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.