trf2  (Novus Biologicals)

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Purification, Mobility Shift

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 promotes BLM-mediated unwinding of telomere R-loops. ( A ) (Top) Schematic of the oligo-based telomere R-loop unwinding assay. Telomere R-loop substrates were generated by hybridizing 32 P-labeled TERRA and two telomere DNA fragments (TDR2 and TDR3). TRF2 and/or RAP1 were pre-incubated with the R-loops and then BLM was added to the reaction, and the complex was resolved by 10% native polyacrylamide gel electrophoresis to monitor for R-loop unwinding. Displacement of the invading radiolabeled TERRA from R-loops indicates that R-loop unwinding. (Bottom) The TRF2–RAP1 complex promotes BLM-mediated unwinding of telomere R-loops. The effects of TRF2 alone (40, 80 nM) or in combination with RAP1 (20, 40, 80 nM) on the ability of BLM (20 nM) to unwind telomere R-loops were examined. 32 P-labeled TERRA and R-loops were resolved by native-PAGE and shown in lanes 1 and 2. ( B ) Quantification of BLM-mediated R-loop unwinding reactions in panel (A). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .9485); **** P < .0001. ( C ) The TRF2 basic domain is required for efficient unwinding of telomere R-loops. The effect of WT TRF2, TRF2 ΔB , TRF2 ΔB,L288R , and RAP1 to enhance BLM-mediated telomere R-loop unwinding was examined. The sizes of 32 P-labeled TERRA and R-loops were resolved by native-PAGE, as shown in lanes 1 and 2. ( D ) Quantification of BLM-mediated R-loop unwinding reactions in panel (C). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. **** P < .0001. ( E ) The TRF2–BLM interaction enhances telomere R-loop unwinding. The effect of TRF2–RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomere R-loops was tested as in Fig. . 32 P-labeled TERRA and R-loops were loaded as size markers (lanes 1 and 2) and resolved by native-PAGE. ( F ) Quantification of the percentages of unwound R-loops in panel (E) as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .98); **** P < .0001.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Mutagenesis

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: BLM preferentially releases TERRA over ssDNA from telomere D/R-loops. ( A ) Schematic of the assay used to measure how TRF2–RAP1 promotes BLM-mediated unwinding of RAD51/ssDNA and RAD51AP1/TERRA-generated telomeric D/R-loops. Telomere D/R-loops were generated by incubating RAD51 with IRDye-700-labeled telomere ssDNA (red), RAD51AP1 with IRDye-800-labeled TERRA (green), and telomere plasmids together as described in Fig. . Native plasmid-sized telomere D/R-loops were obtained after deproteinization and column purification. BLM with or without TRF2–RAP1 was then incubated with these D/R-loops, and ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. ( B ) BLM preferentially releases TERRA over ssDNA from telomere D/R-loop. BLM (20, 40, 80 nM) was tested for its ability to unwind telomere D/R-loops or TRF2–RAP1-bound D/R-loops. ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. The unwinding of telomere D/R-loops by BLM was enhanced by TRF2–RAP1. ( C ) Quantification of the amount of D- and R-loops relative to the negative control (no proteins, lane 1). Data were plotted as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. * P = .02282; ** P = .001278; *** P = .0007284; **** P < .0001. ( D ) The effects of TRF2–RAP1 on WT BLM, the helicase-dead BLM K695R or BLM mutants on D/R-loop unwinding were tested as in panel (B). In contrast to WT BLM, TRF2–RAP1 cannot enhance BLM ’s ability to unwind telomere D/R-loops. D/R-loop unwinding was analyzed by 1% agarose gels. ( E ) Quantification of the relative amounts of D-loops or R-loops to the control without proteins (lane 1) is shown as mean ± SD from three independent experiments. ANOVA test was used to evaluate statistical differences. ns: non-significant ( P = .15; .4147; .8026); ** P = .001193; *** P = .000158.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Plasmid Preparation, Purification, Incubation, Negative Control, Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1–BLM is required to resolve telomere R-loops in U2OS cells. ( A ) U2OS cells expressing TRF2 ΔB, L288R were treated with shControl, shBLM, or shTRF2. Immunofluorescence-FISH analysis of cells containing UTs (PNA telomere probe, red) co-localized with R-loops (S9.6 antibody, green) and DAPI-stained nuclei (blue). White arrow: co-localization of R-loops on UTs. U2OS cells expressing shBLM-resistant WT BLM cDNA and indicated BLM mutants were treated with shBLM, shTRF2, and TRF2 ΔB, L288R . IF-FISH analysis was performed to detect UT/R-loop co-localization. White arrow: co-localization of R-loops on UTs. ( C ) Quantification of data from Fig. and , showing the number of UT/R-loop colocalizations per U2OS cell. Data from three independent experiments is shown as mean ± SEM from minimum 200 nuclei per experiment. Statistical evaluation was performed by one-way ANOVA test. ns: non-significant ( P > .9999); ** P = .0032; .0035; .0062; .0092; .0052; **** P < .0001. ( D ) Model showing that TRF2–RAP1 inhibits telomere HDR by promoting BLM-mediated telomere R-loop removal. RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation. The TRF2–RAP1 complex and TRF2–BLM interaction are required to promote BLM helicase-mediated unwinding of telomere R-loops and then D-loops. The RAP1–TRF2–BLM complex represses HDR on telomeres by removing R-loops to inhibit telomere D-loop formation.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Schematic of the fusion-protein constructs. Top: full constructs contain a TetON doxycycline (DOX)-inducible promoter, a Myc-tagged-TRF1 fused to a chromatin modifier (here, KRAB), a T2A, and mCherry-NES (nuclear export signal). (B) Western blot showing expression of the constructs (anti-Myc antibody) and H3K9me3 global levels in U2OS after 3 days of DOX induction. (C) Representative chromatin immunoprecipitation of H3, H3K9me3, and HP1α at telomeres and ALU repeats in U2OS after 3 days of DOX induction (T-KRAB: TRF1-KRAB). (D) Quantification of (C). Data represent mean ± SEM of n = 3 independent biological replicates. (E) Representative images of IF-FISH assessing PML colocalization with telomeres (TTAGGG) in U2OS after 3 days of DOX induction. Scale bars, 10 μm. (F–I) Quantification of (E). (F) Number of telomere foci per cell. (G) Standard deviation of the telomere foci area within each cell. (H) Number of total (small and large) telomere-PML colocalizations (TPF). (I) percentage of cells with at least 5 APBs. (F–H) Data represent mean ± SEM of all cells analyzed over three biological replicates. n (number of cells analyzed) = 110 (WT), 115 (TRF1), and 113 (TRF1-KRAB). (I) Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (J) Representative C-circle assay in U2OS after 3 days of DOX induction. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates. (K) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in U2OS after 3 days of DOX induction. Scale bars, 10 μm. Right: quantification. Data represent mean ± SEM of 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (D and F–I), ordinary one-way ANOVA. For (J and K), Welch’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Construct, Western Blot, Expressing, Chromatin Immunoprecipitation, Standard Deviation

