Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Biphasic TRF2 recruitment to non-telomeric damage sites in nuclei of HeLa cells. (A) PAR stimulation by PARG inhibition (PARGi) promotes GFP–TRF2 accumulation at low input-power damage sites (indicated by arrowheads). Box plot shows quantification of the relative increase of GFP signals at damage sites. (B) Time-course analysis of GFP–TRF2 recruitment to laser-induced DNA damage sites (between arrowheads). (C) Quantification of GFP signals at damage sites in B. N=16. (D) Detection of endogenous TRF2 at damage sites. PARP inhibition (PARPi) suppresses phase I, but has no effect on phase II, TRF2 recruitment. (E) Quantification of the effects of PARP inhibitors (NU1025 and olaparib) on immediate (1 min, phase I) and late (30 min, phase II) GFP–TRF2 recruitment. (F) Time course analysis of the effect of IDP depletion on dispersion of TRF2 at damage sites in HeLa cells transfected with control siRNA (siControl) or FET siRNAs (siFET). Left: quantification of signal intensity changes of GFP–TRF2 (blue) and dark line (red). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Inhibition, Dispersion, Transfection, Control

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Distinct TRF2 domain requirement for phase I and II recruitment. (A) Schematic diagrams of TRF2 deletion mutants. (B) Time course analysis of damage site localization (between arrowheads) of wild-type and TRF2 deletion mutants in the nuclei of HeLa cells. (C) Box plot shows quantification of TRF2 mutant GFP signals at damage sites at 1 min (phase I) and 30 min (phase II) post-damage induction. (D) Box plot shows quantification on the effects of the N-terminal amino acid substitutions on phase I recruitment of TRF2. Arginine-to-alanine mutations (RA), arginine-to-lysine substitution (RK) and the HJ binding mutation (H31A) were tested. WT, wild type. Amino acid sequences of N-terminal domain mutations are shown on the right. In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: Phase II recruitment is affected by TERT and is dependent on the iDDR region in the hinge domain of TRF2. (A) Box plot shows that TERT depletion using siRNA inhibits phase II recruitment of TRF2 to DNA damage sites in nuclei of HeLa cells. (B) Schematic diagrams of chimeric TRF1/2 mutants (as previously described in Okamoto et al., 2013) used in the experiments represented in panels C–F. (C) Left: representative cell images of the recruitment of chimeric mutants to damage sites (between arrowheads) at ∼1 min (phase I) and ∼30 min (phase II) after damage induction. Right: box plots show quantification of the GFP–TRF2 signal increase at phase I and phase II at damage sites. (D) Comparison of the GFP signal at damage sites in HeLa cells expressing iDDR and TIN2 deletion mutants at 30 min after damage induction. (E) The effect of MRE11 and NBS1 siRNA (siMRE11 and siNBS1) depletion on phase I and II recruitment of GFP–TRF2 was examined comparing to control siRNA (siControl). HeLa cells were fixed and stained with anti-MRE11 antibody (red) to confirm the depletion. Box plot shows quantification of the GFP–TRF2 signal increase at damage sites in control or MRE11 and NBS1 siRNA-treated cells. (F) ChIP-qPCR analysis of GFP–TRF2 binding at I-PpoI cut sites. TRF2 binding was examined in the absence or presence of I-PpoI, and with and without MMS as indicated. Cells were further treated with DMSO or PARP inhibitor (PARPi) (left panel). Cells expressing GFP only were used as a negative control. Alternatively, cells were transfected with control (siCon) or MRE11 and NBS1 siRNA in the presence of I-PpoI with or without MMS (right panel). In box plots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers represent the lowest datum still within 1.5× IQR (inter-quartile range) of the lower quartile, and the highest datum still within 1.5× IQR of the upper quartile. Bar graphs show mean±s.d., *P<0.01, **P<0.001, ***P<0.0001. Scale bars: 10 μm.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Comparison, Expressing, Control, Staining, ChIP-qPCR, Binding Assay, Negative Control, Transfection

