The Functions of the Telomeric Protein TRF1

描述（申请人提供）：线性染色体的末端，端粒，是染色体稳定，老化和肿瘤抑制所必需的结构，由序列TTAGGG和相互作用蛋白组成。所有端粒的最末端都由富含G的3'链的单链组成，长度可达300个碱基。这个3'端侵入端粒的双链部分，形成保护环。端粒是由端粒酶维持的，端粒酶是一种将TTAGGG重复序列复制到染色体末端的逆转录酶。端粒酶在大多数人体细胞中被抑制，导致端粒不断缩短，从而产生抑制肿瘤的作用。两个端粒重复结合因子TRF1和TRF2结合到端粒的双链部分。这些蛋白质与许多不同的伙伴相互作用，导致形成独立的复合物。这两种复合物都与端粒长度调节有关，TRF2复合物起保护作用。TRF1的靶向缺失导致胚胎早期致死，其特征指向细胞凋亡，由此我们提出TRF1是染色体末端保护所必需的假设。本提案的重点是验证这一假设。此外，我们将了解TRF1在胚胎发育中的作用。在AIM1中，我们将生成体内和体外工具，通过创建有条件的删除基因来分析TRF1功能。缺失将在小鼠和组织培养细胞中进行，这些系统连同囊胚培养将用于分析TRF1缺失对细胞生长、活力和染色体结构的影响。AIM2侧重于TRF1的结构功能分析。我们将使用TRF1突变体来挽救由TRF1缺失引起的表型。这些实验将在组织培养系统中进行，并转移到小鼠身上进行救援实验。在AIM3中，我们计划分离与TRF1缺失引起的表型相关的TRF1相互作用因子。TRF1“敲除”是第一个可用的端粒蛋白的靶向删除。这项研究的结果有望揭示端粒复合物的完整性，从而有助于了解衰老和癌症的发展。

DESCRIPTION (provided by applicant): The ends of linear chromosomes, telomeres, are structures essential for chromosome stability, aging and tumor suppression, made up of the sequence TTAGGG and interacting proteins. The very end of all telomeres consists of a single stranded overhang of the G rich 3' strand that can be up to 300 bases long. This 3' end invades the double stranded portion of the telomere, forming protective loops. Telomeres are maintained by telomerase, a reverse transcriptase that copies TTAGGG repeats to the chromosome ends. Telomerase is suppressed in most human somatic cells, leading to constant telomere shortening, giving rise to the tumor suppressive effect. The two telomeric repeat binding factors TRF1 and TRF2 bind to the double stranded portion of the telomere. The proteins interact with a number of different partners, leading to the formation of independent complexes. Both complexes have been implicated in telomere length regulation, and the TRF2 complex plays a protective role. Targeted deletion of TRF1 led to early embryonic lethality with features pointing at apoptosis, giving rise to our hypothesis that TRF1 is necessary for chromosome end protection. This proposal focuses on testing this hypothesis. Additionally, we will gain understanding in the role of TRF1 in the embryonic development. In AIM1 we will generate in vivo and in vitro tools to analyze TRF1 functions by creating a conditional deletion of the gene. The deletion will be made in the mouse and in tissue culture cells, and these systems together with blastocyst cultures will be used to analyze the effect of the TRF1 deletion on cell growth, viability, and chromosome structure. AIM2 focuses on structure function analysis of TRF1. We will use TRF1 mutants to rescue phenotypes resulting from the TRF1 deletion. These experiments will be performed in tissue culture systems, and moved into mice for rescue experiments. In AIM3 we plan to isolate TRF1 interacting factors that are involved in phenotypes derived from the deletion of TRF1. The TRF1 'knockout' is the first targeted deletion of a telomeric protein available. Results from this study are expected to shed light onto the integrity of the telomeric complex, and therefore contribute to the understanding of aging and cancer development.