RESUMEN
Telomere maintenance requires the extension of the G-rich telomeric repeat strand by telomerase and the fill-in synthesis of the C-rich strand by Polα/primase. At telomeres, Polα/primase is bound to Ctc1/Stn1/Ten1 (CST), a single-stranded DNA-binding complex. Like mutations in telomerase, mutations affecting CST-Polα/primase result in pathological telomere shortening and cause a telomere biology disorder, Coats plus (CP). We determined cryogenic electron microscopy structures of human CST bound to the shelterin heterodimer POT1/TPP1 that reveal how CST is recruited to telomeres by POT1. Our findings suggest that POT1 hinge phosphorylation is required for CST recruitment, and the complex is formed through conserved interactions involving several residues mutated in CP. Our structural and biochemical data suggest that phosphorylated POT1 holds CST-Polα/primase in an inactive, autoinhibited state until telomerase has extended the telomere ends. We propose that dephosphorylation of POT1 releases CST-Polα/primase into an active state that completes telomere replication through fill-in synthesis.
Asunto(s)
ADN Polimerasa I , Complejo Shelterina , Proteínas de Unión a Telómeros , Telómero , Humanos , Microscopía por Crioelectrón , ADN Polimerasa I/metabolismo , ADN Primasa/metabolismo , ADN Primasa/genética , Modelos Moleculares , Fosforilación , Complejo Shelterina/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Although telomeres are essential for chromosome stability, they represent fragile structures in our genome. Telomere shortening occurs during aging in cells lacking telomerase due to the end replication problem. In addition, recent work uncovered that the bulk of telomeric DNA poses severe hurdles for the semiconservative DNA replication machinery, requiring the assistance of an increasing number of specialized factors that prevent accidental telomere loss or damage events. In this issue of Genes & Development, Yang and colleagues (pp. 956-969) discover that TFIIH, a basic component of the PolII transcription initiation and nucleotide excision repair machinery, facilitates telomere replication. TFIIH is recruited to telomeres by the shelterin component TRF1, taking on at telomeres a moonlighting function.
Asunto(s)
Telomerasa , Proteína 1 de Unión a Repeticiones Teloméricas , Telómero/genética , Telómero/metabolismo , Acortamiento del Telómero , Proteínas de Unión a Telómeros/metabolismo , Telomerasa/metabolismo , Complejo ShelterinaRESUMEN
Telomeric DNA challenges the replisome and requires TRF1 for efficient duplication. TRF1 recruits the BLM helicase, but BLM loss does not explain the extensive telomere fragility, ATR signaling, and sister telomere associations (STAs) induced by TRF1 deletion. Here, we document that Helix2 of the TRFH domain and Helix1 of the Myb domain of TRF1 are required for efficient telomere replication. Mutation of both helices generated a TRF1 separation-of-function mutant (TRF1-E83K/LW-TI) that induced severe telomere replication defects but no ATR signaling or STAs. We identified the transcription and nucleotide excision repair (NER) factor TFIIH as a critical effector of TRF1. Loss of TFIIH subunits, but no other NER factors, caused the same telomere replication phenotypes as the TRF1-E83K/LW-TI mutant independent of the effects on TRF1 expression. TFIIH subunits coimmunoprecipitated with wild-type TRF1 but not with TRF1-E83K/LW-TI. These results establish that the major mechanism by which TRF1 ensures telomere replication involves a noncanonical function of TFIIH.
Asunto(s)
Telómero , Proteína 1 de Unión a Repeticiones Teloméricas , Telómero/genética , Telómero/metabolismo , Replicación del ADN/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN/metabolismoRESUMEN
The mammalian telomeric shelterin complex-comprised of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-blocks the DNA damage response at chromosome ends and interacts with telomerase and the CST complex to regulate telomere length. The evolutionary origins of shelterin are unclear, partly because unicellular organisms have distinct telomeric proteins. Here, we describe the evolution of metazoan shelterin, showing that TRF1 emerged in vertebrates upon duplication of a TRF2-like ancestor. TRF1 and TRF2 diverged rapidly during vertebrate evolution through the acquisition of new domains and interacting factors. Vertebrate shelterin is also distinguished by the presence of an HJRL domain in the split C-terminal OB fold of POT1, whereas invertebrate POT1s carry inserts of variable nature. Importantly, the data reveal that, apart from the primate and rodent POT1 orthologs, all metazoan POT1s are predicted to have a fourth OB fold at their N termini. Therefore, we propose that POT1 arose from a four-OB-fold ancestor, most likely an RPA70-like protein. This analysis provides insights into the biology of shelterin and its evolution from ancestral telomeric DNA-binding proteins.
