Human Telomerase: Abundance, Activity and Activation
Telomerase is the ribonucleoprotein enzyme that elongates telomeric DNA to compensate for the attrition occurring during DNA replication. It has two key components, telomerase RNA (TR) and telomerase reverse transcriptase (TERT). Its activity is required by stem cells and most cancer cells to facilitate indefinite proliferation. Knowing the levels of telomerase in cells and the mechanisms controlling these levels is important for understanding telomerase biology and cellular immortality. This thesis presents methods to quantify cellular levels of human telomerase, investigations of how specific cancer-associated TERT promoter mutations affect telomerase levels and genome-editing strategies to modify the endogenous TERT gene.
Quantitative analysis on the immortalized human cell lines HEK 293T and HeLa indicated TR and TERT are present at much lower levels than previously reported. The data also suggested the existence of unassembled subpopulations of TR and TERT within these cells, which was supported by the observation that overexpressing either subunit increased total telomerase activity. The specific activity of human telomerase was determined. It was demonstrated that the overexpressed “super telomerase” is as catalytically active as the endogenous telomerase and is thus a good model for biochemical studies.
The recent identification of highly recurrent TERT promoter mutations in human cancers suggested a new model for telomerase activation during tumorigenesis. Quantitative methods established here were applied to analyze telomerase levels in a panel of 23 urothelial cancer cell lines. The results indicated that these mutations correlate with higher levels of TERT mRNA, TERT protein, telomerase activity and telomere length.
To further test the causality between the TERT promoter mutations and increased telomerase levels, protocols were developed to modify the endogenous TERT promoter with CRISPR-Cas9-mediated genome editing. To overcome the low editing efficiency at the TERT locus, a two-step “pop-in/pop-out” strategy was employed. A similar protocol was also designed to fuse an N-terminal FLAG-SNAP-tag to the endogenous TERT protein, which enabled reliable detection by immunoblot, immunoprecipitation and subcellular localization of TERT.
In summary, this thesis addresses several important questions regarding telomerase and its expression. Some techniques developed here also have the potential to be widely applicable in other research fields.