assay, Control

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: A In state P, UbP is displaced towards BRE and interacts with Ins-2. Sharpened cryo-EM density and fitted model of the State P active site in BRCA1-A:polyUb ATA (State C + P). BRCC36’ (mauve) and UbD’ (teal) retain the State C conformation, but UbP’ (cyan) is displaced towards BRE (gold), pulling the ATA probe (gold, ball representation) out of the active site. Unmodelled Ins-2 density (grey) overlays UbP’ and the UbP’:BRE interface. B State P is likely cleavage incompetent. Space-filling representation of the State P active site viewed along the UbD C-terminus axis. Displacement of UbP’ relative to state C exposes the ATA thiol (mimicking the catalytic water) to bulk solvent, likely perturbing its activation despite correct active site alignment. Colours as in ( A ). C Active sites cannot be simultaneously in state C. Both active sites of BRCA1-A:PolyUb ATA (State C + P) are shown: BRCC36 (State C, right) and BRCC36’ (State P, left). If left active site were also in state C, its UbP’ (yellow transparent surface)would clash with the opposing UbP. Colours as in ( A ). D A longer Ins-2 is not necessary for catalysis. Chain cleavage assay comparing activity of BRCA1-A WT and BRCA1-A iso2 (assembled with BRCC36 isoform 2, with short Ins-2) on K63 polyUb (Coomassie). Lane E: enzyme control. O/N: overnight. E BRCA1-A activity is not influenced by BRCC36 isoform choice. Densitometry quantification of ( D ). No difference between BRCA1-A bearing BRCC36 isoform 1 (black) or isoform 2 (red). Ub10+ indicated by red bar. Log₂ time scale; time 0 (plotted at 2 -4 min) denoted as (0); overnight approximated to 1024 min. A.u., arbitrary units.

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Cryo-EM Sample Prep, Solvent, Activation Assay, Cleavage Assay, Activity Assay, Control

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: A Ub elbow binds via Ile44 face. Focused 3D classification and local refinement on particles from BRCA1-A:polyUb ATA (State C + P and double State P), enriching for Ub elbow density. Models for BRE and Ub fitted as rigid bodies. Sharpened cryo-EM density and cartoon of the Ub elbow (pink)–BRE (gold) interface; key interacting residues highlighted. EMDB: EMD-55052. B Ub wrist binds via an interface centred on G35. As in ( A ), focused on Ubwrist. Models real-space refined in Phenix . PDB: 9SMS; EMDB: EMD-55041. C The BRE wrist site accounts for most of the binding affinity of BRE for ubiquitin. SPR binding curves for monoUb association with immobilised GST-BRE, GST-BRE mut(wrist) or GST-BRE mut(wrist+elbow) . Robust K d fitting was not possible; GST-BRE mut(wrist+elbow) shows a greater binding defect than GST-BRE mut(wrist) . D Ubiquitin chains require at least six moieties to span the BRCA1-A arms. Modelled shortest-path chain connectivity (white ubiquitins) superimposed on the polyUb ATA State C + P structure (coloured). A minimum of six ubiquitins spans the wrist-to-active-site path in State P; seven are required in State C. Each chain engages the wrist and elbow of one arm and feeds into the active site of the opposite arm. Chain path indicated by dashed outlines. E Schematic representation of the structure in D. Each chain binds along one arm and feeds into the BRCC36 active site of the opposite arm. The chain bound to BRCC36’ (State P) is denoted with primes and engages BRE from the non-prime pentamer (State C), and vice versa. F BRCA1-A binds chains through avidity. SPR measurement of K d for wild-type BRCA1-A and BRCA1-A mut(elbow+wrist) binding to monoUb or K63 polyUb (immobilisation: anti-His antibody). K d ± Standard Error. The K d for polyUb is based on an estimated maximum chain concentration of 5 µM and represents a lower bound on affinity (true affinity may be higher). G Avidity mutants are impaired in catalysis. Time course of K63 polyUb cleavage by 1 nM BRCA1-A WT or BRCA1-A mut(elbow+wrist) (Coomassie). Lane E: enzyme control (separate gel, non-contiguous lanes). O/N: overnight. mut(e + w): mut(elbow+wrist). H BRCA1-A mut(elbow+wrist) displays a 2-4 fold catalysis defect. Densitometry quantification of ( G ). Peak maxima for BRCA1-A^mut(elbow+wrist)^ (red) are delayed by one to two log₂ time points relative to wild-type (black) for all species except Ub 2 , corresponding to a ~ 2 to 4-fold reduction in catalytic efficiency. All curves normalised to their respective minima and maxima. Log₂ time scale; time 0 (plotted at 2 –4 min) denoted as (0); overnight approximated to 1024 min.

