Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

ABSTRACT

Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.

KEYWORDS:

• genome editing
• telomerase
• telomeres
• pluripotent stem cells
• CRISPR/Cas9
• embryonic stem cells

ACKNOWLEDGMENTS

We thank Kartoosh Heydari for technical assistance and the Collins and Hockemeyer laboratories for helpful discussion.

K. Collins, J. M. Vogan, and X. Zhang are supported by US National Institutes of Health grant R01-HL079585. R. A. Wu and K. Collins were supported by NIH RO1-GM054198. K. Chiba is supported by a Nakajima foundation fellowship. D. Hockemeyer is a New Scholar in Aging of the Ellison Medical Foundation and a Pew-Stewart Scholar in the Cancer Research. The work in the Hockemeyer laboratory is supported by the Glenn Foundation, the Shurl and Kay Curci Foundations, the Siebel Stem Cell Center, and NIH R01-CA196884.

K. Chiba conducted the experiments in hESCs. J. M. Vogan and X. Zhang conducted all experiments in HCT116 cells. K. Chiba and R. A. Wu conducted the oligonucleotide-based telomerase purification and direct extension assays. M. S. Gill prepared several DNA constructs. K. Collins and D. Hockemeyer supervised research. K. Collins and D. Hockemeyer wrote the manuscript with the assistance of all other authors.