Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Transfected Flag-TRF2 ΔB forms a complex that binds selectively to telomeric DNA fragments that carry a 3′-overhang. ( A ) Graphical representation of probes 4M-3′, 4M-bl and 4M-5′. Shaded areas represent telomeric DNA. The probes carried four binding sites for the Myb domain of TRF2 (ds-TTAGGGTTA motifs numbered 1–4) followed by different end structures: 3′-telomeric overhang (4M-3′), blunt (4M-bl) or 5′-telomeric overhang (4M-5′). [ 32 P]-phosphate was at the 5′-end of the top strand. ( B ) TRF2 constructs used in transient transfection. Full-length TRF2 contains a basic domain that binds ss-DNA independently of sequence (Basic), a TRF homology region (TRFH/Dimerization) that serves as dimerization interface, a domain of interaction with TIN2 (aa 350–367), and a Myb domain that binds to ds-TTAGGGTTA (Myb). Interaction with TIN2 allows the recruitment of TRF2 in Shelterin complexes. In these complexes, TPP1 and TIN2 act scaffolds linking TRF2 to the ss-DNA binding protein POT1, which binds to 5′-TTAGGGTTAG-3′. TRF2 ΔB lacks amino acids 2-44 containing the sequence-independent ss-DNA binding domain of TRF2. TRF2 ΔB (R361P) contains an additional mutation that blocks the association of TRF2 ΔB with TIN2. All constructs were tagged at the N-terminus with the Flag epitope. ( C ) Western blot analysis of HeLa cells transiently transfected with the different TRF2 ΔB constructs. Whole cell extracts prepared for EMSA (50 µg) were probed with the anti-Flag M2 antibody. ( D ) Transfected TRF2 ΔB is in a complex that binds selectively to telomeric DNA fragments carrying a 3′-overhang. Top panel: WCEs from mock-transfected (lane 1) and TRF2 ΔB -transfected HeLa cells (lanes 2–4) were incubated with the indicated [ 32 P]-probes (4M-3′, 4M-blunt, 4M-5′) and the protein/DNA complexes that formed were resolved by electrophoresis in TAM buffer. T2, TRF2-containing complex T2. NS, non-specific band. Bottom panel: after incubation of the extracts with the indicated probes, protein/DNA complexes containing Flag-TRF2 ΔB were captured using magnetic beads coated with an anti-Flag antibody, were eluted with an excess of 3XFLAG peptide, and were subsequently resolved by electrophoresis in TAM buffer. ( E ) Effects of R361P mutation on formation of complex T2. HeLa cells were mock-transfected or transfected with Flag-TRF2 ΔB or Flag-TRF2 ΔB (R361P). WCEs were prepared and incubated with probe 4M-3’, after which point protein/DNA complexes were resolved by electrophoresis in a native polyacrylamide gel containing TAM buffer. Complex T2 failed to be detected in extracts of HeLa cells transfected with the R361P mutant.

Article Snippet: Mouse monoclonal antibodies used in western blots were against the Flag tag (M2, Stratagene, La Jolla, CA, USA) and TPP1 (1D8-1B6, Novus Biologicals).

Techniques: Transfection, Binding Assay, Construct, Sequencing, Mutagenesis, FLAG-tag, Western Blot, Incubation, Electrophoresis, Magnetic Beads

Journal: Nucleic Acids Research

Article Title: Characterization of the DNA binding specificity of Shelterin complexes

doi: 10.1093/nar/gkr665

Figure Lengend Snippet: Complex T2 formed by Flag-tagged TRF2 is a member of the Shelterin family. ( A ) Western blot analysis of HT1080-TRF2 and HT1080-Vector cells. WCEs prepared for EMSA (50 µg each) were analyzed by western blotting to detected total TRF2 (anti-TRF2 antibody) and the stably transfected Flag-TRF2 (anti-Flag M2 antibody). Membranes were re-probed with an antibody against β-actin. ( B ) Complex T2 formed by Flag-TRF2 contains the Shelterin scaffolding component TIN2. Extracts of HT1080-TRF2 cells were incubated with the indicated probes, after which point protein/DNA complexes were resolved by native electrophoresis in a composite gel containing TAM buffer. Five minutes prior to loading, the indicated antibodies were added to samples in lanes 4–8 (1 µg each of anti-Flag M2 antibody, normal mouse IgG, or antibodies against TIN2, c-Fos, or Vimentin). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( C ) Loss of Shelterin components prevents formation of complex T2. HT1080-TRF2 cells were transiently transfected with siRNA smartpools against TPP1, TIN2, POT1 or TRF1 or with non-targeting (NT) siRNA. Nuclear extracts prepared from HT1080-Vector cells (Vector) or from HT1080-TRF2 cells (TRF2) transfected with the different siRNA were incubated with probe 4M-3′, after which protein/DNA complexes were separated by native electrophoresis in composite gel containing TAM buffer. As expected, complex T2 was more abundant in cells expressing Flag-TRF2 (lane 1 versus 2), was selective for telomeric DNA fragments that carried a 3′-overhang (lane 7 versus 8), and was supershifted by the anti-Flag antibody (lane 9 versus 10). Complex T2 was reduced in HT1080-TRF2 cells transfected with siRNA smartpools directed against TIN2, TPP1 or POT1 (lanes 3–5 versus lane 2). Short arrow indicates positions of the supershifted complex T2. NS, non-specific band. ( D ) Relative abundance of the TPP1, TIN2, POT1 and TRF1 mRNA in the siRNA-transfected cells. For each transfection, abundance of the targeted mRNA was measured by real-time PCR, comparing cells transfected with the targeting and non-targeting (NT) siRNA. For each mRNA, abundance in cells treated with the non-targeting siRNA was set to 1. To show that targeting was specific, TRF2 and GAPDH mRNAs were also measured, both of which found to be unaffected by the different siRNA treatments.

Article Snippet: Mouse monoclonal antibodies used in western blots were against the Flag tag (M2, Stratagene, La Jolla, CA, USA) and TPP1 (1D8-1B6, Novus Biologicals).

Techniques: Western Blot, Plasmid Preparation, Stable Transfection, Transfection, Scaffolding, Incubation, Electrophoresis, Expressing, Real-time Polymerase Chain Reaction