PROPERTIES OF THE TELOMERE-BINDING PROTEIN TIN2

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。人类端粒包含两个相关的端粒DNA结合蛋白，TRF 1和TRF 2。TRF 1复合物包含TRF 1相互作用伴侣，TIN 2，以及PIP 1和POT 1，并调节端粒长度稳态。TRF 2复合物主要参与端粒保护，并包含TRF 2相互作用伴侣human（h）Rap 1以及参与DNA损伤反应的几种因子。先前的报道显示，鼠TRF 1的条件性缺失减少了端粒上TRF 2的存在。在这里，我们发现，TRF 2也失去了从人类端粒TRF 1耗尽与小干扰RNA促使TRF 1和TRF 2复合物之间的连接的搜索。使用质谱和免疫共沉淀，我们发现TRF 1，TIN 2，PIP 1和POT 1与TRF 2-hRap 1复合物相关。凝胶过滤鉴定了含有TIN 2和POT 1但不含TRF 1的TRF 2复合物，表明TRF 1不是这种相互作用所需的。免疫共沉淀，远Western分析和双杂交分析表明，TIN 2，而不是POT 1或PIP 1，直接与TRF 2相互作用。此外，发现TIN 2同时结合TRF 1和TRF 2，表明TIN 2可以连接这些端粒蛋白。这种连接似乎稳定了端粒上的TRF 2，因为用TIN 2小干扰RNA处理细胞导致染色体末端TRF 2和hRap 1的存在减少。TRF 1和TRF 2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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human telomeres contain two related telomeric DNAbinding 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.