Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase, TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. A minimal TPP1 OB-fold serves to inhibit telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify specific loop residues within the TPP1 OB-fold necessary for association with telomerase, and critical residues in TERT, including those mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.

Summary Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2–3 months. As a proof-of-principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high throughput manner and for investigating candidates arising from human genome-wide studies.

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease1–6. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita (DC) is characterized by defective maintenance of blood, pulmonary tissue, and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis7,8. Short telomeres, a hallmark of DC, impairs tissue stem cell function in mouse models, suggesting that a tissue stem cell defect underlies the pathophysiology of DC9,10. Here, we show that even in the undifferentiated state, iPSCs from DC patients harbor the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked DC severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of DC caused by mutations in TCAB1, telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, suggesting that a similar process occurs in tissue stem cells in DC patients. These findings in iPSCs from DC patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell culture-based system for the development of targeted therapeutics.

Mechanisms of epithelial cell renewal remain poorly understood in the mammalian kidney, particularly in the glomerulus, a site of cellular damage in chronic kidney disease. Within the glomerulus, podocytes – differentiated epithelial cells critical for filtration – are thought to lack significant capacity for regeneration. Here, we show that podocytes rapidly lose differentiation markers and enter cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression induces marked upregulation of Wnt signaling and disrupts glomerular structure resulting in a collapsing glomerulopathy resembling those in humans, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased levels of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Either silencing transgenic TERT expression or inhibition of Wnt signaling through systemic expression of the Wnt-inhibitor Dkk1 in TERT transgenic mice results in marked normalization of podocytes, including rapid cell cycle exit, re-expression of differentiation markers and improved filtration barrier function. These data reveal an unexpected property of podocytes to reversibly enter cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt/β-catenin pathways in podocyte proliferation and disease.

Ribonucleoprotein enzymes require dynamic conformations of their RNA constituents for regulated catalysis. Human telomerase employs a non-coding RNA (hTR) with a bipartite arrangement of domains-a template-containing core and a distal three-way junction (CR4/5) that stimulates catalysis through unknown means. Here, we show that telomerase activity unexpectedly depends upon the holoenzyme protein TCAB1, which in turn controls conformation of CR4/5. Cells lacking TCAB1 exhibit a marked reduction in telomerase catalysis without affecting enzyme assembly. Instead, TCAB1 inactivation causes unfolding of CR4/5 helices that are required for catalysis and for association with the telomerase reverse-transcriptase (TERT). CR4/5 mutations derived from patients with telomere biology disorders provoke defects in catalysis and TERT binding similar to TCAB1 inactivation. These findings reveal a conformational "activity switch" in human telomerase RNA controlling catalysis and TERT engagement. The identification of two discrete catalytic states for telomerase suggests an intramolecular means for controlling telomerase in cancers and progenitor cells.