Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: p300 acetylates TRF2 in vivo and the two proteins form a stable complex. ( A ) Endogenous TRF2 is acetylated by p300. HEK293 cells were transfected with p300-HA or Flag-PCAF and subjected to immunoprecipitation with anti-TRF2 or normal IgG antibodies, followed by immunoblotting with anti-acetylated lysine antibody (Ac-Lys). ( B ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-p53 and either p300-HA or p300ΔHAT-HA and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. The arrowheads mark the positions of p300-HA and p300ΔHAT-HA. ( C ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-p53 and either p300-HA or p300ΔHAT-HA and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-Flag antibody. ( D ) HEK293 cells were transfected with Flag-TRF2 or Flag-TRF1 and subjected to immunoprecipitation with anti-TRF2, anti-TRF1 or anti-Flag antibodies, followed by immunoblotting with anti-p300 antibody. ( E ) HEK293 cells were subjected to immunoprecipitation with either anti-TRF2 or anti-TRF1 antibodies, followed by immunoblotting with anti-p300 antibody. IgG antibody was used as a negative control. ( F ) HEK293 cells were analysed by indirect immunofluorescence for co-localization of TRF2 with p300. Immunofluorescence was used to detect endogenous TRF2 (red) and p300 (green). DNA was stained with DAPI (blue).

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: In Vivo, Transfection, Immunoprecipitation, Western Blot, Negative Control, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Identification of the interaction domain of TRF2 with p300. ( A ) Schematic representation of TRF2 and its deletion variants used in this study. ( B ) HEK293 cells were co-transfected with p300-HA and various TRF2-Myc and subjected to immunoprecipitation with anti-HA antibody, followed by immunoblotting with anti-Myc antibody. The TRF2-Myc proteins were detected by immunoblotting with anti-Myc antibody. ( C ) Schematic representation of TRF2 and its deletion variants (GAR, TRFH, Hinge and Myb). ( D ) The various GST-TRF2 proteins were affinity-purified and incubated with lysates prepared from cells expressing p300-HA, followed by detecting p300-HA. The purified GST fusion proteins were visualized by Coomassie staining. The arrowhead indicates the degradation product of GST-Hinge. Molecular mass makers are shown in kilodaltons.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, Affinity Purification, Incubation, Expressing, Purification, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Identification of the acetylation site on TRF2. ( A ) HEK293 cells were co-transfected with p300-HA and various TRF2-Myc and subjected to immunoprecipitation with anti-Myc antibody, followed by immunoblotting with anti-acetylated lysine antibody. The various TRF2-Myc proteins were detected by immunoblotting with anti-Myc antibody. ( B ) The in vitro TRF2 acetylation assay was carried out using the various GST-TRF2 proteins. The acetylated proteins were detected by immunoblotting with anti-acetylated lysine antibody. The purified GST fusion proteins were visualized by Coomassie staining. Molecular mass makers are shown in kilodaltons. The arrowheads indicate the degradation product of GST-Hinge. ( C ) The various GST-TRF2 proteins were subjected to the in vitro acetylation assay using GST-p300 in the presence of [ 14 C] acetyl coenzyme A. The acetylated proteins were detected by autoradiography. The purified GST fusion proteins were visualized by Coomassie staining. Molecular mass makers are shown in kilodaltons. The arrowheads indicate the degradation product of GST-Hinge. ( D ) MS/MS spectra corresponding to the acetylated peptide (AAFKacTLSGAQDSEAAFAK) from TRF2. HEK293 cells were co-transfected with Flag-TRF2 and p300-HA and subjected to immunoprecipitation with anti-Flag antibody. Immunoprecipitated TRF2 proteins were recovered from the gel, and analysed by LC-MS/MS. In gel, digestion was performed as described previously . The peak at 126.09 m /z (immonium ion) is due to the presence of the acetylated lysine residue. ( E ) HEK293 cells were co-transfected with p300-HA and various point mutant constructs of Flag-TRF2 and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. ( F ) HEK293 cells were co-transfected with p300-HA and either Flag-TRF2 or Flag-K293R and subjected to immunoprecipitation with anti-HA or anti-Flag antibodies, followed by immunoblotting with anti-Flag or anti-HA antibodies.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Transfection, Immunoprecipitation, Western Blot, In Vitro, Acetylation Assay, Purification, Staining, Autoradiography, Tandem Mass Spectroscopy, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Construct

