UC Berkeley

Along with the evolution of linear chromosomes, so did the problems associated with chromosome ends. DNA polymerases can only move in the 5’ to 3’ direction and rely on RNA primers to begin. In most cases, these primers are removed when the replication fork of an upstream origin of replication reaches the primer replacing it with DNA and ligating the two fragments together. The lagging strand chromosome end does not have an upstream Okazaki fragment to replace its RNA primer leading to DNA sequence loss. This loss is known as the end replication problem. In addition to the end replication problem, without a mechanism to obscure the end of linear chromosomes, these ends can be recognized by the cellular DNA damage response (DDR) machinery as double strand breaks (DSBs), leading to activation of the p53, senescence and/or cell death. Cells that bypass p53 will attempt to repair these aberrant DSBs leading to chromosome fusions and genomic instability. This is known as the end protection problem.In response to these pressures, a ribonucleoprotein, telomerase, evolved. Telomerase consists of a protein subunit TERT (hTERT in humans) and an RNA subunit TR (hTR in humans). TERT is a reverse transcriptase that uses TR as a template to add sequence-specific non-coding tandem repeats to the end of linear chromosomes. This sequence serves as a buffer for terminal sequence loss. The sequence specificity allows for binding the telomere protein complex, shelterin. Together the sequence and the shelterin complex are known as the telomere. The binding of shelterin and the generation of a 3’ single stranded DNA overhang allows the telomeric DNA to bend back on itself and invade into the double stranded sections of the telomeric DNA thus sequestering the telomere end from the DDR. In humans, the expression hTERT is limited to early developmental cells and some adult stem cells. The down regulation of hTERT following differentiation leads to telomere shortening accompanying each cell division. This mechanism limits the replicative potential of most human somatic cells. This limitation acts as a tumor suppression mechanism which must be overcome by cancer in order to achieve their immortal phenotype. Most cancers accomplish this by reactivating and upregulating hTERT expression. This reactivation must occur before telomere loss triggers senescence or cell death. Aberrant telomere length set point in the stem cell state, can lead to a range of diseases. Longer telomere set point allows cells more “time” to accumulate oncogenic mutations increasing the risk of developing cancer. On the other hand, short telomeres can lead to a spectrum of diseases that present as tissue failure and depletion of one or more stem cell compartments. The most common among these diseases, dyskeratosis congenita (DC), presents with nail dysplasia, abnormal skin pigmentation, and oral leukoplakia. Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1-S) and the amino acid L104 outside the TEL patch, TPP1’s telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1-S (TPP1-S KO), as well as the complete TPP1 KO cell lines, show telomere shortening. However, TPP1-S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1-S KO. In contrast to TPP1S KO cells, TPP1L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point. While the study of TPP1 concentrates on the mechanisms underlying telomere length regulation and maintenance in the context of telomerase recruitment and elongation, we have developed a tool to study the switch to C-strand fill in synthesis. Following replication, cellular exonucleases process telomere ends allowing for telomerase to add de novo sequence and establishing the overhang. Following telomerase synthesis, the CST complex is recruited to the telomere, blocking further action by telomerase, and recruiting polymerase α (POLα). POLα then primes and fills in the C-strand of the telomere. This mechanism is also active in telomerase negative cells and internal DSBs. Several mutations in CTC1, a member of the CST complex, have been associated with DC. Here we have developed a series of human embryonic stem cell (hESC) lines harboring floxed CTC1 conditional alleles in order to study the phenotypes associated with pathogenic CTC1 alleles. Loss of CTC1 increases stem cells’ sensitivity to single cell passaging requiring finer temporal control over the loss of the gene. To establish this control, we used a tamoxifen-inducible Cre system the control to loss of CTC1. This control enabled us to get around the increased sensitivity following single cell passaging and validate the system. Our system is able to recapitulate the overhang and deprotection phenotypes associated with CTC1. These lines can be used to complement disease-associated alleles and perform more detailed structure-function analysis of CTC1.