Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: Assembly of PML bodies is essential for telomere synthesis in BRCA2-depleted ALT-like cells.( A ) Comparison of PML mRNA (left) and protein (right) levels in telomerase-positive (+ mTR ) and telomerase-negative (– mTR ) MEFs, in the presence or absence of Brca2 . Fibroblasts from BI or telomerase-null TBI mice were utilized to conditionally deplete Brca2 . Tamoxifen (4-OHT) treatment selectively depletes the Brca2 F11/F11 allele. The dot graph is the result of MEFs from three different mice each to avoid individual difference. RT–qPCR was conducted in two technical replicates (left) and western blot was performed in one sample from three different animals each (right, ). ( B ) Representative images of APB in BI and TBI fibroblasts, in the presence or absence of Brca2. PML, green; telomere, red. Enlarged images of APB from the white square are shown on the right. Scale bar, 5 μm. ( C ) The number and intensity of telomere foci from (B) were scored. Graphs are from three independent experiments. More than 100 cells were scored to assess telomere number in each experiment. More than 500 foci from 50 different cells each were scored per experiment to assess the intensity. ( D ) Fibroblasts were transfected with two different siRNAs ( siPML #3 and siPML #4) targeting PML , with or without Brca2 , then subjected to analysis of telomere synthesis in G 2 phase. (Left) Images representing the degree of EdU incorporation at telomeres in Brca2 and telomerase double knockout TBI fibroblasts. Fibroblasts were treated with 18 μM CDK1 inhibitor RO-3306 to arrest cells in G 2 phase, then subjected to assay of EdU incorporation at telomeres as described in the Materials and Methods. Enlarged images of EdU-positive telomeres in the control are shown on the left. Scale bar, 5 μm. (Right) Cells with more than three EdU-positive telomeres were scored. The graph is the result of three independent experiments (each dot corresponds to a single experiment). More than 100 BI (+ mTR ) and >200 TBI (– mTR ) fibroblasts were scored in each experiment. Western blot analysis to evaluate the efficiency of PML knockdown after siRNA transfection is shown at the bottom. * P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Comparison, Quantitative RT-PCR, Western Blot, Transfection, Double Knockout

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: LLPS plays a pivotal role in telomere synthesis of ALT-like MEFs.( A ) Illustration of TRF1-IDR -expressing constructs. The IDR domains from two different IDR-containing proteins, the N-terminus of FUS ( FUS N ) and the N-terminus of DDX4 ( DDX4 N ), respectively, were fused to the TRF1 expression construct. mCherry (mCh) fluorescence protein-expressing cDNA was inserted after TRF1 . ( B ) Representative time-lapse captured images of TRF1–FUS movements in wild-type fibroblasts. Telomere foci forming into droplets are marked with arrows (a, b) and the enlarged images are shown below. Scale bar, 10 μm. ( C ) Integrated fluorescence intensity of TRF1-IDR clusters in the nucleus per cell are shown as arbitrary units on the y -axis. More than 400 cells were scored. x/y, integrated intensity of TRF1 foci/mean intensity of mCherry in the nucleus. Black, TRF1–mCh; red, TRF1–FUS; pink, TRF1–DDX4. ( D ) Fixed images of telomere clustering after transduction of TRF1-IDR-expressing lentivirus to wild-type fibroblasts. Cells were fixed and subjected to T-FISH coupled with immunofluorescence with anti-mCherry antibody. Note the telomere clusters in TRF1–FUS- or TRF1–DDX4-expressing fibroblasts in enlarged images. The number of telomere foci decreased upon clustering in TRF1–FUS (average foci number, 76.7) or TRF1–DDX4 (77.2) expression, compared with control (TRF1–mCherry, 82.3). Telomere, green; mCherry, red. Scale bar, 5 μm. ( E and F ) TBI fibroblasts were transfected with siPML in the presence (+) or absence (–) of Brca2, followed by transduction with the indicated lentivirus: M, TRF1–mCh; F, TRF1–FUS; D, TRF1–DDX4. ( E ) Percentage of cells with >3 EdU-positive telomeres. The graph is the result of three independent experiments. More than 120 cells were scored in each set. ( F ) Comparison of telomere length. Telomere lengths were measured using T-FISH from metaphase chromosomes and are shown as arbitrary units of fluorescent intensity. More than 5000 telomere foci from > 50 metaphase spreads were scored in each experimental set. ( G and H ) U2OS cells were transfected with siLuc (control, +) or siPML (–), followed by transduction with the indicated lentivirus: M; F; D. ( G ) (Top) Representative images of EdU (pink) incorporation at telomeres (green) in G 2 phase of each experimental set are shown. (Bottom) Representative images of BLM (red) at telomeres (green) are shown. Scale bar, 5 μm. ( H ) (Left) EdU-positive telomeres in U2OS cells. More than 100 cells were counted in each experimental set. (Right) BLM-positive telomeres in U2OS cells. More than 120 cells were scored in each set. * P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Expressing, Construct, Fluorescence, Transduction, Immunofluorescence, Transfection, Comparison