Journal: Journal of Cell Science

Article Title: Biphasic recruitment of TRF2 to DNA damage sites promotes non-sister chromatid homologous recombination repair

doi: 10.1242/jcs.219311

Figure Lengend Snippet: TRF2 specifically promotes non-sister chromatid HR repair. (A) The effect of TRF2 depletion on DSB repair using the I-SceI HR system. Complementation analysis of TRF2-depleted cells was performed using the wild type and chimeric TRF1/2 mutants. BRCA1 depletion was used as a positive control. Comparable expression levels of the recombinant TRF2 proteins were confirmed using western blot analysis (right). Histone H3 serves as a loading control. (B) The effect of TRF2 depletion on different DSB repair pathways was examined using SCE, classic NHEJ (C-NHEJ) and alternative NHEJ (Alt-NHEJ) assays. (C) Schematic showing similarity between strand invasion in D-loop formation at telomeres and at DSB sites by TRF2. (D) Schematic showing biphasic mechanism of TRF2 recruitment to damage sites. Phase I involves PARP-dependent recruitment through the basic domain. Phase II is mediated by the MYB/SANT domain, which is also dependent on the iDDR region and the Mre11 complex. Bar graphs show mean±s.d.

Article Snippet: Mouse monoclonal antibodies specific for PAR polymers [BML-SA216–0100, Enzo Life Sciences; 1:500 dilution for immunofluorescence staining (IF)], TRF2 (NB100–56506, Novus Biologicals; 1:500 for IF), MRE11 [GTX70212, GeneTex; 1:500 for IF, 1:1000 for western blotting (WB)], GFP (632592, Takara Bio; 1:500 for ChIP), Actin (A4700, Sigma; 1:1000 for WB), FLAG (F3165, Sigma; 1:1000 for WB), and BRCA1 (GTX70111, GeneTex; 1:1000 for WB), as well as rabbit polyclonal antibodies specific for γH2AX (GTX628789, GeneTex; 1:500 for IF), EWSR1 (GTX114069, GeneTex; 1:1000 for WB), TAF15 (GTX103116, GeneTex; 1:1000 for WB), H3 (14-411, Upstate Bio; 1:1000 for WB) and Rad21 ( Kong et al., 2014 ; 1:2000 for WB) were used.

Techniques: Positive Control, Expressing, Recombinant, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: AURKB localization at telomere is linked to stem cell pluripotency. ( A ) AURKB localizes to the telomeres of mitotic mouse ES129.1 cells (arrowheads in (i)), but is lost in ES129.1 cells subjected to retinoic acid treatment differentiation (ii). Note that AURKB localization at pericentric heterochromatin is not lost in differentiated cells (arrows in Ai-ii). ( B ) AURKB localizes to the pericentric heterochromatin (arrows) but not to the telomeres of somatic, non-ESCs including mouse NIH3T3 (i) and human HT1080 (ii), telomerase-negative SKLU1 ALT cancer (iii) and telomerase overexpressing HT1080 (iv) cells. In mouse cells, TERF1 was used as a telomere marker. In human cells, TERF2 antibody was used as the telomere marker as the TERF1 antibody did not work in human cell types. Scalebars represent 5μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Marker

Journal: Nucleic Acids Research

Article Title: Aurora Kinase B, a novel regulator of TERF1 binding and telomeric integrity

doi: 10.1093/nar/gkx904

Figure Lengend Snippet: Loss of AURKB activity in ESCs results in the formation of MTS. ( A ) Examples of MTS (obtained with APH treatment) shown. ( B ) Representative metaphase images of untreated control mouse ES129.1 cells (i) and those treated with either 0.2 µM APH (ii) or 1 µM AURKB inhibitor ZM447439 (iii) for 24 h. TEL-FISH analyses indicated that 24 h of 1 µM ZM447439 treatment resulted in an increase in MTS formation from an average of 2.3 of MTS/metaphase in untreated control cells to 8.5 MTS/metaphase in ZM447439 treated cells ( P < 0.0001; N = 1000 chromosomes from three biological replicates), compared to an average of 7.3 MTS/metaphase in cells treated with 0.2 µM APH ( P < 0.0001; N = 1000 chromosomes from three biological replicates) (iv and v). ( C ) Western blot analyses of AURKB and actin in ES129.1 cells subjected to scramble control siRNA and siRNA depletion of TERF1, TERF2 and AURKB, respectively (i). Representative images of metaphase ES129.1 cells subjected to scramble control siRNA (ii; negative control), 72 h of AURKB (iii) and TERF1 siRNA depletion (iv), respectively. About 72 h of AURKB depletion resulted in aberrant MTS formation, increasing from an average of 2.3 MTS/metaphase in cells subjected to scramble control siRNA depletion to 5.1 MTS/metaphase in AURKB-depleted cells ( P = 0.0006, N = 1200 chromosomes from three biological replicates) (v and vi). As a comparison, 72 h of TERF1 siRNA depletion caused an average of 19.95 MTS/metaphase ( P < 0.0001; Cv and vi). Magnified images of the boxed chromosomes in B and C are shown in the inset, with examples of MTS indicated by the arrowheads. Each point in scatterplots (Biv and Cv) represents of the number of MTS in a single metaphase spread, with error bars showing Q1, Q2 and Q3 values. P -values are indicated in column graphs (Biv and Cv). Scalebars represent 5 μm.