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: p300 regulates the levels of endogenous TRF2. ( A ) HEK293 cells expressing p300-HA or p300ΔHAT-HA were subjected to immunoblotting as indicated. ( B ) HEK293 cells expressing p300 siRNA (sip300) or scrambled control siRNA (siControl) were subjected to immunoblotting as indicated. ( C ) HEK293 cells expressing p300-HA or p300 siRNA were treated with 100 μg/ml cycloheximide and together with, or without, 10 μM MG132 for the indicated times, followed by immunoblotting with anti-TRF2 or anti-actin antibodies. ( D ) Graphical representation of the relative TRF2 levels normalized against the β-actin loading control. The TRF2 expression levels were quantified with the average and standard deviation from three independent experiments. ( E ) HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R, TIN2-V5 and p300-HA, and subjected to immunoprecipitation with anti-V5 or anti-Flag antibodies, followed by immunoblotting with anti-Flag or anti-V5 antibodies. The p300 expression was detected with anti-p300 or anti-HA antibodies.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Expressing, Western Blot, Standard Deviation, Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Acetylation inhibits ubiquitin-dependent proteolysis of TRF2. ( A ) HEK293 cells expressing Flag-TRF2 or Flag-K293R were treated with 100 μg/ml cycloheximide and together with, or without, 10 μM MG132 for the indicated times, followed by immunoblotting with anti-Flag or anti-actin antibodies. ( B ) Graphical representation of the relative TRF2 levels normalized against the β-actin loading control. The TRF2 expression levels were quantified with the average and standard deviation from three independent experiments. ( C ) HEK293 cells were co-transfected with HA-ubiquitin and either Flag-TRF2 or Flag-K293R and treated with, or without, 10 μM MG132 for 2 h as specified. Immunoprecipitation was carried out with anti-Flag antibody before probing with anti-HA antibody. ( D ) ChIP of telomeric DNA by ectopically expressed TRF2. HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R and together with p300-HA or p300 siRNA and subjected to ChIP with anti-Flag antibody. Total DNA and immunoprecipitated DNA were applied to nitrocellulose in a slot blot manifold. Duplicate slot blots were hybridized with a telomeric probe or an Alu probe. Input DNA represents 10% of total DNA. HEK293 cells were co-transfected with Flag-TRF2 or Flag-K293R and together with p300-HA or p300 siRNA and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblotting with anti-acetylated lysine antibody. The p300 expression was detected with anti-p300 or anti-HA antibodies. ( E ) Quantification of blots shown in D. Histogram values represent the expressed TRF2 telomeric ChIP signal normalized to input signal. The percentage of telomeric DNA in ChIP is shown with the average and standard deviation from three independent experiments. ( F ) ChIP of telomeric DNA by endogenous TRF2. HEK293 cells were transfected with p300-HA or p300 siRNA and subjected to ChIP with anti-TRF2 antibody. HEK293 cells expressing p300-HA or p300 siRNA were subjected to immunoprecipitation with anti-TRF2 antibody, followed by immunoblotting with anti-acetylated lysine antibody. The endogenous TRF2 expression was detected with anti-TRF2 antibody. ( G ) Quantification of blots shown in F. Histogram values represent the endogenous TRF2 telomeric ChIP signal normalized to input signal. The percentage of telomeric DNA in ChIP is shown with the average and standard deviation from three independent experiments.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Expressing, Western Blot, Standard Deviation, Transfection, Immunoprecipitation, Dot Blot

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R induces a DNA-damage signal at telomeres. ( A ) HT1080 cells were transfected with TRF2-Myc or K293R-Myc, and multiple independent stable clones were isolated, followed by immunoblotting with anti-TRF2 or anti-actin antibodies. ( B ) HT1080 cells stably expressing TRF2-Myc or K293R-Myc were analysed by indirect immunofluorescence for co-localization of 53BP1 foci (green) with telomeric sites marked by TTAGGG-specific FISH probe (red). Representative fluorescence images of nuclei showing a large number of 53BP1 foci are shown as indicated. DNA was stained with DAPI (blue). A subset of 53BP1 foci co-localized with TTAGGG probe is indicated by arrows. ( C ) Quantification of the induction of 53BP1 foci by K293R overexpression. The average percentage of cells with either 6–10 or >10 53BP1 foci is shown. For each condition, at least 200 cells were counted. ( D ) Quantification of the induction of TIFs by K293R overexpression. The average percentage of cells with either 4–6 or >6 TIFs is shown. Cells with four or more DNA-damage foci co-localized with TTAGGG probe were scored as TIF positive. ( E ) The average percentage of 53BP1 foci located at telomeres was determined in TIF-positive nuclei.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Transfection, Clone Assay, Isolation, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Fluorescence, Staining

