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1.
Cell ; 174(1): 218-230.e13, 2018 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-29804836

RESUMEN

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.


Asunto(s)
ARN no Traducido/química , Telomerasa/metabolismo , Biocatálisis , Línea Celular , Células HeLa , Humanos , Chaperonas Moleculares , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismo , Telomerasa/antagonistas & inhibidores , Telomerasa/química , Telomerasa/genética , Telómero/metabolismo
2.
Cell ; 160(5): 1013-1026, 2015 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-25684364

RESUMEN

VIDEO ABSTRACT: 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.


Asunto(s)
Peces Killi/fisiología , Envejecimiento , Animales , Secuencia de Bases , Sistemas CRISPR-Cas , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Técnicas Genéticas , Humanos , Peces Killi/genética , Masculino , Modelos Animales , Datos de Secuencia Molecular , Telomerasa/genética , Telomerasa/metabolismo , Vertebrados/fisiología
3.
Cell ; 150(3): 481-94, 2012 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-22863003

RESUMEN

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. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are 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.


Asunto(s)
Telomerasa/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Línea Celular , Línea Celular Tumoral , Cuerpos Enrollados/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Complejo Shelterina , Telomerasa/química , Telomerasa/genética , Proteínas de Unión a Telómeros/genética
4.
Genes Dev ; 29(23): 2420-34, 2015 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-26584619

RESUMEN

Telomerase inactivation causes loss of the male germline in worms, fish, and mice, indicating a conserved dependence on telomere maintenance in this cell lineage. Here, using telomerase reverse transcriptase (Tert) reporter mice, we found that very high telomerase expression is a hallmark of undifferentiated spermatogonia, the mitotic population where germline stem cells reside. We exploited these high telomerase levels as a basis for purifying undifferentiated spermatogonia using fluorescence-activated cell sorting. Telomerase levels in undifferentiated spermatogonia and embryonic stem cells are comparable and much greater than in somatic progenitor compartments. Within the germline, we uncovered an unanticipated gradient of telomerase activity that also enables isolation of more mature populations. Transcriptomic comparisons of Tert(High) undifferentiated spermatogonia and Tert(Low) differentiated spermatogonia by RNA sequencing reveals marked differences in cell cycle and key molecular features of each compartment. Transplantation studies show that germline stem cell activity is confined to the Tert(High) cKit(-) population. Telomere shortening in telomerase knockout strains causes depletion of undifferentiated spermatogonia and eventual loss of all germ cells after undifferentiated spermatogonia drop below a critical threshold. These data reveal that high telomerase expression is a fundamental characteristic of germline stem cells, thus explaining the broad dependence on telomerase for germline immortality in metazoans.


Asunto(s)
Células Madre Adultas/enzimología , Regulación Enzimológica de la Expresión Génica , Espermatogonias/enzimología , Telomerasa/genética , Telomerasa/metabolismo , Animales , Diferenciación Celular/genética , Células Madre Embrionarias/enzimología , Citometría de Flujo , Técnicas de Sustitución del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genética
5.
Nature ; 474(7351): 399-402, 2011 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-21602826

RESUMEN

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 disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour 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 dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), 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, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita 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.


Asunto(s)
Disqueratosis Congénita/genética , Disqueratosis Congénita/patología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Telómero/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular , Reprogramación Celular , Fibroblastos , Regulación de la Expresión Génica , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN/genética , Telomerasa/genética , Telomerasa/metabolismo , Telómero/enzimología , Telómero/genética , Telómero/metabolismo
7.
Elife ; 82019 08 27.
Artículo en Inglés | MEDLINE | ID: mdl-31452512

RESUMEN

Only a subset of cancer patients respond to T-cell checkpoint inhibitors, highlighting the need for alternative immunotherapeutics. We performed CRISPR-Cas9 screens in a leukemia cell line to identify perturbations that enhance natural killer effector functions. Our screens defined critical components of the tumor-immune synapse and highlighted the importance of cancer cell interferon-γ signaling in modulating NK activity. Surprisingly, disrupting the ubiquitin ligase substrate adaptor DCAF15 strongly sensitized cancer cells to NK-mediated clearance. DCAF15 disruption induced an inflamed state in leukemic cells, including increased expression of lymphocyte costimulatory molecules. Proteomic and biochemical analysis revealed that cohesin complex members were endogenous client substrates of DCAF15. Genetic disruption of DCAF15 was phenocopied by treatment with indisulam, an anticancer drug that functions through DCAF15 engagement. In AML patients, reduced DCAF15 expression was associated with improved survival. These findings suggest that DCAF15 inhibition may have useful immunomodulatory properties in the treatment of myeloid neoplasms.


Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/genética , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/patología , Línea Celular Tumoral , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Leucemia Mieloide Aguda/mortalidad , Análisis de Supervivencia
8.
Stem Cell Reports ; 10(2): 553-567, 2018 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-29337115

RESUMEN

Undifferentiated spermatogonia comprise a pool of stem cells and progenitor cells that show heterogeneous expression of markers, including the cell surface receptor GFRα1. Technical challenges in isolation of GFRα1+ versus GFRα1- undifferentiated spermatogonia have precluded the comparative molecular characterization of these subpopulations and their functional evaluation as stem cells. Here, we develop a method to purify these subpopulations by fluorescence-activated cell sorting and show that GFRα1+ and GFRα1- undifferentiated spermatogonia both demonstrate elevated transplantation activity, while differing principally in receptor tyrosine kinase signaling and cell cycle. We identify the cell surface molecule melanocyte cell adhesion molecule (MCAM) as differentially expressed in these populations and show that antibodies to MCAM allow isolation of highly enriched populations of GFRα1+ and GFRα1- spermatogonia from adult, wild-type mice. In germ cell culture, GFRα1- cells upregulate MCAM expression in response to glial cell line-derived neurotrophic factor (GDNF)/fibroblast growth factor (FGF) stimulation. In transplanted hosts, GFRα1- spermatogonia yield GFRα1+ spermatogonia and restore spermatogenesis, albeit at lower rates than their GFRα1+ counterparts. Together, these data provide support for a model of a stem cell pool in which the GFRα1+ and GFRα1- cells are closely related but show key cell-intrinsic differences and can interconvert between the two states based, in part, on access to niche factors.


Asunto(s)
Diferenciación Celular/genética , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Espermatogénesis/genética , Espermatogonias/citología , Animales , Antígeno CD146/genética , Linaje de la Célula/genética , Factores de Crecimiento de Fibroblastos/genética , Citometría de Flujo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Masculino , Ratones , Transducción de Señal/genética , Espermatogonias/crecimiento & desarrollo , Nicho de Células Madre/genética , Células Madre/citología , Testículo/citología
9.
Nat Med ; 18(1): 111-9, 2011 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-22138751

RESUMEN

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 crucial for filtration--are thought to lack substantial capacity for regeneration. Here we show that podocytes rapidly lose differentiation markers and enter the cell cycle in adult mice in which the telomerase protein component TERT is conditionally expressed. Transgenic TERT expression in mice induces marked upregulation of Wnt signaling and disrupts glomerular structure, resulting in a collapsing glomerulopathy resembling those in human disease, including HIV-associated nephropathy (HIVAN). Human and mouse HIVAN kidneys show increased expression of TERT and activation of Wnt signaling, indicating that these are general features of collapsing glomerulopathies. Silencing transgenic TERT expression or inhibiting Wnt signaling through systemic expression of the Wnt inhibitor Dkk1 in either TERT transgenic mice or in a mouse model of HIVAN 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 capacity of podocytes to reversibly enter the cell cycle, suggest that podocyte renewal may contribute to glomerular homeostasis and implicate the telomerase and Wnt-ß-catenin pathways in podocyte proliferation and disease.


Asunto(s)
Nefropatía Asociada a SIDA/metabolismo , Glomérulos Renales/metabolismo , Riñón/metabolismo , Podocitos/citología , Telomerasa/metabolismo , Vía de Señalización Wnt , Nefropatía Asociada a SIDA/genética , Animales , Diferenciación Celular , Proliferación Celular , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/citología , Glomérulos Renales/citología , Glomérulos Renales/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Podocitos/metabolismo , Telomerasa/genética
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