Article Snippet: Primary antibodies used were as follows: rabbit polyclonal antisera against mouse TERF1 ( ); mouse monoclonal antisera against AURKB (BD Transduction Laboratories, #611082); mouse monoclonal antisera against GFP (Roche, #11814460001), rabbit polyclonal antisera against phosphorylated H3.3 serine 31 (Active Motif, #39637), mouse monoclonal antisera against TERF2 (Santa Cruz, #sc-47693) and rat monoclonal antisera against hemagglutinin (HA) tag (Roche, #11867423001).

Techniques: Activity Assay, Control, Western Blot, Negative Control, Comparison

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Representative images of immune biomarkers and TRF2 staining, and their cell detection mask overlays used in the digital image analysis. Original magnification, x 200

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Staining

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Correlative analysis between the clinical and histomolecular characteristics of the patients and the analyzed biomarkers in the metastatic melanoma cohort. *χ2-test, Student’s t-test or ANOVA test were used to investigate difference between groups.

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques: Mutagenesis

Journal: Oncoimmunology

Article Title: Association of TRF2 expression and myeloid-derived suppressor cells infiltration with clinical outcome of patients with cutaneous melanoma

doi: 10.1080/2162402X.2021.1901446

Figure Lengend Snippet: Multivariate analysis for overall survival in the cohort population

Article Snippet: Formalin-fixed paraffin-embedded (FFPE) serial 4 μm tissue sections were freshly cut, deparaffinized, pre-treated, and stained with monoclonal antibodies (Abs) directed against CD33 (clone SP266, ready-to-use, Roche, Tucson, AZ, USA), CD14 (clone EP128, dilution 1/200, Epitomics, Burlingame, CA, USA), CD15 (clone MMA, ready-to-use, Roche, Tucson, AZ, USA), and TRF2 (clone 4A794.15, dilution 1/500, OriGene, Rockville, MA, USA) on a BenchMark ULTRA autostainer (Ventana Medical Systems, Tucson, AZ, USA).

Techniques:

Journal: Molecular Biology of the Cell

Article Title: Telomeric DNA Mediates De Novo PML Body Formation

doi: 10.1091/mbc.E09-04-0309

Figure Lengend Snippet: Endogenous PML, Sp100 and Hausp accumulate at telomeric sites but not at centromeres in U2OS cells recovering from MMS treatment. (A) Immunofluorescence image of a U2OS cell treated with MMS, fixed and stained with anti-PML (green) and anti-TRF2 (red) antibodies. (B) Image of a U2OS cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). (C) Image of a HeLa cell that recovers from MMS treatment and is stained with antibodies against PML (green) and TRF2 (red). Arrows in B and C indicate the positions where PML colocalize or associate with TRF2 foci. (D) Localization of Sp100 at telomeric sites in a U2OS cell that recovers from MMS treatment. During recovery from MMS treatment, U2OS cells were fixed and stained with anti-Sp100 and anti-TRF2 antibodies. (E) Immunofluorescence image of a U2OS cell that recovers from MMS treatment. Sites where Hausp colocalize with telomeric DNA are indicated by arrows. (F) PML does not colocalize with centromeres in a U2OS cell that recovers from MMS treatment. After MMS treatment, U2OS cells were incubated in fresh medium, fixed and stained with anti-PML (green) and anti-CENPA (red) antibodies. All cell nuclei are counterstained with DAPI (blue).

Article Snippet: The following antibodies were used for immunofluorescence staining: mouse mAb 5E10 against PML (gift from R. van Driel, Amsterdam, The Netherlands), rabbit polyclonal antibody against PML (1130 directed against sequence: MEPAPARSPRPQQDP), rabbit polyclonal antibody against SP100 (ab1380, Chemicon, Temecula, CA), rabbit polyclonal antibody against Daxx (sc-7152, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibody against Hausp (A300–033A, Bethyl Laboratories, Montgomery, TX), mouse mAb against TRF1 (ab10579–50, Abcam, Cambridge, MA), mouse mAb against TRF2 (IMG-124, Imgenex, San Diego, CA), human autoimmune serum against centromeres (Antibodies Incorporated, Davis, CA), rabbit polyclonal antibody against γH2AX (A300–081A, Bethyl Laboratories), rabbit polyclonal antibody against 53BP1 (NB100–304, Novus Biologicals, Littleton, CO), and rabbit polyclonal antibody against SMC5 (A300–236A, Bethyl Laboratories).

