title: 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.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Kunitoshi
authors.initials: K
authors.lastname: Chiba
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Vogan
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Wu
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Manraj S
authors.initials: MS
authors.lastname: Gill
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Xiaozhu
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA hockemeyer@berkeley.edu.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hockemeyer
mesh_terms: D015139:Blotting, Southern; D000939:Epitopes / Q000378:metabolism*; D000072669:Gene Editing; D005838:Genotype; D000066449:Human Embryonic Stem Cells / Q000378:metabolism; D006801:Humans; D010641:Phenotype; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D019098:Telomerase / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*; D059505:Telomere Homeostasis
chemical_list: D000939:Epitopes; D019098:Telomerase
keywords: CRISPR/Cas9; embryonic stem cells; genome editing; pluripotent stem cells; telomerase; telomeres