Asunto(s)
Proteína 2 de Unión a Repeticiones Teloméricas , Tripeptidil Peptidasa 1 , Animales , Mamíferos/genética , Complejo Shelterina , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Telomere repeat binding factor 2 (TRF2) is an essential component of the telomeres and also plays an important role in a number of other non-telomeric processes. Detailed knowledge of the binding and interaction of TRF2 with telomeric nucleosomes is limited. Here, we study the binding of TRF2 to in vitro-reconstituted kilobasepair-long human telomeric chromatin fibres using electron microscopy, single-molecule force spectroscopy and analytical ultracentrifugation sedimentation velocity. Our electron microscopy results revealed that full-length and N-terminally truncated TRF2 promote the formation of a columnar structure of the fibres with an average width and compaction larger than that induced by the addition of Mg2+, in agreement with the in vivo observations. Single-molecule force spectroscopy showed that TRF2 increases the mechanical and thermodynamic stability of the telomeric fibres when stretched with magnetic tweezers. This was in contrast to the result for fibres reconstituted on the 'Widom 601' high-affinity nucleosome positioning sequence, where minor effects on fibre stability were observed. Overall, TRF2 binding induces and stabilises columnar fibres, which may play an important role in telomere maintenance.
Asunto(s)
Cromatina , Complejo Shelterina , Proteína 2 de Unión a Repeticiones Teloméricas , Humanos , Nucleosomas , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Papillary thyroid cancer (PTC) is the most common endocrine malignancy. 10% to 15% of individuals show familial clustering with three or more affected members, but the factors underlying this risk are unknown. In a group of recently studied individuals with POT1 pathogenic variants and ultra-long telomere length, PTC was the second most common solid tumor. We tested whether variants in POT1 and four other telomere-maintenance genes associated with familial cancer underlie PTC susceptibility. Among 470 individuals, we identified pathogenic or likely pathogenic variants in three genes encoding telomere-binding proteins: POT1, TINF2, and ACD. They were found in 4.5% and 1.5% of familial and unselected cases, respectively. Individuals harboring these variants had ultra-long telomere length, and 15 of 18 (83%) developed other cancers, of which melanoma, lymphoma, and sarcoma were most common. Among individuals with PTC and melanoma, 22% carried a deleterious germline variant, suggesting that a long telomere syndrome might be clinically recognizable. Successive generations had longer telomere length than their parents and, at times, developed more cancers at younger ages. Tumor sequencing identified a single oncogenic driver, BRAF p.Val600Glu, in 10 of 10 tumors studied, but no telomere-maintenance mechanism, including at the TERT promoter. These data identify a syndromic subset of PTCs with locus heterogeneity and telomere lengthening as a convergent mechanism. They suggest these germline variants lower the threshold to cancer by obviating the need for an acquired telomere-maintenance mechanism in addition to sustaining the longevity of oncogenic mutations.