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: Stabilization of telomere G4 is associated with the increase of TERRA-R-loops, which leads to the assembly of telomeric PML bodies and telomere synthesis. ( A–F ) Experiments were performed in BI and TBI fibroblasts. For conditional depletion of Brca2, fibroblasts were treated with tamoxifen (4-OHT) and the indicated characteristics were analyzed everyday (B-F). ( A ) Western blot analysis of Brca2 deletion and the creation of the Brca2 F11 allele upon 4-OHT treatment. ( B ) Assessing G4-positive telomeres. (Top) Representative images of G4 (green) and telomere (red) co-localization after 4 days of tamoxifen treatment. Fibroblasts were fixed and subjected to immunofluorescence with BG4 (anti-DNA G4 monoclonal antibody; green), followed by T-FISH with the TelC-PNA probe (red). Enlarged images of the white squares are shown on the right. (Bottom) Percentage of cells with more than three G4-positive telomeres is shown at the bottom. More than 100 cells each were scored in all sets. The graph is the result of three independent experiments. Scale bar, 5 μm. ( C ) Assessing TERRA RNA levels with TERRA-FISH and RNA dot-blot. (Top) TERRA RNA-FISH coupled with immunofluorescence assay. Immunofluorescence with anti-TRF1 antibody (red) was determined after 4 days of tamoxifen treatment, followed by TERRA RNA detection with TelC-PNA hybridization at 37°C (green). Scale bar, 5 μm. (Middle) Total RNA was extracted from fibroblasts after 4 days of Brca2 depletion, then dot-blotted. Radiolabeled TERRA and 18S rRNA probes were employed for hybridization. (Bottom) Quantification of TERRA RNA measured by RNA-FISH, shown as arbitrary units of fluorescent intensity. More than 500 TERRA foci from 40 cells each were scored. ( D ) Assessing R-loops at telomeres via immunofluorescence with S9.6 (anti-DNA:RNA hybrid antibody, green), coupled with T-FISH (red). Analysis was performed 4 days post-Brca2 depletion. Enlarged images from the white square are shown on the right. The graph represents the percentage of cells with >4 R-loop-positive telomeres. More than 120 cells each were scored. ( E ) Quantification of APBs per cell. More than 100 cells each were scored. ( F ) Telomere lengths were measured using T-FISH from metaphase spreads and are shown as arbitrary units of fluorescent intensity. More than 3000 telomere foci from > 50 metaphase chromosome spreads were measured. ( G ) Abundance of TERRA RNA levels in BJ fibroblasts and HeLa LT TERC KO cell lines with or without BRCA2. Both HeLa LT TERC KO and BJ cells were transfected with siLuc (+) or siBRCA2 (–) for 3 days prior to fixation. Representative images of TRF1 (green) and TERRA (red). Scale bar, 2.5 μm. (Right) RNA dot-blot hybridized with TERRA or 18S rRNA probes. All the graphs are from three independent experiments. * P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Western Blot, Immunofluorescence, Dot Blot, RNA Detection, Hybridization, Transfection