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Cryo-EM Sample Prep, Binding Assay, Ubiquitin Proteomics, Concentration Assay, Control

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: A BRCA1-A adopts both open and closed conformations. CryoEM maps and fitted atomic models for the open (top) and closed (bottom) forms of BRCA1-A with indicated substrates. A closed conformation is detected in all datasets independent of ubiquitin chain type, except diUb ATA . BRE W353 Cα–Cα distances (Ub wrist interface) indicated for high-resolution models. Active site states and EMDB/PDB IDs indicated. “Apo”: intact but unoccupied active sites with Ub wrist density only. B Structure and cartoon representation of a closed conformation in State C*. Focused 3D classification of closed BRCA1-A:oligoUb ATA particles on BRCC36 and UbD; sharpened density and model of the best-resolved subclass shown. Both active sites contain UbD; one UbP adopts a State C-like conformation (State C*); the second UbP is disordered. EMD-55053; PDB: 9SO9. C Superposition of Open and Closed state reveals complex transition. Open BRCA1-A:polyUb ATA (State C + P, white) and closed BRCA1-A:oligoUb ATA (State C*, colour) superposed on BRE (red dot; backbone traces; ubiquitins omitted). Arm closure brings each BRCC36 active site closer to the opposing ABRAXAS, bends the coiled coil, and moves the BRE elbows inward. Dashed lines: disordered ABRAXAS regions in the closed form. D ABRAXAS controls BRCA1-A arm shape. Backbone trace of one BRCA1-A pentamer. The ABRAXAS MPN domain and C-terminal RAP80-binding region are rigidly connected via BRE. Upon engagement of the ABRAXAS-finger with the opposing BRCC36, movement of the ABRAXAS MPN pulls on the arm end and bends the coiled coil upwards. Two conserved prolines (yellow) act as hinges between the fore and aft halves of the BRCC36 coiled coil. E Arm closure shifts ubiquitin engagement at the elbow. Focused 3D classification of closed BRCA1-A:polyUb ATA on the left arm, enriching for Ub elbow occupancy. Additional density appears above the elbow (Ub supra-elbow ); Ub elbow shifts slightly forward (Supplementary Fig. ). EMD-55055. BRCA1-A subunits, UbP and UbD fitted from model in ( B ); Ub wrist and Ub elbow modelled using AlphaFold3 model (ModelArchive52: ma-yb0lm) as reference. F Arm closure advances the ubiquitin chain toward the opposing active site. Open (State C + P, grey) and closed (State C*, colour) BRCA1-A:polyUbATA superposed on BRE (red dot). Ubiquitins feeding into the State C/C* active site shown as solids; Lys63 of Ub elbow marked yellow. Arm closure displaces the chain toward the active site. W: Ub wrist ; E: Ub elbow ; P: UbP; D: UbD. G Closure brings Ub elbow within one ubiquitin length of UbP. Close-up of ( F ). BRCA1-A as surface; Ub elbow in cartoon (pink: open; magenta: closed); Lys63 as sticks. Ub bridge (white), manually placed to connect UbP to Ub elbow , shows three ubiquitin conformations with termini suitably positioned to confirm reachability upon closure.

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Ubiquitin Proteomics, Binding Assay

Journal: Nature Communications

Article Title: A ubiquitin chain-feeding mechanism for BRCA1-A

doi: 10.1038/s41467-026-75797-w

Figure Lengend Snippet: Proposed mechanism for processive polyUb cleavage by BRCA1-A. (1) Two polyUb chains are bound without slack: active site 1 (S1) is in State C and active site 2 (S2) in State P. Ubiquitin moieties are represented as circles, with positions indicated as W/w (Ubwrist), E/e (Ubelbow), P/p (UbP), D/d (UbD), or numbered by position relative to UbD. Moieties from chain A (bound to arm 1, feeding into S1) are labelled with uppercase letters and Roman numerals; moieties from chain B (bound to arm 2, feeding into S2) are labelled with lowercase letters and Arabic numerals. (2) UbD is cleaved from chain A at S1, releasing it from the active site but not from the arm due to avidity. (3) Arm closure enables UbP from chain B (p) to transition from State P to State C*, entering S2. Meanwhile, chain A re-engages S1, with its former UbP and Ub III now serving as UbD and UbP, respectively. (4) As the arms reopen, chain A and S1 return to State P. For S2 to re-enter State C, chain B — now one Ub too short — must dissociate from arm 2 and rebind further downstream. The complex returns to the initial configuration with active site states reversed. Repeated cycles of (2)–(4) enable processive cleavage. Slack in one or both chains would permit multiple cleavage cycles without requiring arm closure. Animated in Supplementary Movie .

Article Snippet: The BRCA1-A complex was expressed by simultaneously infecting SF9 cells (1 million cells/mL in ESF 921 medium, Expression Systems) with pFASTDual:ABRAXAS:BRCC36, pFASTDUal:BRE:His-3C-BABAM1 and NKI2.9:StrpII-3C-RAP80 260-413 , either wild type or mutants where appropriate.

Techniques: Ubiquitin Proteomics

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