UC Berkeley

Telomeres are the DNA-protein complexes that cap the ends of linear chromosomes and protect the genome. Due to the end replication problem, telomeres shorten after each replication until they become critically short and trigger replicative senescence. Therefore, telomere length determines the number of times a cell can divide and acts as a molecular clock of cellular aging. To overcome such proliferative limits and maintain tissue homeostasis, most stem cells express telomerase, a reverse transcriptase that de novo synthesizes telomeric repeats. This allows stem cells to maintain a long telomere sequences to support the proliferative demand of their differentiated progenies. Telomere length is determined by the interaction between the telomeric DNA sequences and a six-protein telomere-binding shelterin complex called shelterin. When this interaction is balanced, telomeres are at ‘telomere length homeostasis’ in which the average telomere length is maintained within a set range. On the contrary, when the interaction is defective, pathological telomere shortening and stem cell depletion can occur. During my doctoral work, I studied the two aspects of telomere length regulation that are mediated by shelterin proteins; telomere length homeostasis and defective telomere maintenance that leads to dyskeratosis congenita (DC), a rare inherited bone marrow failure syndrome.In human pluripotent stem cells, telomeres are maintained within a set length range of 9-12 kilobases. This requires that short telomeres are preferentially elongated by telomerase so none of the telomeres shorten too far below the set range. Previous works have shown that short telomeres can be preferentially elongated by telomerase, indicating that there is an unknown mechanism that distinguishes short telomeres from long ones. Studies using overexpression or knockdown of shelterin proteins have demonstrated that average telomere length of a cell population could be altered by modulating the expression levels of the proteins. Such approaches have uncovered some important roles of shelterin proteins in telomere length regulation. However, since such approaches perturb telomere length set points of a cell population, they are not suitable for studying how short and long telomeres within a cell are marked differently to maintain telomere length homeostasis. Therefore, the exact mechanism by which the shelterin proteins ensure preferential elongation of short telomeres and maintain telomere length homeostasis is unknown. In this study, I aim to study the molecular signatures of one of the shelterin proteins, TPP1, on short and long telomeres in cells at telomere length homeostasis. For this, I genetically engineered human embryonic stem cells to express endogenously tagged TPP1. This system allowed visualization of TPP1 on telomeres of different lengths within cells with unperturbed telomere length homeostasis. I used various imaging techniques to investigate the modes of TPP1-telomere interaction in this cell model. I observed that a subset of short telomeres within a nucleus exhibited higher amount of bound TPP1 per unit length of telomeres, indicating that the density of TPP1 was higher on those telomeres. Also, a cell line that was engineered to have reduced telomerase expression and shorter telomere length set point compared to the wild-type cells showed higher average TPP1 density. This indicates the density of TPP1 bound on telomeres could be a potential molecular signature that distinguishes short telomeres from long telomeres. Defective telomere maintenance leads to a reduced proliferative capacity of cells and a spectrum of diseases called ‘telomeropathies’. Telomeropathies exhibit a large variety of symptoms, age of onset, and severity, which all correlate with the extent of premature telomere shortening. An example of telomeropathy is DC, a rare inherited bone marrow failure syndrome. DC is associated with mutations in genes involved in telomere maintenance and the second most mutated gene in DC is TINF2, encoding the shelterin protein TIN2. The mutations in TIN2 result in a more severe form of DC compared to DC-causing mutations in other genes but the mechanism of the rapid telomere shortening in TIN2-DC patients is unknown. In this work, I developed two novel, endogenous, isogenic models to study the mechanism of TIN2-DC mutations. Human embryonic stem cells targeted to carry a missense TIN2-DC mutation exhibited short telomeres compared to the wild-type cells, and the differentiated cells had reduced proliferative capacity, recapitulating DC patient phenotypes. Our data indicated that the TIN2-DC mutation did not lead to unprotected telomeres and the disease mutation-related telomere shortening occurred through the telomerase pathway, not through an unknown telomere degradation mechanism. Furthermore, we demonstrated that a targeted disruption of the mutant TIN2 expression using genome editing led to restoration of telomere length and proliferative capacity of mutant cells. To test the effect of the target gene disruption in a more disease-relevant cell type, I developed an in vivo model of TIN2-DC using genome-edited human hematopoietic stem cells. The data in this study demonstrate similarities and differences in the phenotypes that the two stem cells models exhibit in response to same target gene modifications. The comparison between the two stem cell models highlights the importance of using a proper disease model to assess potential therapeutic gene modifications.