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: Telomeric R-loops drive LLPS formation. ( A–E ) Telomerase-positive BI and telomerase-negative TBI fibroblasts were transduced with lentivirus encoding RNase H1-GFP ( RNH1–GFP ). Fibroblasts expressing ectopic RNH1 were selected with puromycin. ( A ) (Top) Number of APBs per cell. More than 100 cells each were scored. (Bottom) Western blot analysis to assess deletion and creation of the Brca2 F11 allele after 4-OHT treatment. The effect of Brca2 depletion and/or RNH1–GFP overexpression was assessed. The same blot was re-probed with anti-PML and anti-POLD3 antibodies. ( B ) Representative fluorescent images showing APB in TBI fibroblasts with or without RNH1–GFP expression in the presence (+) or absence (–) of Brca2. PML, anti-PML immunofluorescence (green); telomere, T-FISH (red). Enlarged images of APBs showing the telomere clustering from the white square are shown below. Scale bar, 5 μm. ( C ) Effect of R-loop formation and LLPS in G 2 telomere synthesis. TBI fibroblasts expressing RNH1–GFP were transduced with lentivirus expressing TRF1–mCh (M), –FUS (F) and –DDX4 (D), respectively, then subjected to G 2 telomere synthesis assay. The graph showing the percentage of TBI cells with >3 EdU-positive telomeres is from three independent experiments. More than 100 cells each were scored. ( D ) Effect of G4 stabilization in telomere R-loop accumulation. BI or TBI fibroblasts, treated with 4-OHT (–) or left untreated (+) to deplete Brca2 , were exposed to 5 μM G4 stabilizer pyridostatin (PDS) for 2 h. Cells untreated with PDS were included as control. The percentage of cells with >5 R-loop-positive telomeres is marked. More than 120 cells each were scored. ( E ) Effect of PDS and/or RNH1 on APB formation. More than 100 cells each were counted. ( F ) HeLa LT TERC KO cells were transfected with siLuc (control) or siBRCA2 , and simultaneously transfected with mCherry - or RNH1–mCherry -expressing constructs. (Top) Representative images of R-loop (green) and telomere (red) co-localization. Representative images of PML (green) and telomere (red) co-localization. Enlarged images from the white arrow are shown. Scale bar, 2.5 μm. (Bottom) Quantification of R-loop-positive telomeres and APBs from images on the top. More than 120 cells each were scored. ( G ) Model for how the absence of BRCA2 instigates ALT-like telomere synthesis. Brca2 depletion provokes stabilization of G4, and subsequently increases TERRA-R-loops. R-loops trigger LLPS at telomeres, which contain the protein complex required for BIR . All graphs are the result of three independent experiments. * P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Transduction, Expressing, Western Blot, Over Expression, Immunofluorescence, Transfection, Construct

