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1.
Nature ; 574(7779): 571-574, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31645724

RESUMEN

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Inestabilidad Genómica , Conformación de Ácido Nucleico , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/deficiencia , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
2.
Nucleic Acids Res ; 45(8): 4281-4293, 2017 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-28334750

RESUMEN

To prevent progressive telomere shortening as a result of conventional DNA replication, new telomeric DNA must be added onto the chromosome end. The de novo DNA synthesis involves elongation of the G-rich strand of the telomere by telomerase. In human cells, the CST complex (CTC1-STN1-TEN1) also functions in telomere replication. CST first aids in duplication of the telomeric dsDNA. Then after telomerase has extended the G-rich strand, CST facilitates fill-in synthesis of the complementary C-strand. Here, we analyze telomere structure after disruption of human CTC1 and demonstrate that functional CST is essential for telomere length maintenance due to its role in mediating C-strand fill-in. Removal of CTC1 results in elongation of the 3΄ overhang on the G-rich strand. This leads to accumulation of RPA and telomeric DNA damage signaling. G-overhang length increases with time after CTC1 disruption and at early times net G-strand growth is apparent, indicating telomerase-mediated G-strand extension. In contrast, C-strand length decreases continuously, indicating a deficiency in C-strand fill-in synthesis. The lack of C-strand maintenance leads to gradual shortening of the telomeric dsDNA, similar to that observed in cells lacking telomerase. Thus, telomerase-mediated G-strand extension and CST-mediated C-strand fill-in are equally important for telomere length maintenance.


Asunto(s)
ADN/química , Telomerasa/genética , Homeostasis del Telómero , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , ADN/genética , ADN/metabolismo , Daño del ADN , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , Replicación del ADN , Eliminación de Gen , Regulación de la Expresión Génica , Células HCT116 , Células HEK293 , Humanos , Telomerasa/metabolismo , Telómero/ultraestructura , Acortamiento del Telómero , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/metabolismo
3.
Mol Cell ; 39(4): 606-17, 2010 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-20619712

RESUMEN

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.


Asunto(s)
Replicación del ADN , Fibroblastos/enzimología , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Exodesoxirribonucleasas , Fase G2 , Fusión Génica , Genotipo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Conformación de Ácido Nucleico , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Fase S , Telómero/química , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Factores de Tiempo , Proteínas Supresoras de Tumor/metabolismo
4.
Dev Biol ; 418(2): 258-67, 2016 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-27565025

RESUMEN

Plexins (Plexs) comprise a large family of cell surface receptors for semaphorins (Semas) that function as evolutionarily conserved guidance molecules. GTPase activating protein (GAP) activity for Ras family small GTPases has been implicated in plexin signaling cascades through its RasGAP domain. However, little is known about how Ras family GTPases are controlled in vivo by plexin signaling. Here, we found that Drosophila Rap1, a member of the Ras family of GTPases, plays an important role controlling intersegmental nerve b motor axon guidance during neural development. Gain-of-function studies using dominant-negative and constitutively active forms of Rap1 indicate that Rap1 contributes to axonal growth and guidance. Genetic interaction analyses demonstrate that the Sema-1a/PlexA-mediated repulsive guidance function is regulated positively by Rap1. Furthermore, neuronal expression of mutant PlexA robustly restored defasciculation defects in PlexA null mutants when the catalytic arginine fingers of the PlexA RasGAP domain critical for GAP activity were disrupted. However, deleting the RasGAP domain abolished the ability of PlexA to rescue the PlexA guidance phenotypes. These findings suggest that PlexA-mediated motor axon guidance is dependent on the presence of the PlexA RasGAP domain, but not on its GAP activity toward Ras family small GTPases.


Asunto(s)
Orientación del Axón/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/fisiología , Proteínas de Unión al GTP Monoméricas/fisiología , Proteínas del Tejido Nervioso/fisiología , Receptores de Superficie Celular/fisiología , Proteínas de Unión a Telómeros/fisiología , Proteínas Activadoras de ras GTPasa/fisiología , Animales , Animales Modificados Genéticamente , Orientación del Axón/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes de Insecto , Proteínas de Unión al GTP Monoméricas/deficiencia , Proteínas de Unión al GTP Monoméricas/genética , Neuronas Motoras/fisiología , Mutagénesis , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Regulación hacia Arriba , Proteínas Activadoras de ras GTPasa/deficiencia , Proteínas Activadoras de ras GTPasa/genética
5.
Mol Cell ; 33(5): 559-69, 2009 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-19285940

