| Abstract: |
Human telomeres contain two related telomeric DNA-binding proteins, TRF1 and TRF2. The TRF1 complex contains the TRF1 interacting partner, TIN2, as well as PIP1 and POT1 and regulates telemere-length homeostasis. The TRF2 complex is primarily involved in telomere protection and contains the TRF2 interacting partner human (h)Rap1 as well as several factors involved in the DNA damage response. A prior report showed that conditional deletion of marine TRF1 reduced the presence of TRF2 on telomeres. Here we showed that TRF2 is also lost from human telomeres upon TRF1 depletion with small interfering RNA prompting a search for the connection between the TRF1 and TRF2 complexes. Using mass spectrometry and co-immunoprecipitation, we found that TRF1, TIN2, PIP1, and POT1 are associated with the TRF2-hRap1 complex. Gel filtration identified a TRF2 complex containing TIN2 and POT1 but not TRF1 indicating that TRF1 is not required for this interaction. Co-immunoprecipitation, Far-Western assays, and two-hybrid assays showed that TIN2, but not POT1 or PIP1, interacts directly with TRF2. Furthermore, TIN2 was found to bind TRF1 and TRF2 simultaneously, showing that TIN2 can link these telomeric proteins. This connection appeared to stabilize TRF2 on the telomeres as the treatment of cells with TIN2 small interfering RNA resulted in a decreased presence of TRF2 and hRap1 at chromosome ends. The TIN2-mediated cooperative binding of TRF1 and TRF2 to telomeres has important implications for the mechanism of telomere length regulation and protection. |
| Keywords: |
signal transduction; controlled study; unclassified drug; dna binding protein; human cell; gene deletion; nonhuman; mass spectrometry; proteins; protein analysis; animal cell; phenotype; telomere; dna damage; protein depletion; protein protein interaction; small interfering rna; protein binding; protein stability; rna, small interfering; rna interference; hela cells; telomerase; animalia; telomeric repeat binding factor 2; rna; blotting, western; dna; membrane glycoproteins; escherichia coli; co-immunoprecipitation; glutathione transferase; immunoprecipitation; green fluorescent proteins; cell adhesion molecules; protein structure, tertiary; cell nucleus; chromosome protein; two hybrid system; chromosomes; telomeric repeat binding factor 1; chromatography, gel; antigens, surface; electrophoresis, polyacrylamide gel; two-hybrid system techniques; beta-galactosidase; dna protein complex; telomeric repeat binding protein 2; gel filtration; filtration; rap protein; structure (composition); human telomeres; humans; human; priority journal; article; telomere-length homeostasis; protein pip1; protein pot1; protein tin2; far western blotting; telomeric repeat binding protein 1
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