Elucidating the role of TIN2L at the telomere and in dyskeratosis congenita

描述（由申请人提供）：先天性角化不良症（DC）是一种骨髓衰竭和癌症易感性综合征。与一般人群相比，DC患者患急性髓性白血病和舌癌的风险特别高，分别为200倍和1100倍。潜在的DC是端粒维持的缺陷，端粒是位于线性染色体末端的基本核蛋白结构。反映了这一点，在DC中突变的7个基因中有6个编码端粒酶组装或活性所需的成分或因子，端粒酶在DNA复制后补充末端端粒重复序列。15%的DC患者有第七个基因TINF2突变，该基因编码端粒结合复合物庇护蛋白的中心成员TIN2。绝大多数TINF2突变是从头发生的，与其他DC患者相比，这些患者的端粒长度更短，发病年龄更早，强调了这些突变所赋予的端粒功能的严重破坏。TIN2的短异构体（TIN2S）和长异构体（TIN2L）在人细胞中均有表达。TIN2L与端粒和核基质相关，这表明它不太可能与TIN2S具有相同的功能。所有与dc相关的TINF2突变都是杂合的，并映射到一个意义不明确的区域（dc -簇），位于其已知结合区域和TIN2L特有的C端延伸之间。该项目的目标是确定dc相关的TIN2突变如何导致如此严重的端粒缩短。我们假设它们特异性地影响TIN2L的一种独特功能，而这种功能在TIN2S中是不存在的。这一假设得到了初步免疫共沉淀研究的支持，该研究表明，与TIN2S相比，TIN2L与庇护蛋白复合物成员TRF2的相互作用更强，而且这种增强的相互作用需要dc -簇区域和TIN2L的保守区域。我们将使用质谱（MS）来鉴定与TIN2L结合但不与TIN2S结合的蛋白质，并确定dc -簇在这些相互作用中的作用。为了确定分子间/分子内相互作用所需的TIN2L区域，我们将设计一个包含TIN2L的重叠肽阵列。该阵列将被TIN2L和TIN2S探测，以确定C端和dc簇突变的TIN2L特有的分子内相互作用，以确定这些分子内相互作用对dc簇的依赖性。为了确定与TRF2增加相互作用和与通过质谱鉴定的蛋白质结合所必需的区域，我们将用这些蛋白质探测阵列。为了表征通过MS和端粒酶鉴定的两种TIN2亚型TRF2蛋白在DC表型中的作用，我们将使用慢病毒在DC患者来源的淋巴母细胞系中稳定过表达这些蛋白，并确定对端粒长度和群体倍增时间的影响。通过研究TIN2S和TIN2L的野生型功能以及DC-簇在这些功能中的作用，我们希望阐明TIN2不仅在这种破坏性的端粒疾病中发挥作用，而且在DC患者的癌症发病机制中发挥作用。这可能最终会对散发性癌症产生影响。

DESCRIPTION (provided by applicant): Dyskeratosis congenita (DC) is a bone marrow failure and cancer predisposition syndrome. Patients with DC have a particularly high risk of developing acute myeloid leukemia and tongue cancer in comparison to the general population, 200 and 1100 fold respectively. Underlying DC is a defect in the maintenance of telomeres, the essential nucleoprotein structures located at the ends of linear chromosomes. Reflecting this, six of the seven genes mutated in DC encode components or factors required for the assembly or activity of telomerase, the enzyme that replenishes terminal telomeric repeats following DNA replication. Fifteen percent of patients with DC have mutations in the seventh gene, TINF2, which encodes TIN2, a central member of the telomeric binding complex shelterin. The vast majority of TINF2 mutations are de novo, and these patients have shorter telomere lengths and earlier age of disease onset in comparison to other DC patients, underscoring the severe disruption in telomere function conferred by these mutations. Both a short (TIN2S) and a long (TIN2L) isoform of TIN2 are expressed in human cells. TIN2L associates with the telomeres and the nuclear matrix, indicating that it is unlikely to have identical function to TIN2S. All DC-associated TINF2 mutations are heterozygous and map to a region of unclear significance (the DC-cluster), located between its known binding regions and a C- terminal extension unique to TIN2L. The goal of this project is to determine how DC-associated TIN2 mutations result in such severe telomere shortening. We hypothesize that they specifically impact on a unique function of TIN2L, that is absent from TIN2S. This hypothesis is supported by preliminary co-immunoprecipitation studies which showed that TIN2L interacts more robustly with TRF2, a member of the shelterin complex, than TIN2S and that this increased interaction requires the DC-cluster region and a conserved region of TIN2L. We will use mass spectrometry (MS) to identify proteins which bind to TIN2L but not TIN2S, and to determine the role of the DC-cluster in these interactions. To identify the regions of TIN2L necessary for inter/intramolecular interactions we will design an overlapping peptide array containing TIN2L. This array will be probed with TIN2L and TIN2S to identify intramolecular interactions unique to the C terminus and TIN2L with a DC-cluster mutation to determine the dependency of these intramolecular interactions on the DC-cluster. To determine the regions necessary for the increased interaction with TRF2 and binding to the proteins identified via MS, we will probe the array with these proteins. To characterize the role of the two TIN2 isoforms, TRF2, proteins identified via MS and telomerase in the DC phenotype, we will use lentivirus to stably overexpress these proteins in DC patient derived lymphoblastic cell lines, and the effects on telomere length and population doubling time will be determined. Through the study of the wild type function of TIN2S and TIN2L and the role of the DC-cluster in these functions we hope to elucidate the role of TIN2 not only in this devastating telomeric disease, but also in the pathogenesis of cancer in DC patients. This may ultimately have implications for sporadic cancer.