Techniques: Immunofluorescence, Staining, Incubation

Journal: The EMBO Journal

Article Title: SMCHD 1 promotes ATM ‐dependent DNA damage signaling and repair of uncapped telomeres

doi: 10.15252/embj.2019102668

Figure Lengend Snippet: Loss of TRF2 leads to t‐loop unwinding. In wild‐type cells, SMCHD1 may remodel the telomeric chromatin which promotes ATM activation by the MRN complex (arrows). ATM activation is required for NHEJ at TRF2‐depleted telomeres. In SMCHD1 knockout cells, ATM activation and DNA damage signaling are attenuated resulting in inefficient 3′‐overhang processing and impaired telomere end‐to‐end fusions. SMCHD1 loss and lack of ATM activation can be compensated for by ATR (not depicted). Only the subset of events and factors are depicted which are directly relevant to this paper.

Article Snippet: Antibodies The following primary antibodies were used: TRF2 (#05‐521, Millipore, mouse, dilution 1:1,000, used for Western blots (WB)), γH2AX (Millipore, #05‐636, mouse, dilution 1:1,000, used for WB and IF), hnRNPA1 (4B10, #sc‐32301, Santa Cruz Biotechnology, mouse, dilution 1:3,000, used for WB), vinculin (#ab129002, Abcam, rabbit monoclonal [EPR8185], dilution 1:3,000, used for WB), 53BP1 (#NB100‐304, Novus Biologicals, rabbit, dilution 1:2,000, used for IF), phospho‐ATM‐Ser1981 (#ab81292, Abcam, rabbit, dilution 1:1,000, used for WB and IF), ATM (#ab32420, Abcam, rabbit monoclonal [Y170], dilution 1:1,000, used for WB), SMCHD1 (#A302‐871A, Bethyl Laboratories, N‐terminal, rabbit, dilution 1:2,000, used for WB and ChIP), SMCHD1 (#A302‐872A, Bethyl Laboratories, C‐terminal, rabbit, dilution 1:2,000, used for WB and ChIP), POLQ (a generous gift from Dr JS Hoffmann, mouse, dilution 1:1,000), 53BP1 (A300‐272A.

Techniques: Activation Assay, Knock-Out

Journal: Journal of Nucleic Acids

Article Title: A ssDNA Aptamer That Blocks the Function of the Anti-FLAG M2 Antibody

doi: 10.4061/2011/720798

Figure Lengend Snippet: 3XABA oligonucleotide blocks the interaction of Flag-tagged proteins with the M2 antibody. (a)-(b) The 3XABA oligo blocks the binding of Flag-TRF2 ΔB to M2-coated beads. Magnetic beads coated with the M2 antibody were incubated in the absence (No Comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). In vitro translated [ 35 S]-labeled Flag-TRF2 ΔB was then added and the amount captured by the beads was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (a). In a second experiment done in triplicate, the amount of [ 35 S]-labeled protein captured was counted by scintillation (b). The amount of [ 35 S]-labeled protein captured in the absence of competitor (No Comp) was arbitrarily set to 100%. In both experiments, beads coated with normal mouse IgG were included as negative control for the capture (IgG). Data represent the mean ± S.D. ( n = 3). (c)-(d) The 3XABA oligo elutes the Flag-TRF2 ΔB proteins already bound to M2-coated beads. The [ 35 S]-Flag-TRF2 ΔB protein was first captured by magnetic beads coated with the M2 antibody. The beads were then incubated in the absence (No comp) or presence of the indicated competitor (3XABA, 3XCTR, 3XFLAG). The amount of [ 35 S]-Flag-TRF2 ΔB released was determined by SDS-PAGE electrophoresis and exposure to a PhophorImager cassette (c). In a second experiment done in triplicate, the amount of [ 35 S]-labeled protein released was counted by scintillation (d). The amount of [ 35 S]-labeled protein released by the boiling (total) was arbitrarily set to 100%. In both experiments, beads boiled to release to all of the captured [ 35 S]-labeled protein were included as positive control for the elution (Total). Data represent the mean ± S.D. ( n = 3).

