| Telomeres are the repetitive sequences at the ends of linear chromosomes. The key functions of telomeres are to protect the cells from losing genomic information and to prevent chromosome ends from being repaired by the double strand break repair machinery. To counteract loss of telomeric DNA, cells can express a reverse transcriptase, telomerase, that synthesizes telomeric repeats de novo. In humans, telomerase activity is mostly restricted to germ and stem cells, so the telomeres of most somatic cells progressively shorten with each cell division. Once telomeres become critically short, they are recognized as sites of DNA damage and cells cease to proliferate. By this mechanism, telomere shortening functions as a tumor suppression mechanism. TERT, the protein component of telomerase, becomes silenced once stem cells differentiate. However, in 90% of cancer cells, TERT is transcriptionally re-activated. Thus, telomerase regulation is crucial for our understanding of telomere length regulation in stem cell maintenance and tumorigenesis. To understand how telomerase acts on telomeres, I attempted to endogenously tag telomerase. To do this I inserted epitope tags at the endogenous TERT locus in hESCs using genome editing. However, I found that all the tested tags cause defects in telomere maintenance, which was previously not appreciated in experiments using exogenous overexpression.;Recently, point mutations in the TERT promoter were identified as the most frequent non-coding mutations in cancer. To elucidate the role of TERT promoter mutations (TPMs) in tumorigenesis, I genetically engineered these TPMs into human embryonic stem cells (hESCs) using genome editing. Using the resulting isogenic hESC lines, I demonstrated that TPMs lead to a failure of TERT silencing upon differentiation from stem into somatic cells. To understand role of TPMs in tumorigenesis, I monitored long-term telomere maintenance and proliferation in human fibroblasts engineered to carry TPMs. I found that TPMs immortalize cells but do not prevent telomere shortening and telomere fusions. In vitro, around the time when telomere fusions occurred, TERT expression was gradually increased. Thus, TPMs are required, but not sufficient, for cancer cell immortality and contribute to tumorigenesis in two steps. First, TPMs expand proliferation capacity of a cell by elongating only the shortest telomeres but do not prevent overall telomere shortening. In the second step, TPMs fuel tumorigenesis by not fully suppressing genomic instability. In order for cells to immortalize they need to upregulate TERT during this second step. |