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Schematic of the fusion-protein constructs. Here, Myc-TRF1 is fused to HP1α V22M, a mutation that abrogates H3K9me3 binding. (B) Western blot showing the expression of the constructs (anti-Myc antibody) in U2OS after 3 days of DOX induction. (C)Representative chromatin immunoprecipitation of H3, H3K9me3, and HP1α at telomeres and ALU repeats in U2OS after 3 days of DOX induction (T-HP1α: TRF1-HP1α). (D) Quantification of (C). Data represent mean ± SEM of n = 3 independent biological replicates. (E) Representative images of IF-FISH assessing PML colocalization with telomeres (TTAGGG) in U2OS after 3 days of DOX induction. Scale bars, 10 μm. (F) Quantification of cells with at least 5 APBs from (E). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (G) Representative C-circle assay in U2OS after 3 days of DOX induction. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates. (H) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in U2OS after 3 days of DOX induction. Left: representative images. Scale bars, 10 μm. Right: quantification. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (I) Quantification from (E) of the standard deviation between telomeric foci area within each cell. Data represent mean ± SEM of all cells from 3 independent biological replicates. n (number of cells analyzed) = 110 (WT), 115 (TRF1), and 100 (TRF1-HP1α). (J) Representative image from experiment in (E) of a cell with ultra-bright APBs (left) and cells with a telomeric bridge (right) in U2OS + TRF1-HP1α. Scale bar: 10 μm. (K and L) Percentage of cells with ultra-bright APBs (K) and entanglements (L). Data represent mean ± SEM of 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (D, F, I, and K–L), ordinary one-way ANOVA. For (G and H), Welch’s t test. * p < 0.05, ** p < 0.01, and ** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Construct, Mutagenesis, Binding Assay, Western Blot, Expressing, Chromatin Immunoprecipitation, Standard Deviation

Journal: Cell reports

Article Title: Local heterochromatin enrichment promotes telomere clustering and PML nuclear body assembly at telomeres

doi: 10.1016/j.celrep.2026.117004

Figure Lengend Snippet: (A) Quantification of basal levels of telomere-PML colocalization (TPF) in HeLa ST and HeLa LT. Data represent mean ± SEM of 3 independent biological replicates. (B) Western blot showing siRNA-mediated knockdown of HP1α in HeLa LT control cells and expressing TRF1-KRAB (T-KRAB). (C) Western blot showing Myc (TRF1-KRAB), HP1α, H3K9me3, and actin in indicated cell lines. (D) Quantification of TPF in indicated cell lines. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (E) Representative images of IF-FISH assessing PML (IF) colocalization with telomeres (TTAGGG) in HeLa LT with indicated constructs after 3 days of DOX induction. Scale bars, 10 μm. (F) Quantification of TPF from (E). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (G) C-circle assay in HeLa LT after 3 days of DOX induction. Data represent mean ± SEM of n = 3 independent biological replicates. (H) G2 telomere synthesis: colocalization of TRF2 with G2-incorporated EdU in HeLa LT after 3 days of DOX induction. Scale bars, 10 μm. (I) Quantification of (H). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. (J) Representative image and quantification of entanglements in HeLa LT with indicated constructs. Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. Scale bar: 10 μm. (K) Representative images of IF-FISH assessing PML (IF) colocalization with telomeres (TTAGGG) in HeLa ST with indicated constructs after 3 days of DOX induction. Scale bars, 10 μm. (L and M) Quantification of TPF (L) and entanglements (M) from (K). Data represent mean ± SEM of n = 3 independent biological replicates, with at least 30 cells analyzed per replicate. Statistical analyses: for (B, D, F, I, and L), ordinary one-way ANOVA. For (G, J, and M), Welch’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. ns, non-significant. See also .

Article Snippet: Coverslips were stained for rabbit- anti -TRF2 (Novus, #NB110-57130 dilution 1:200) immunofluorescence as described above.

Techniques: Western Blot, Knockdown, Control, Expressing, Construct