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Predisposición Genética a la Enfermedad , Mutación de Línea Germinal , Complejo Shelterina , Homeostasis del Telómero , Proteínas de Unión a Telómeros , Telómero , Cáncer Papilar Tiroideo , Neoplasias de la Tiroides , Humanos , Proteínas de Unión a Telómeros/genética , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , Mutación de Línea Germinal/genética , Masculino , Femenino , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Homeostasis del Telómero/genética , Telómero/genética , Persona de Mediana Edad , Adulto , Proteínas Proto-Oncogénicas B-raf/genética , Anciano , Melanoma/genética , Melanoma/patología , LinajeRESUMEN
Protecting chromosome ends from misrecognition as double-stranded (ds) DNA breaks is fundamental to eukaryotic viability. The protein complex shelterin prevents a DNA damage response at mammalian telomeres. Mammalian shelterin proteins TRF1 and TRF2 and their homologs in yeast and protozoa protect telomeric dsDNA. N-terminal homodimerization and C-terminal Myb-domain-mediated dsDNA binding are two structural hallmarks of end protection by TRF homologs. Yet our understanding of how Caenorhabditis elegans protects its telomeric dsDNA is limited. Recently identified C. elegans proteins TEBP-1 (also called DTN-1) and TEBP-2 (also called DTN-2) are functional homologs of TRF proteins, but how they bind DNA and whether or how they dimerize is not known. TEBP-1 and TEBP-2 harbor three Myb-containing domains (MCDs) and no obvious dimerization domain. We demonstrate biochemically that only the third MCD binds DNA. We solve the X-ray crystal structure of TEBP-2 MCD3 with telomeric dsDNA to reveal the structural mechanism of telomeric dsDNA protection in C. elegans. Mutagenesis of the DNA-binding site of TEBP-1 and TEBP-2 compromises DNA binding in vitro, and increases DNA damage signaling, lengthens telomeres, and decreases brood size in vivo. Via an X-ray crystal structure, biochemical validation of the dimerization interface, and SEC-MALS analysis, we demonstrate that MCD1 and MCD2 form a composite dimerization module that facilitates not only TEBP-1 and TEBP-2 homodimerization but also heterodimerization. These findings provide fundamental insights into C. elegans telomeric dsDNA protection and highlight how different eukaryotes have evolved distinct strategies to solve the chromosome end protection problem.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión a Telómeros , Animales , Proteínas de Unión a Telómeros/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Dimerización , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Unión Proteica , Telómero/genética , Telómero/metabolismo , Complejo Shelterina , ADN/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas , Mamíferos/genéticaRESUMEN
For more than a decade, it has been known that mammalian cells use shelterin to protect chromosome ends. Much progress has been made on the mechanism by which shelterin prevents telomeres from inadvertently activating DNA damage signaling and double-strand break (DSB) repair pathways. Shelterin averts activation of three DNA damage response enzymes [the ataxia-telangiectasia-mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) kinases and poly(ADP-ribose) polymerase 1 (PARP1)], blocks three DSB repair pathways [classical nonhomologous end joining (c-NHEJ), alternative (alt)-NHEJ, and homology-directed repair (HDR)], and prevents hyper-resection at telomeres. For several of these functions, mechanistic insights have emerged. In addition, much has been learned about how shelterin maintains the telomeric 3' overhang, forms and protects the t-loop structure, and promotes replication through telomeres. These studies revealed that shelterin is compartmentalized, with individual subunits dedicated to distinct aspects of the end-protection problem. This review focuses on the current knowledge of shelterin-mediated telomere protection, highlights differences between human and mouse shelterin, and discusses some of the questions that remain.
Asunto(s)
Reparación del ADN/genética , Reparación del ADN por Recombinación/genética , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Cromosomas , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Humanos , Ratones , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
Telomere elongation through telomerase enables chromosome survival during cellular proliferation. The conserved multifunctional shelterin complex associates with telomeres to coordinate multiple telomere activities, including telomere elongation by telomerase. Similar to the human shelterin, fission yeast shelterin is composed of telomeric sequence-specific double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1, and Tpz1. Here, we report the crystal structure of the fission yeast Tpz1475-508-Poz1-Rap1467-496 complex that provides the structural basis for shelterin bridge assembly. Biochemical analyses reveal that shelterin bridge assembly is a hierarchical process in which Tpz1 binding to Poz1 elicits structural changes in Poz1, allosterically promoting Rap1 binding to Poz1. Perturbation of the cooperative Tpz1-Poz1-Rap1 assembly through mutation of the "conformational trigger" in Poz1 leads to unregulated telomere lengthening. Furthermore, we find that the human shelterin counterparts TPP1-TIN2-TRF2 also assemble hierarchically, indicating cooperativity as a conserved driving force for shelterin assembly.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Proteínas de Unión a Telómeros/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN , Humanos , Estructura Cuaternaria de Proteína , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Human shelterin is a six-subunit complex-composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1-that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide-binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.