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R increases the occurrence of telomeric signal-free chromatid ends. ( A ) Representative telomere FISH analysis on metaphase spreads for telomere defects. HT1080 cells stably expressing the empty vector TRF2-Myc or K293R-Myc were processed for telomeric FISH. Telomere signal-free chromatid ends are indicated by arrows. ( B ) Quantification of telomere signal-free chromatid ends in HT1080 cells stably expressing the empty vector, TRF2-Myc or K293R-Myc.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: p300-mediated acetylation of TRF2 is required for maintaining functional telomeres

doi: 10.1093/nar/gks1354

Figure Lengend Snippet: Overexpression of K293R limits cell proliferation. ( A ) Cell growth curves of HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. Stable cells were replated every 3 days to maintain log-phase growth and calculate the growth rate, with day 0 representing the first day after blasticidin selection. ( B ) Cell growth curves of DU145 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. ( C ) HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc, at the indicated PDs were photographed after staining for SA-β-Gal activity. ( D ) The percentage of total cells that are positive for SA-β-Gal activity is shown in four HT1080 cell lines. For quantitation of senescent cells, a total of 200 cells were counted in each field and four fields were examined. ( E ) Flow cytometric analysis of HT1080 cell lines stably expressing the empty vector, TRF2-Myc or K293R-Myc. Cell lines at the indicated PDs were stained with propidium iodide, followed by fluorescence-activated cell sorter analysis. The percentage of total cells in each phase of the cell cycle is shown. Results are representative of three separate experiments. ( F ) The percentage of total cells in the aneuploidy fraction is shown with the average and standard deviation from three experiments being present in E.

Article Snippet: Immunoprecipitation and immunoblotting were performed using anti-TRF2 (Upstate Biotechnology, Waltham, MA, USA), anti-TRF1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-acetylated lysine (Cell Signaling Technology, Danvers, MA, USA), anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma, St. Louis, MO, USA), anti-V5 (Invitrogen, Carlsbad, CA, USA), anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-TRF2 (T188) (Upstate Biotechnology, Waltham, MA, USA), anti-ATM (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ATM (S1981) (Cell Signaling Technology, Danvers, MA, USA) and anti-actin (Sigma, St. Louis, MO, USA) antibodies.

Techniques: Over Expression, Stable Transfection, Expressing, Plasmid Preparation, Selection, Staining, Activity Assay, Quantitation Assay, Fluorescence, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

doi: 10.1083/jcb.200408181

Figure Lengend Snippet: No detectable localization of hRif1 protein at telomeres in normal cells or in cells depleted of hRap1, TRF1, or TRF2 protein. (A) Western blot analysis of whole cell extracts from LOX cells treated with lentiviruses expressing hairpin siRNAs against TRF1, TRF2, or hRap1. (B) Immunofluorescence analysis of hRif1 protein in LOX melanoma cells. LOX cells were infected with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, or TRF2. 3 d after infection, cells were fixed and stained with hRif1 antibody PAB2857 (red) and TRF1 or TRF2 antibodies (green). A microscope (model Eclipse E600; Nikon) with a 100× objective and a Coolsnap fx charge-coupled device camera and software (Roper Scientific) was used to visualize the image. (C) Histogram showing the average numbers of hRif1 foci per nucleus in LOX cells after treatment with lentiviruses expressing hairpin siRNAs against hRap1, TRF1, TRF2, or with lentiviruses expressing hTER template mutant 47A. For each treatment, hRif1 foci in ∼50 interphase cells were counted after immunofluorescence analysis and the average numbers per nucleus were presented.

Article Snippet: In brief, 1mg of total protein of each whole cell extract was incubated with antibodies against TRF1 (Novus), TRF2 (BD Transduction Laboratories) and hRap1 (Bethyl Laboratories) and protein A or G beads (Sigma-Aldrich) in a total volume of 500 μl for 4 h at 4°C.

Techniques: Western Blot, Expressing, Immunofluorescence, Infection, Staining, Microscopy, Software, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules

doi: 10.1083/jcb.200408181

Figure Lengend Snippet: hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.

Article Snippet: In brief, 1mg of total protein of each whole cell extract was incubated with antibodies against TRF1 (Novus), TRF2 (BD Transduction Laboratories) and hRap1 (Bethyl Laboratories) and protein A or G beads (Sigma-Aldrich) in a total volume of 500 μl for 4 h at 4°C.

Techniques: Synthesized, Mutagenesis, Microscopy, Expressing, Infection, Staining