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: Telomeric R-loops are required for telomere H3K27 tri-methylation inside the liquid condensaste.( A ) (Left) BI (+ mTR ) and TBI (– mTR ) fibroblasts depleted of Brca2 (–) or left untouched (+) were subjected to ChIP against H3K27me3, H3K9me3 and histone H3, followed by hybridization with telomere probes. (Right) Relative H3K27me3 level at telomeres, normalized to histone H3. Four different BI and TBI MEFs, respectively, originating from different animals were employed in the analysis. ( B ) (Left) BJ fibroblasts transfected with siLuc (control) or siBRCA2 were subjected to ChIP against H3K27me3 and histone H3. (Right) Abundance of H3K27me3 at telomeres, normalized to H3. The graph is the result of three independent experiments. ( C ) RNH1 -expressing BI and TBI fibroblasts, in the presence or absence of Brca2, were subjected to ChIP against anti-H3K27me3 and -H3 antibodies, respectively, followed by hybridization with telomere probes. (Right) Level of H3K27me3 at telomeres, normalized to histone H3. The data represent three independent experiments. ( D and E ) BI and TBI fibroblasts were depleted of PRC2 core components EZH2, SUZ12 and EED proteins via siRNA transfection, in the presence (+) or absence (–) of Brca2. ( D ) Depleting the PRC2 complex abolished telomere LLPS. More than 100 cells each were counted. ( E ) Depletion of PRC2 markedly reduces telomeric R-loop generation. More than 100 cells each were counted. ( F ) Oligonucleotide pull-down assay to analyze the PRC2-recruiting region in telomere R-loops. Cell lysates were incubated with the indicated biotinylated oligonucleotides and subjected to western blot analysis with the indicated antibodies. RNA oligonucleotides (CCCUAA) 8 and (UUAGGG) 8 represent TERRA antisense and TERRA, respectively. The hybrid of (TTAGGG) 8 + (CCCTAA) 8 represents a double-stranded telomere, and (UUAGGG) 8 + (CCCTAA) 8 represents DNA:RNA hybrids (R-loops). (UUAGGG) 8 + (CCCTAA) 5 and (UUAGGG) 8 + (CCCTAA) 3 represent DNA:RNA hybrids with three and five repeats of exposed TERRA, respectively. (UUAGAG) 8 + (CCCTAA) 3 and (UUAGAA) 8 + (CCCTAA) 3 represent non-G4-forming sequences. ( G ) Illustration of the telomeric chromatin remodeling essential for ALT-like telomere synthesis inside the liquid condensate. TERRA RNA protruding from the R-loop recruits the PRC2 complex, which catalyzes H3K27me3 at telomeres. All results are from three independent experiments. * P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Methylation, Hybridization, Transfection, Expressing, Pull Down Assay, Incubation, Western Blot

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: BRCA2-deficient ALT-like cells exhibit selective sensitivity towards G4 stabilizer and EZH2 inhibitor. ( A ) Relative growth of BI and TBI fibroblasts, with or without Brca2, upon PDS (left) or EPZ-6438 (right, EZH2i) treatment. The numbers of cells counted were normalized to non-treated cells to compare the rate of cell growth. Black line with circle, wild-type control; blue line with square, BI and –Brca2; black dotted line with open circle, TBI; red dotted line with open square, TBI and –Brca2. ( B ) Relative growth of TBI fibroblasts, with or without Brca2, upon combination of 2.5 μM PDS and 1 μM EZH2i. Cells were counted every 2 days and were normalized to non-treated cells to compare the growth rate. Black line with circle, TBI + EZH2i; blue line with square, TBI + EZH2i + PDS; black dotted line with open circle, TBI and –Brca2 + EZH2i; red dotted line with open square, TBI and –Brca2 + EZH2i + PDS. ( C and D ) Cell viability analysis of HeLa LT TERC KO cell lines with and without BRCA2 upon PDS and/or EPZ-6438 (EZH2i) treatment. HeLa LT TERC KO cells were transfected with siLuc (control) or siBRCA2 for 24 h, then seeded in a 96-well plate and treated with the drug at the indicated concentrations. Three days later, cell viability was measured via MTT assay. ( C ) Graph showing the result of PDS treatment from 0 to 2.5 μM for 3 days. Black line with circle, siLuc; red dotted line with square, siBRCA2. ( D ) Graph showing the response to EZH2i and combined treatment with PDS and EZH2i in HeLa LT TERC KO cells, with or without BRCA2. Cells treated with 1 μM PDS were co-treated with 0–10 μM EPZ-6438. Black line with circle, control (siLuc + EZH2i); black dotted line with square, siLuc + EZH2i + PDS; red line with open circle, siBRCA2 + EZH2i; red dotted line with open square, siBRCA2 + EZH2i + PDS. The experiments were repeated three times independently. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Transfection, MTT Assay