Article Snippet: Normal mouse IgG (cat. # sc-2025) and anti-vimentin mouse monoclonal antibody (IgG 1 clone sc-6260) were obtained from Santa Cruz (Santa Cruz, CA), as was the rabbit polyclonal antibody against TRF2 (cat. # H-300).

Techniques: Binding Assay, Magnetic Beads, Incubation, In Vitro, Labeling, SDS Page, Electrophoresis, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Kinetics of shelterin genes expression during HHV-6A/B infection. HSB-2 cells (A-G) and Molt3 cells (H-M) were respectively infected with HHV-6A or HHV-6B. At various time post infection, total RNA was extracted and analyzed by reverse transcriptase QPCR for TRF1, TRF2, POT1, RAP1, TIN2, TPP1, GAPDH and U90 genes expression. Shelterin genes expression was normalized relative to GAPDH gene expression while U90 was analyzed to demonstrate infection. Results represent data from 4-6 independent experiments expressed as mean +/-SD gene expression relative to that of uninfected cells. *p<0.05.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, Reverse Transcription

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: TRF2 expression during productive HHV-6A/B infections. Mock, HHV-6A- or HHV-6B-infected cells were analyzed for TRF2 expression by flow cytometry. Uninfected and 5 days old HHV-6A-infected HSB-2 cells (A-B) and HHV-6B-infected Molt3 cells (C) were fixed, permeabilized and stained for TRF2, P41 and gp102 proteins expression. Numbers in the top and bottom left quadrants indicate mean relative TRF2 fluorescence intensities. Results are representative of two independent experiments. D) Western blot analysis of TRF2 expression in HHV-6A/B infected. Tubulin was used as loading controls and IE1 to demonstrate HHV-6A/B infection. Numbers represent TRF2 expression levels relative to mock-infected cells after normalization with tubulin.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, Flow Cytometry, Staining, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Increased TRF2 expression in HHV-6A-infected U2OS cells. U2OS cells were infected with HHV-6A and analyzed for TRF2 and IE2 expression at 24h, 48h and 72h post-infection by dual color immunofluorescence. A) Representative TRF2 and IE2 expression in bystander and IE2 expressing cells at 48h post infection. B) Mean relative TRF2 expression + SD in uninfected (white), IE2-(green-uninfected bystander) or IE2+ (red-infected) cells at 24h, 48h and 72h post infection. Each symbol represents the relative TRF2 expression from a single cell.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Expressing, Infection, Immunofluorescence

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Binding of TRF2 to HHV-6 viral DNA. Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled telomeric dsDNA (A) and binding was assessed by EMSA. Excess of unlabeled telomeric and non-telomeric dsDNA were added as competitors. Samples were migrated on non-denaturing acrylamide gel, dried and exposed to X-ray films. B) Recombinant MBP or MBP-TRF2 were incubated with 32 P-labeled non-telomeric dsDNA and binding was assessed by EMSA. C) Recombinant MBP and MBP-TRF2 were coated to the wells of a 96 well-plate and incubated with HaeIII digested DIG-labeled HHV-6A DNA (25 ng/condition) in the presence or absence of competitors. After washing, bound DNA was quantified by adding peroxidase-labeled anti-DIG antibodies and substrate. Results are expressed as mead absorbance +SD of triplicate values. Experiment is representative of two additional experiments. *** P<0.001.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Binding Assay, Recombinant, Incubation, Labeling, Acrylamide Gel Assay

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Colocalization of shelterin complex proteins and HHV-6A IE2 protein at viral and cellular telomeres. A) U2OS cells were infected for 48h with HHV-6A after which cells were processed for IF-FISH. Telomeres were labeled in blue, p41 in green and IE2 in red. These images demonstrate colocalization of IE2 with P41, a viral protein that associates with viral DNA during infection, and diffuse telomeric signals (arrows). B) Telomeres were labeled in magenta, TRF2 in green and IE2 in red. The panels in the middle row show images of cells productively infected (minority of cells) with HHV-6A. Large diffuse telomeric signals (viral replication compartments) where TRF2 and IE2 accumulates (rectangles) are represented. The panels in the third row represent infected cells that do not actively replicate viral DNA with TRF2 and IE2 colocalizing (dashed squares) at distinct telomeres. C) Colocalization of HHV-6A IE2 protein at telomeres in the absence of viral DNA. U2OS cells were transfected with an empty vector or an IE2 expression vector. Forty-eight hours later cells were processed for dual color immunofluorescence. TRF2 was labeled in green and IE2 in red. Examples of IE2 colocalizing with TRF2 are presented (dashed squares). D) U2OS cells were transfected with WT IE2 or IE2 Δ1290-1500 expression vectors. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labeled in cyan, IE2 in red and nuclei in blue. Examples of IE2 colocalizing with TRF2 are presented (dashed squares). E) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector or a myc tagged TRF1 expression vector. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labels in cyan, TRF1 in green and IE2 in red. Examples of TRF1 localizing at telomeres (dashed squares) in uninfected cells are shown in the top row. Examples of IE2 colocalizing with TRF1 and telomeres in infected cells are presented in the bottom row (dashed squares). F) Uninfected and HHV-6A-infected U2OS cells were transfected with an empty vector or a myc tagged POT1 expression vector. Forty-eight hours later cells were processed for IF-FISH. Telomeres were labels in cyan, POT1 in green and IE2 in red. Examples of POT1 localizing at telomeres (dashed squares) in uninfected cells are shown in the top row. Examples of IE2 colocalizing with POT1 and telomeres in infected cells are presented in the bottom row (dashed squares).