Asunto(s)
Complejo Shelterina , Humanos , Espectrometría de Masas , Microscopía Electrónica , Dominios Proteicos , Complejo Shelterina/químicaRESUMEN
Telomeres are protected by shelterin, a six-subunit protein complex that represses the DNA damage response (DDR) at chromosome ends. Extensive data suggest that TRF2 in shelterin remodels telomeres into the t-loop structure, thereby hiding telomere ends from double-stranded break repair and ATM signaling, whereas POT1 represses ATR signaling by excluding RPA. An alternative protection mechanism was suggested recently by which shelterin subunits TRF1, TRF2, and TIN2 mediate telomeric chromatin compaction, which was proposed to minimize access of DDR factors. We performed superresolution imaging of telomeres in mouse cells after conditional deletion of TRF1, TRF2, or both, the latter of which results in the complete loss of shelterin. Upon removal of TRF1 or TRF2, we observed only minor changes in the telomere volume in most of our experiments. Upon codeletion of TRF1 and TRF2, the telomere volume increased by varying amounts, but even those samples exhibiting small changes in telomere volume showed DDR at nearly all telomeres. Upon shelterin removal, telomeres underwent 53BP1-dependent clustering, potentially explaining at least in part the apparent increase in telomere volume. Furthermore, chromatin accessibility, as determined by ATAC-seq (assay for transposase-accessible chromatin [ATAC] with high-throughput sequencing), was not substantially altered by shelterin removal. These results suggest that the DDR induced by shelterin removal does not require substantial telomere decompaction.
Asunto(s)
Daño del ADN , Telómero/ultraestructura , Proteína 1 de Unión a Repeticiones Teloméricas/fisiología , Proteína 2 de Unión a Repeticiones Teloméricas/fisiología , Animales , Células Cultivadas , Cromatina/fisiología , Ratones , Microscopía Fluorescente , Proteína 1 de Unión al Supresor Tumoral P53/fisiologíaRESUMEN
The six-subunit shelterin complex binds to mammalian telomeres and protects them from triggering multiple DNA damage response pathways. The loss of this protective function by shelterin can have detrimental effects on cells. In this review, we first discuss structural studies of shelterin, detailing the contributions of each subunit and inter-subunit interactions in protecting chromosome ends. We then examine the influence of telomeric chromatin dynamics on the function of shelterin at telomeres. These studies provide valuable insights and underscore the challenges that future research must tackle to attain high-resolution structures of shelterin.
RESUMEN
Biologists aim to explain patterns of growth, reproduction and ageing that characterize life histories, yet we are just beginning to understand the proximate mechanisms that generate this diversity. Existing research in this area has focused on telomeres but has generally overlooked the telomere's most direct mediator, the shelterin protein complex. Shelterin proteins physically interact with the telomere to shape its shortening and repair. They also regulate metabolism and immune function, suggesting a potential role in life history variation in the wild. However, research on shelterin proteins is uncommon outside of biomolecular work. Intraspecific analyses can play an important role in resolving these unknowns because they reveal subtle variation in life history within and among populations. Here, we assessed ecogeographic variation in shelterin protein abundance across eight populations of tree swallow (Tachycineta bicolor) with previously documented variation in environmental and life history traits. Using the blood gene expression of four shelterin proteins in 12-day-old nestlings, we tested the hypothesis that shelterin protein gene expression varies latitudinally and in relation to both telomere length and life history. Shelterin protein gene expression differed among populations and tracked non-linear variation in latitude: nestlings from mid-latitudes expressed nearly double the shelterin mRNA on average than those at more northern and southern sites. However, telomere length was not significantly related to latitude. We next assessed whether telomere length and shelterin protein gene expression correlate with 12-day-old body mass and wing length, two proxies of nestling growth linked to future fecundity and survival. We found that body mass and wing length correlated more strongly (and significantly) with shelterin protein gene expression than with telomere length. These results highlight telomere regulatory shelterin proteins as potential mediators of life history variation among populations. Together with existing research linking shelterin proteins and life history variation within populations, these ecogeographic patterns underscore the need for continued integration of ecology, evolution and telomere biology, which together will advance understanding of the drivers of life history variation in nature.
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Telomeres are inert DNA sequences (TTAGGG) at the end of chromosomes that protect genetic information and maintain DNA integrity. Emerging evidence has demonstrated that telomere alteration can be closely related to occupational exposure and the development of various disease conditions, including cancer. However, the functions and underlying molecular mechanisms of telomere alteration and shelterin dysregulation after welding fume exposures have not been broadly defined. In this study, we analyzed telomere length and shelterin complex proteins in peripheral blood mononuclear cells (PBMCs) and in lung tissue recovered from male Sprague-Dawley rats following exposure by intratracheal instillation (ITI) to 2 mg/rat of manual metal arc-stainless steel (MMA-SS) welding fume particulate or saline (vehicle control). PBMCs and lung tissue were harvested at 30 d after instillation. Our study identified telomere elongation and shelterin dysregulation in PBMCs and lung tissue after welding fume exposure. Mechanistically, telomere elongation was independent of telomerase reverse transcriptase (TERT) activation. Collectively, our findings demonstrated that welding fume-induced telomere elongation was (a) TERT-independent and (b) associated with shelterin complex dysregulation. It is possible that an alteration of telomere length and its regulatory proteins may be utilized as predictive biomarkers for various disease conditions after welding fume exposure. This needs further investigation.
