Date of Award
Doctor of Philosophy (PhD)
de Lange Laboratory
POT1 is a single stranded telomeric DNA binding protein implicated in telomere length regulation in human cells. To address the role of POT1 in telomere protection we used RNAi in human cells. We confirmed that POT1 acts as a negative regulator of telomerase and showed that POT1 is required to protect telomeres. Reduced levels of POT1 elicited a strong telomere DNA damage response and a growth arrest in primary cells; the 3’ telomeric overhang shortened and the sequence of the 5’ terminus changed from its precise sequence (ATC-5’) to a randomized ending. In order to determine the phenotype of complete POT1 loss, we used conditional gene deletion in the mouse. Unexpectedly, we identified two POT1 orthologs in the mouse and rat genomes, whereas other mammals have one. As both proteins (POT1a and POT1b) localized to telomeres based on IF and ChIP, we targeted both genes for Cremediated deletion. POT1a/b double-knockout (DKO) cells exhibited a telomeric DNA damage signal and senescence. DKO cells also displayed a novel telomere dysfunction phenotype, extensive endoreduplication. However, POT1a/b were largely dispensable for repression of telomere fusions, which is a prominent outcome of inhibition of another telomere binding protein, TRF2. Previous structural analysis of POT1 and its binding partner TPP1 predict that they act interdependently. Consistent with this, we found that TPP1 is essential for the telomeric function of both mouse POT1 proteins. Single knockouts and complementation experiments revealed that POT1a and POT1b have distinct functions. POT1a was found to be primarily responsible for repression of the DNA damage signal at telomeres, while POT1b had a unique role in repressing an activity that creates extended telomeric overhangs. POT1b KO cells showed accelerated telomere shortening indicating that POT1b controls exonucleolytic degradation of the C-rich strand. The enhanced telomere shortening of POT1b KO cells explains our finding that POT1b KO mice display aging phenotypes reminiscent of late generation telomerase knockout mice. My results argue that a gene duplication event gave rise to two functionally distinct POT1 proteins in rodents. Such a divergence is unprecedented in chromosome biology and has implications for modeling telomere biology and telomere-related diseasestates in the mouse.
Hockemeyer, Dirk, "The Role of Pot1 in Telomere Protection and Maintenance" (2007). Student Theses and Dissertations. 191.