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Infection, Labeling, Transfection, Plasmid Preparation, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Binding of TRF2 to viral DNA during HHV-6A/B infection. A) Schematic representation of the HHV-6A/B genome. The DR6 probe used for hybridization is shown in red. Uninfected and HHV-6A-infected HSB-2 cells (B-C) or uninfected and HHV-6B-infectd Molt3 cells (D-E) were analyzed for TRF2 binding to viral DNA using ChIP. The input was hybridized with Alu probe to assess quantity of starting material. Anti-IgG (negative control) or TRF2 antibodies were used for immunoprecipitation. Eluted DNA was serially diluted and hybridized with 32 P-labeled telomeric (TTAGGG) 3 or HHV-6 (DR6) probes. After hybridization the membranes were washed and exposed to X-ray films. The quantity of TRF2 bound to telomeric and viral DNA is measured relative to the input. Results are of 3 independent experiments.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Binding Assay, Infection, Hybridization, Negative Control, Immunoprecipitation, Labeling

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: IE2 localized to VRC in the absence of TRF2. U2OS cells were transduced with a lentiviral vector coding for a Dox inducible shRNA against TRF2. Transduced cells were selected with puromycin for a week. A) Half of the cultures was treated with Dox for seven days to induce TRF2 knockdown (KD), as determined by western blot. B) Control (-Dox) and TRF2 KD (+Dox) cells were infected with HHV-6A for 48h and processed for IF-FISH. Telomeres were labeled in cyan, TRF2 in green and IE2 in red. As show in the–Dox condition, TRF2 colocalized with IE2 as well as diffuse (dashed square) and punctate (dashed circle) telomeric signals. In the +Dox condition, TRF2 KD was confirmed with IE2 colocalizing with diffuse telomere signals (dashed squares). C) DDR at telomeres as a consequence of TRF2 knockdown. U2OS cells were treated or not with Dox and infected with HHV-6A as in . Cells were then processed for IF-FISH. Telomeres were labeled in cyan, IE2 in red, 53BP1 (as marker of DDR) in green and nuclei in blue.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Infection, Labeling, Marker

Journal: bioRxiv

Article Title: Modulation and recruitment of TRF2 at viral telomeres during human herpesvirus 6A/B infection

doi: 10.1101/514075

Figure Lengend Snippet: Knockdown of TRF2 does not affect HHV-6A/B replication. SUP-T1 cells were transduced with a lentiviral vector coding for a Dox inducible shRNA against TRF2. A) Transduced cells were selected with puromycin for two weeks. TRF2 knockdown (KD) was induced by adding Dox to the culture medium for three weeks and confirmed by western blot. B-C) Control (-Dox) and TRF2 KD (+Dox) SUP-T1 cells were infected with HHV-6A (B) or HHV-6B (C). Whole cell DNA was isolated at various time points and the relative number of HHV-6A/B genomes determined and normalized against cellular DNA.

Article Snippet: The following primary antibody were used: rabbit-α-IE1-Alexa-488 , mouse-α-IE2-Alexa-568 (Arsenault et al, 2003, JCV), mouse-α-P41 (NIH AIDS Reagent Program), rabbit-α-TRF2 (NB100-56694, Novus Biologicals), rabbit-α-53BP1 (H-300, Santa Cruz Biotechnology), mouse-α-γH2AX (Ser139, clone JBW301, EMD Millipore) and mouse-α-PML (PG-M3, Santa Cruz Biotechnology).

Techniques: Transduction, Plasmid Preparation, shRNA, Western Blot, Infection, Isolation