Asunto(s)
Pulmón , Ratas Sprague-Dawley , Acero Inoxidable , Telomerasa , Soldadura , Animales , Masculino , Ratas , Contaminantes Ocupacionales del Aire/toxicidad , Exposición por Inhalación/efectos adversos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Acero Inoxidable/toxicidad , Telomerasa/genética , Telomerasa/metabolismo , Telómero/efectos de los fármacos , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
The shelterin protein TPP1 is involved in both recruiting telomerase and stimulating telomerase processivity in human cells. Assessing the in vivo significance of the latter role of TPP1 has been difficult, because TPP1 mutations that perturb telomerase function tend to abolish both telomerase recruitment and processivity. The Saccharomyces cerevisiae telomerase-associated Est3 protein adopts a protein fold similar to the N-terminal region of TPP1. Interestingly, a previous structure-guided mutagenesis study of Est3 revealed a TELR surface region that regulates telomerase function via an unknown mechanism without affecting the interaction between Est3 and telomerase [T. Rao et al., Proc. Natl. Acad. Sci. U.S.A. 111, 214-218 (2014)]. Here, we show that mutations within the structurally conserved TELR region on human TPP1 impaired telomerase processivity while leaving telomerase recruitment unperturbed, hence uncoupling the two roles of TPP1 in regulating telomerase. Telomeres in cell lines containing homozygous TELR mutations progressively shortened to a critical length that caused cellular senescence, despite the presence of abundant telomerase in these cells. Our findings not only demonstrate that telomerase processivity can be regulated by TPP1 in a process separable from its role in recruiting telomerase, but also establish that the in vivo stimulation of telomerase processivity by TPP1 is critical for telomere length homeostasis and long-term viability of human cells.
Asunto(s)
Mutación , Complejo Shelterina/metabolismo , Telomerasa/metabolismo , Homeostasis del Telómero , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Humanos , Complejo Shelterina/genética , Telomerasa/genética , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.
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Ensamble y Desensamble de Cromatina/fisiología , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Ensamble y Desensamble de Cromatina/genética , Heterocromatina/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Mutación , Unión Proteica , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
Shelterin, a protective complex at telomeres, plays essential roles in cancer. In addition to maintain telomere integrity, shelterin functions in various survival pathways. However, the detailed mechanisms of shelterin regulation in cancer remain elusive. Here, we perform a comprehensive analysis of shelterin in 9125 tumor samples across 33 cancer types using multi-omic data from The Cancer Genome Atlas, and validate some findings in Chinese Glioma Genome Atlas and cancer cell lines from Cancer Cell Line Encyclopedia. In the genomic landscape, we identify the amplification of TRF1 and POT1, co-amplification/deletion of TRF2-RAP1-TPP1 as the dominant alteration events. Clustering analysis based on shelterin expression reveals three cancer clusters with different degree of genome instability. To measure overall shelterin activity in cancer, we derive a shelterin score based on shelterin expression. Pathway analysis shows shelterin is positively correlated with E2F targets, while is negatively correlated with p53 pathway. Importantly, shelterin links to tumor immunity and predicts response to PD-1 blockade immune therapy. In-depth miRNA analysis reveals a miRNA-shelterin interaction network, with p53 regulated miRNAs targeting multiple shelterin components. We also identify a significant amount of lncRNAs regulating shelterin expression. In addition, we find shelterin expression could be used to predict patient survival in 24 cancer types. Finally, by mining the connective map database, we discover a number of potential drugs that might target shelterin. In summary, this study provides broad molecular signatures for further functional and therapeutic studies of shelterin, and also represents a systemic approach to characterize key protein complex in cancer.
