Journal: Nucleic Acids Research

Article Title: SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage

doi: 10.1093/nar/gkw1202

Figure Lengend Snippet: TRF2 interacts with SIRT6. ( A ) BJ-hTERT were treated with 0.2 μM CPT for 2 h and then processed for the WB analysis of the expression levels of SIRT6 and TRF2. The expression levels of the two proteins were reported in the histograms (upper panel) as fold changes in treated versus untreated samples, after β-actin normalization. All histograms show the mean values of three independent experiments. Bars indicate means ± SD. ( B ) Representative IF images of SIRT6 from BJ-hTERT fibroblasts treated as in (A). Original magnification, 63x. Scale bar 50 μm. ( C ) Representative confocal IF images of untreated BJ-hTERT showing the colocalization between TRF2 and SIRT6. a, TRF2 staining; b, SIRT6 staining; c, merge; d, SIRT6/TRF2 colocalizations showed as white spots after software analysis. Scale bar 10 μm. ( D ) Pull-down assay of HeLa nuclear extracts with His-tagged SIRT6 protein conjugated with Ni-NTA magnetic beads. The eluted product was analyzed by WB using an antibody against TRF2. The ponceau staining is shown as beads loading control. ( E ) In vitro GST pull-down assay with GST alone or GST-TRF2 in the presence or absence of His-SIRT6. Following the pull-down, samples were analyzed by WB using antibodies against TRF2 or SIRT6. ( F ) Anti-Myc immunoprecipitation (IP) followed by WB with anti-SIRT6 or -Myc antibody in nuclear extracts of HCT116 cells infected with Myc-TRF2 or Empty retroviral vectors. ( G ) Anti-SIRT6 IP in nuclear extracts of HCT116 cells expressing Myc-TRF2. Anti-rabbit IgG IP was used as a negative control. WB was carried out with anti-TRF2 or anti-SIRT6 antibody. ( H ) Anti-TRF2 IP in nuclear extracts of HCT116 cells. Anti-mouse IgG IP was used as a negative control. WB was carried out with anti-TRF2 or anti-SIRT6 antibody. ( I ) Nuclear extracts of HCT116 cells infected with Myc-TRF2wt or Empty retroviral vectors were immunoprecipitated with anti-Myc antibody. The IP was performed in presence or in absence of 50 μg/ml EtBr, followed by WB with anti-SIRT6 or -Myc antibody. ( J ) Schematic representation of TRF2 and its deletion variants used in this study. ( K ) The various GST-TRF2 proteins or GST alone were affinity-purified and incubated with lysates prepared from HeLa cells, followed by detection with anti-SIRT6 antibody. The purified GST fusion proteins (indicated by the asterisks) were visualized by ponceau staining. Molecular mass markers are expressed in kilodaltons (KDa).

Article Snippet: Empty-, TRF2 wt - or TRF2 cT -overexpressing cells were obtained by infecting with amphotrophic retroviruses generated by transient transfection of retroviral vectors (pBabe-puro-Empty, pBabe-puro-mycTRF2 and pLPC-Myc-TRF cT ; the last one was a gift from Eros Lazzerini Denchi, Addgene plasmid #44573; ) into Phoenix amphotropic packaging cells with JetPEI (Polyplus, New York, NY, USA), according to the manufacturer's instructions.

Techniques: Expressing, Staining, Software, Pull Down Assay, Magnetic Beads, Control, In Vitro, Immunoprecipitation, Infection, Retroviral, Negative Control, Affinity Purification, Incubation, Purification

Journal: Nucleic Acids Research

Article Title: SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage

doi: 10.1093/nar/gkw1202

Figure Lengend Snippet: TRF2 is deacetylated by SIRT6. ( A ) Lower panel: Representation of TRF2 organization in four domains. Upper panel: Footprinting graph showing profile of in vitro acetylation of TRF2 lysines by sulfosuccinimidyl acetate (in green) and TRF2 lysines still acetylated upon SIRT6 addition (in red). The mass spectrometry analysis was done using a nanoLC-MSMS giving both the peptide mass and sequence allowing the determination of the specific acetylated aminoacids. Mass spectrometry analysis gives acetylation profiles of the protein before and after incubation with SIRT6 showing TRF2 lysines deacetylated by SIRT6. ( B ) Positions of lysines deacetylated upon SIRT6 addition (in yellow), on the 3D structure of the TRFH domain of TRF2 (PDB:3BUA). The third lysine identified, K190 is located on a region of structural disorder, represented as broken lines. ( C ) Schematic representation of the TRF2 cT variant obtained through the domain swapping approach between TRF1 and TRF2. ( D ) HCT116 cells infected with Myc-TRF2 wt , Myc-TRF2 cT or Empty retroviral vectors were analyzed by western blot for the expression of Myc-tagged TRF2 variants or SIRT6, after 72 h of siGFP or siSIRT6 transfection. ( E ) TRF2-overexpressing HCT116 cells were subjected to IP with anti-TRF2 antibody after silencing (siSIRT6) or overexpression (overSIRT6) of SIRT6. Immunoprecipitated TRF2 proteins were loaded on SDS-PAGE and the regions around 60 kDa were cut and, separately, digested as described in material and methods section, to be then analyzed by LC–MS/MS. The relative abundance of the ions of peptide 174–192 mono- (+1KAc) and diacetylated (+2KAc) species was calculated integrating the peak area in each LC–MSMS run and reported in the histograms. ( F ) After transient transfection with ubiquitin-HA vector, TRF2-overexpressing HCT116 cells were either transfected with siSIRT6 siRNA or control siGFP. Protein lysates prepared from cells after 6 h by the end of treatment with 2 μM CPT, as well as those from untreated cells as negative controls, in presence or not of MG132, were used in immunoprecipitation with anti-TRF2 antibody, followed by western blot with anti-HA. The effectiveness of SIRT6 siRNA and the expression of TRF2 was confirmed by immunoblot using input samples. The levels of β-actin were used as a loading control.

Article Snippet: Empty-, TRF2 wt - or TRF2 cT -overexpressing cells were obtained by infecting with amphotrophic retroviruses generated by transient transfection of retroviral vectors (pBabe-puro-Empty, pBabe-puro-mycTRF2 and pLPC-Myc-TRF cT ; the last one was a gift from Eros Lazzerini Denchi, Addgene plasmid #44573; ) into Phoenix amphotropic packaging cells with JetPEI (Polyplus, New York, NY, USA), according to the manufacturer's instructions.

Techniques: Footprinting, In Vitro, Mass Spectrometry, Sequencing, Incubation, Variant Assay, Infection, Retroviral, Western Blot, Expressing, Transfection, Over Expression, Immunoprecipitation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Plasmid Preparation, Control