RESUMEN
Genome-wide association studies (GWAS) have become well-powered to detect loci associated with telomere length. However, no prior work has validated genes nominated by GWAS to examine their role in telomere length regulation. We conducted a multi-ancestry meta-analysis of 211,369 individuals and identified five novel association signals. Enrichment analyses of chromatin state and cell-type heritability suggested that blood/immune cells are the most relevant cell type to examine telomere length association signals. We validated specific GWAS associations by overexpressing KBTBD6 or POP5 and demonstrated that both lengthened telomeres. CRISPR/Cas9 deletion of the predicted causal regions in K562 blood cells reduced expression of these genes, demonstrating that these loci are related to transcriptional regulation of KBTBD6 and POP5. Our results demonstrate the utility of telomere length GWAS in the identification of telomere length regulation mechanisms and validate KBTBD6 and POP5 as genes affecting telomere length regulation.
Asunto(s)
Estudio de Asociación del Genoma Completo , Homeostasis del Telómero , Telómero , Humanos , Telómero/genética , Telómero/metabolismo , Células K562 , Homeostasis del Telómero/genética , Polimorfismo de Nucleótido Simple , Regulación de la Expresión Génica , Sistemas CRISPR-CasRESUMEN
Overcoming replicative senescence is an essential step during oncogenesis, and the reactivation of TERT through promoter mutations is a common mechanism. TERT promoter mutations are acquired in about 75% of melanomas but are not sufficient to maintain telomeres, suggesting that additional mutations are required. We identified a cluster of variants in the promoter of ACD encoding the shelterin component TPP1. ACD promoter variants are present in about 5% of cutaneous melanoma and co-occur with TERT promoter mutations. The two most common somatic variants create or modify binding sites for E-twenty-six (ETS) transcription factors, similar to mutations in the TERT promoter. The variants increase the expression of TPP1 and function together with TERT to synergistically lengthen telomeres. Our findings suggest that TPP1 promoter variants collaborate with TERT activation to enhance telomere maintenance and immortalization in melanoma.
Asunto(s)
Melanoma , Regiones Promotoras Genéticas , Complejo Shelterina , Neoplasias Cutáneas , Telomerasa , Homeostasis del Telómero , Proteínas de Unión a Telómeros , Humanos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Melanoma/genética , Mutación , Regiones Promotoras Genéticas/genética , Complejo Shelterina/genética , Neoplasias Cutáneas/genética , Telomerasa/genética , Telómero/genética , Telómero/metabolismo , Homeostasis del Telómero/genética , Proteínas de Unión a Telómeros/genética , Activación TranscripcionalRESUMEN
Telomere length regulation is essential for cell viability in eukaryotes. While many pathways that affect telomere length are known, we do not yet have a complete understanding of the mechanism of length regulation. To identify new pathways that might regulate telomere length, we carried out a genetic screen in yeast and identified the cyclin-dependent kinase complex Bur1/2 as a regulator of telomere length. Mutations in either BUR1 cyclin-dependent kinase or the associated BUR2 cyclin resulted in short telomeres. This regulation did not function through the known role of BUR1 in regulating histone modification as bur1∆ set2∆ and bur2∆ set2∆ double mutants rescued cell growth but did not rescue the telomere shortening effects. We found that both bur1∆ and bur2∆ set2∆ were also defective in de novo telomere addition, and deletion of SET2 did also not rescue this elongation defect. The Bur1/2 cyclin-dependent kinase regulates transcription of many genes. We found that TLC1 RNA levels were reduced in bur2∆ set2∆ mutants; however, overexpression of TLC1 restored the transcript levels but did not restore de novo telomere elongation or telomere length. These data suggest that the Bur1/2 kinase plays a role in telomere elongation separate from its role in transcription of telomerase components. Dissecting the role of the Bur1/2 kinase pathway at telomeres will help complete our understanding of the complex network of telomere length regulation.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Quinasas Ciclina-Dependientes/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Telómero/genética , Telómero/metabolismo , Transcripción GenéticaRESUMEN
Previous models suggested that regulation of telomere length in Saccharomyces cerevisiae by Tel1(ATM) and Mec1(ATR) would parallel the established pathways regulating the DNA damage response. Here, we provide evidence that telomere length regulation differs from the DNA damage response in both the Tel1 and Mec1 pathways. We found that Rad53 mediates a Mec1 telomere length regulation pathway but is dispensable for Tel1 telomere length regulation, whereas in the DNA damage response, Rad53 is regulated by both Mec1 and Tel1 Using epistasis analysis with a Tel1 hypermorphic allele, Tel1-hy909, we found that the MRX complex is not required downstream of Tel1 for telomere elongation but is required downstream of Tel1 for the DNA damage response. Our data suggest that nucleolytic telomere end processing is not a required step for telomerase to elongate telomeres.
Asunto(s)
Daño del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Telómero/metabolismo , Secuencias de Aminoácidos , Fosforilación , Proteínas de Saccharomyces cerevisiae/química , Transducción de Señal , Homeostasis del TelómeroRESUMEN
Cancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which trigger the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain-containing protein 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose-dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenance in vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously investigated.
Asunto(s)
Proteínas Nucleares/genética , Homeostasis del Telómero/genética , Acortamiento del Telómero/genética , Factores de Transcripción/genética , Acetanilidas/farmacología , Animales , Azepinas/farmacología , Southern Blotting , Proteínas de Ciclo Celular , Línea Celular , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Células HeLa , Compuestos Heterocíclicos con 3 Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Hibridación Fluorescente in Situ , Ratones , Morfolinas/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Pironas/farmacología , Interferencia de ARN , Telomerasa/genética , Telomerasa/metabolismo , Telómero/efectos de los fármacos , Telómero/enzimología , Telómero/genética , Homeostasis del Telómero/efectos de los fármacos , Acortamiento del Telómero/efectos de los fármacos , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Triazoles/farmacologíaRESUMEN
The regulation of telomere length equilibrium is essential for cell growth and survival since critically short telomeres signal DNA damage and cell cycle arrest. While the broad principles of length regulation are well established, the molecular mechanism of how these steps occur is not fully understood. We mutagenized the RIF2 gene in Saccharomyces cerevisiae to understand how this protein blocks excess telomere elongation. We identified an N-terminal domain in Rif2 that is essential for length regulation, which we have termed BAT domain for Blocks Addition of Telomeres. Tethering this BAT domain to Rap1 blocked telomere elongation not only in rif2Δ mutants but also in rif1Δ and rap1C-terminal deletion mutants. Mutation of a single amino acid in the BAT domain, phenylalanine at position 8 to alanine, recapitulated the rif2Δ mutant phenotype. Substitution of F8 with tryptophan mimicked the wild-type phenylalanine, suggesting the aromatic amino acid represents a protein interaction site that is essential for telomere length regulation.