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Regulación Neoplásica de la Expresión Génica , Tasa de Mutación , Neoplasias/genética , Neoplasias/mortalidad , Proteínas de Unión a Telómeros/genética , Transcriptoma , Análisis por Conglomerados , Bases de Datos Genéticas , Inestabilidad Genómica , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias/metabolismo , Neoplasias/terapia , Pronóstico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Mapas de Interacción de Proteínas/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Complejo Shelterina , Tasa de Supervivencia , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVES: To investigate the potential role of shelterin dysfunction in naïve CD4+ T cells in the pathogenesis of Behçet's disease (BD). METHODS: Naïve CD4+ T cells were isolated from 40 BD patients and 40 sex- and age-matched healthy controls (HC). Senescent profiles, shelterin subunits expression, telomere length, telomerase activity, and critical DNA damage response (DDR) was evaluated. TRF2 silencing was conducted for further validation. RESULTS: Compared to HC, BD patients had significantly decreased naïve CD4+ T cells, increased cell apoptosis, senescence, and productions of TNF-α and IFN-γ upon activation. Notably, BD naïve CD4+ T cells had shortened telomere, impaired telomerase activity, and expressed lower levels of shelterin subunits TRF2, TIN2, and RAP1. Furthermore, BD naïve CD4+ T cells exhibited significantly increased DDR, evidenced by elevated phosphorylated ataxia telangiectasia (AT) mutated (pATM), pp53, and p21. Finally, TRF2-silencing markedly upregulated DDR, apoptosis, and proinflammatory cytokines production in HC naïve CD4+ T cells. CONCLUSION: Our study demonstrated that TRF2 deficiency in BD naïve CD4+ T cells promoted cell apoptosis and senescence, leading to proinflammatory cytokines overproduction. Therefore, restoring TRF2 might be a promising therapeutic strategy for BD.
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Telomeres are chromosome protectors that shorten during eukaryotic cell replication and in stressful conditions. Developing individuals are susceptible to telomere erosion when their growth is fast and resources are limited. This is critical because the rate of telomere attrition in early life is linked to health and life span of adults. The metabolic telomere attrition hypothesis (MeTA) suggests that telomere dynamics can respond to biochemical signals conveying information about the organism's energetic state. Among these signals are glucocorticoids, hormones that promote catabolic processes, potentially impairing costly telomere maintenance, and nucleotides, which activate anabolic pathways through the cellular enzyme target of rapamycin (TOR), thus preventing telomere attrition. During the energetically demanding growth phase, the regulation of telomeres in response to two contrasting signals - one promoting telomere maintenance and the other attrition - provides an ideal experimental setting to test the MeTA. We studied nestlings of a rapidly developing free-living passerine, the great tit (Parus major), that either received glucocorticoids (Cort-chicks), nucleotides (Nuc-chicks) or a combination of both (NucCort-chicks), comparing these with controls (Cnt-chicks). As expected, Cort-chicks showed telomere attrition, while NucCort- and Nuc-chicks did not. NucCort-chicks was the only group showing increased expression of a proxy for TOR activation (the gene TELO2), of mitochondrial enzymes linked to ATP production (cytochrome oxidase and ATP-synthase) and a higher efficiency in aerobically producing ATP. NucCort-chicks had also a higher expression of telomere maintenance genes (shelterin protein TERF2 and telomerase TERT) and of enzymatic antioxidant genes (glutathione peroxidase and superoxide dismutase). The findings show that nucleotide availability is crucial for preventing telomere erosion during fast growth in stressful environments.
Asunto(s)
Passeriformes , Telómero , Humanos , Animales , Adulto , Telómero/genética , Glucocorticoides , Nucleótidos , Passeriformes/genética , Adenosina Trifosfato , Acortamiento del TelómeroRESUMEN
In this work, we present studies on relatively new and still not well-explored potential anticancer targets which are shelterin proteins, in particular the TRF1 protein can be blocked by in silico designed "peptidomimetic" molecules. TRF1 interacts directly with the TIN2 protein, and this protein-protein interaction is crucial for the proper functioning of telomere, which could be blocked by our novel modified peptide molecules. Our chemotherapeutic approach is based on assumption that modulation of TRF1-TIN2 interaction may be more harmful for cancer cells as cancer telomeres are more fragile than in normal cells. We have shown inâ vitro within SPR experiments that our modified peptide PEP1 molecule interacts with TRF1, presumably at the site originally occupied by the TIN2 protein. Disturbance of the shelterin complex by studied molecule may not in short term lead to cytotoxic effects, however blocking TRF1-TIN2 resulted in cellular senescence in cellular breast cancer lines used as a cancer model. Thus, our compounds appeared useful as starting model compounds for precise blockage of TRF proteins.