title: Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.
abstract: Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;
authors.forename: Alec N
authors.initials: AN
authors.lastname: Sexton
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Regalado
authors.affiliation: The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA;
authors.forename: Christine S
authors.initials: CS
authors.lastname: Lai
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California 94804, USA.
authors.forename: Gregory J
authors.initials: GJ
authors.lastname: Cost
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California 94804, USA.
authors.forename: Colleen M
authors.initials: CM
authors.lastname: O'Neil
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California 94804, USA.
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California 94804, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA;
authors.forename: Rudolf
authors.initials: R
authors.lastname: Jaenisch
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA; hockemeyer@berkeley.edu kcollins@berkeley.edu.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Collins
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA; hockemeyer@berkeley.edu kcollins@berkeley.edu.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hockemeyer
mesh_terms: D053595:Embryonic Stem Cells; D004789:Enzyme Activation / Q000235:genetics; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006801:Humans; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D000089804:Shelterin Complex; D019098:Telomerase / Q000235:genetics / Q000378:metabolism*; D016615:Telomere / Q000201:enzymology* / Q000235:genetics; D059505:Telomere Homeostasis / Q000235:genetics*; D034501:Telomere-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000097764:ETS Translocation Variant 6 Protein
chemical_list: D050783:Proto-Oncogene Proteins c-ets; D012097:Repressor Proteins; D000089804:Shelterin Complex; D019098:Telomerase; D034501:Telomere-Binding Proteins; D000097764:ETS Translocation Variant 6 Protein; C495876:ACD protein, human; C440849:POT1 protein, human; C509186:TERT protein, human
keywords: embryonic stem cells; human genome engineering; shelterin; telomerase; telomere maintenance