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、衰老和抑制肿瘤所必需的结构，由序列TTAGGG和相互作用的蛋白质组成。所有端粒的末端都由一条长达300个碱基的富含G的3‘链组成。这个3‘端侵入端粒的双链部分，形成保护环。端粒由端粒酶维持，端粒酶是一种将TTAGGG重复复制到染色体末端的逆转录酶。端粒酶在大多数人类体细胞中被抑制，导致端粒持续缩短，从而产生肿瘤抑制作用。两个端粒重复序列结合因子TRF1和TRF2与端粒的双链部分结合。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体。这两个复合体都参与了端粒长度的调节，而TRF2复合体起到了保护作用。TRF1的靶向缺失会导致早期胚胎死亡，其特征是细胞凋亡，这引发了我们的假设，即TRF1是染色体末端保护所必需的。这项提议的重点是检验这一假设。此外，我们还将了解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，通过创建有条件的基因缺失来分析TRF1的功能。该缺失将在小鼠和组织培养细胞中进行，这些系统将与囊胚培养一起用于分析TRF1缺失对细胞生长、活力和染色体结构的影响。AIM2重点分析了TRF1的结构和功能。我们将使用TRF1突变体来挽救由于TRF1缺失而产生的表型。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缺失所产生的表型相关的TRF1相互作用因子。TRF1‘基因敲除’是第一个可获得的端粒蛋白的靶向缺失。这项研究的结果有望阐明端粒复合体的完整性，从而有助于理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.