Journal: Nucleic Acids Research

Article Title: Disruption of G-quadruplex dynamicity by BRCA2 abrogation instigates phase separation and break-induced replication at telomeres

doi: 10.1093/nar/gkae251

Figure Lengend Snippet: Clinical implication of the Brca2 deficiency and ALT development in breast cancer. ( A ) Approximately half of BRCA2 -mutated human breast cancers display APBs. Representative fluorescence image of APB-positive human breast cancer tissues. Immunofluorescence with anti-PML antibody combined with T-FISH was performed in paraffin-embedded tissue sections. White arrow, APB. Bar graph, the frequency of APB-positive breast cancer specimens. Breast cancers of wild-type BRCA2 (WT) and mutant BRCA2 (Mutation) are compared. Nineteen samples each were employed. x/y, number of APB-positive sections/total number of samples. Scale bars, 2 μm. ( B ) Heterogeneity in telomere lengths (coefficient of variation) was calculated as the standard deviation of telomere length divided by the average telomere length. ( C ) Increase of inflammation-associated gene expression upon Brca2 depletion. Relative mRNA levels of Interferon-α, -β, -γ and ISG15 , respectively, in telomerase-positive (+ mTR ) and telomerase-negative (– mTR ) MEFs were assessed in the presence or absence of Brca2 . The graph is the result of MEFs generated from three different animals. Two technical replicates were employed in every PCR. ( D ) Model of how BIR is triggered for telomere synthesis after BRCA2 abrogation. Disruption of BRCA2 stabilizes telomeric G4, which accumulates TERRA-containing R-loops at telomeres. Increased R-loops provoke telomere phase separation. At the same time, TERRA protruding from R-loops recruits PRC2 and marks telomeres with H3K27me3; BIR machineries are recruited due to the breakage and assembly of the liquid condensates facilitating telomere recombination. H3K27me3-marked telomeres are posed for conservative telomere synthesis, BIR. Student's t -test (mean ± SEM).

Article Snippet: The following antibodies were purchased: anti-PML (MAB3738; Merck Millipore), anti-PML [EPR16792] (ab179466; Abcam), anti-PML [PG-M3] (sc-966; Santa Cruz Biotechnology), anti-mCherry [1C51] (ab125096; Abcam), anti-mCherry (ab167453; Abcam), anti-DNA G-quadruplex structures [BG4] (MABE917; Merck Millipore), anti-DYKDDDDK Tag [D6W5B] (#14793; Cell Signaling Technology), anti-DNA–RNA hybrid [S9.6] (MABE1095; Merck Millipore), anti-POLD3 (21935–1-AP; Proteintech), anti-BLM (A300-110A; Bethyl Laboratories), anti-RNase H1 [N2C3] (GTX117624; GeneTex), anti-BRCA2 [AB-1] (OP95; Merck Millipore), anti-H3K27me3 [C36B11] (#9733; Cell Signaling Technology), anti-H3K9me3 (ab8898; Abcam), anti-histone H3 (ab1797; Abcam), anti-EZH2 [D2C9] (#5246; Cell Signaling Technology), anti-SUZ12 (ab12073; Abcam), anti-EED [EPR23043-5] (ab240650; Abcam), anti-vinculin (sc-73614; Santa-Cruz Biotechnology), and anti-beta actin (ab8226; Abcam).

Techniques: Fluorescence, Immunofluorescence, Mutagenesis, Standard Deviation, Expressing, Generated, Disruption

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: (A) WB analysis of siRNA BRCA2 in Flp-In cells. (B and C) Cytotoxicity of Flp-In WT or PBM1/2 cells transfected with siRNA control or BRCA2 and treated with increasing doses of CPT or olaparib, measured by ATP-dependent luminescence. At least n = 4; *p < 0.05; **p < 0.01; two-way ANOVA. (D) Model for the role of PAR-dependent TIM degradation in DNA replication and stalled fork protection. See for details.

Article Snippet: BRCA2 (Ab-1) , Millipore Sigma , Cat#OP-95; RRID:AB_2067762.

Techniques: Transfection, Control

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: BRCA2 (Ab-1) , Millipore Sigma , Cat#OP-95; RRID:AB_2067762.

Techniques: Control, Virus, Recombinant, TNKS1 Histone Ribosylation Assay, Transfection, Protease Inhibitor, In Situ, DNA Extraction, Mutagenesis, Cell Viability Assay, Staining, Negative Control, Software