Ye Jeffrey Zheng-Sheng, Donigian Jill R, van Overbeek Megan, Loayza Diego, Luo Yan, Krutchinsky Andrew N, Chait Brian T, de Lange Titia
Laboratory for Cell Biology and Genetics, the Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 2004 Nov 5;279(45):47264-71. doi: 10.1074/jbc.M409047200. Epub 2004 Aug 16.
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 telomere-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 murine 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.
人类端粒包含两种相关的端粒DNA结合蛋白，即TRF1和TRF2。TRF1复合体包含TRF1相互作用伴侣TIN2，以及PIP1和POT1，并调节端粒长度的稳态。TRF2复合体主要参与端粒保护，包含TRF2相互作用伴侣人（h）Rap1以及一些参与DNA损伤反应的因子。先前的一份报告显示，条件性缺失小鼠TRF1会降低TRF2在端粒上的存在。在此我们表明，在用小干扰RNA耗尽TRF1后，TRF2也会从人类端粒上消失，这促使我们寻找TRF1和TRF2复合体之间的联系。通过质谱分析和免疫共沉淀，我们发现TRF1、TIN2、PIP1和POT1与TRF2-hRap1复合体相关。凝胶过滤鉴定出一个包含TIN2和POT1但不包含TRF1的TRF2复合体，这表明这种相互作用不需要TRF1。免疫共沉淀、Far-Western分析和双杂交分析表明，直接与TRF2相互作用的是TIN2，而不是POT1或PIP1。此外，发现TIN2能同时结合TRF1和TRF2，这表明TIN2可以连接这些端粒蛋白。这种联系似乎能使TRF2在端粒上稳定存在，因为用TIN2小干扰RNA处理细胞会导致染色体末端TRF2和hRap1的存在减少。TIN2介导的TRF1和TRF2与端粒的协同结合对端粒长度调节和保护机制具有重要意义。