RESUMEN

Genome stability depends upon the RecQ helicases, which are conserved from bacteria to man, but little is known about how their myriad activities are regulated. Fission yeast lacking the telomere protein Taz1 (mammalian TRF1/TRF2 ortholog) lose many hallmarks of telomeres, including accurate replication and local protection from DNA repair reactions. Here we show that the RecQ homolog, Rqh1, is sumoylated. Surprisingly, Rqh1 acts on taz1Delta telomeres in a deleterious way, promoting telomere breakage and entanglement. Mutation of Rqh1 sumoylation sites rescues taz1Delta cells from these hazards without dramatically affecting nontelomeric Rqh1 functions. The prominence of Rqh1 in the etiology of several different telomere defects supports the idea that they originate from a common underlying lesion--aberrant processing of the stalled telomeric replication forks that accumulate in the absence of Taz1. Our work underscores the principle that RecQ helicases are "double-edged swords" whose activity, while necessary for maintaining genome-wide stability, must be vigilantly controlled.


Asunto(s)
ADN Helicasas/metabolismo , Regulación Fúngica de la Expresión Génica , Procesamiento Proteico-Postraduccional , RecQ Helicasas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Telómero/metabolismo , Alelos , Frío , ADN Helicasas/genética , Replicación del ADN , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Inestabilidad Genómica , Genotipo , Mutación , Fenotipo , RecQ Helicasas/genética , Recombinación Genética , Origen de Réplica , Schizosaccharomyces/genética , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/efectos de la radiación , Estrés Fisiológico/genética , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Factores de Tiempo
6.
EMBO J ; 31(8): 2024-33, 2012 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-22425786

RESUMEN

To counteract replication-dependent telomere shortening most eukaryotic cells rely on the telomerase pathway, which is crucial for the maintenance of proliferative potential of germ and stem cell populations of multicellular organisms. Likewise, cancer cells usually engage the telomerase pathway for telomere maintenance to gain immortality. However, in ∼10% of human cancers telomeres are maintained through telomerase-independent alternative lengthening of telomeres (ALT) pathways. Here, we describe the generation and characterization of C. elegans survivors in a strain lacking the catalytic subunit of telomerase and the nematode telomere-binding protein CeOB2. These clonal strains, some of which have been propagated for >180 generations, represent the first example of a multicellular organism with canonical telomeres that can survive without a functional telomerase pathway. The animals display the heterogeneous telomere length characteristic for ALT cells, contain single-stranded C-circles, a transcription profile pointing towards an adaptation to chronic stress and are therefore a unique and valuable tool to decipher the ALT mechanism.


Asunto(s)
Caenorhabditis elegans/enzimología , Caenorhabditis elegans/crecimiento & desarrollo , Telomerasa/deficiencia , Proteínas de Unión a Telómeros/deficiencia , Telómero/metabolismo , Animales , Caenorhabditis elegans/genética , Análisis de Supervivencia
7.
EMBO J ; 31(24): 4576-86, 2012 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-23188080

RESUMEN

Telomeres protect the natural ends of chromosomes from being repaired as deleterious DNA breaks. In fission yeast, absence of Taz1 (homologue of human TRF1 and TRF2) renders telomeres vulnerable to DNA repair. During the G1 phase, when non-homologous end joining (NHEJ) is upregulated, taz1Δ cells undergo telomere fusions with consequent loss of viability. Here, we show that disruption of the fission yeast MRN (Rad23(MRE11)-Rad50-Nbs1) complex prevents NHEJ at telomeres and, as a result, rescues taz1Δ lethality in G1. Neither Tel1(ATM) activation nor 5'-end resection was required for telomere fusion. Nuclease activity of Rad32(MRE11) was also dispensable for NHEJ. Mutants unable to coordinate metal ions required for nuclease activity were proficient in NHEJ repair. In contrast, Rad32(MRE11) mutations that affect binding and/or positioning of DNA ends leaving the nuclease function largely unaffected also impaired NHEJ at telomeres and restored the viability of taz1Δ in G1. Consistently, MRN structural integrity but not nuclease function is also required for NHEJ of independent DNA ends in a novel split-molecule plasmid assay. Thus, MRN acts to tether unlinked DNA ends, allowing for efficient NHEJ.


Asunto(s)
Reparación del ADN por Unión de Extremidades/fisiología , Fase G1/fisiología , Complejos Multiproteicos/metabolismo , Telómero/metabolismo , Supervivencia Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cartilla de ADN/genética , Electroforesis en Gel de Campo Pulsado , Exodesoxirribonucleasas/metabolismo , Reacción en Cadena de la Polimerasa , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Unión a Telómeros/deficiencia
8.
Proc Natl Acad Sci U S A ; 109(20): 7805-10, 2012 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-22547822

RESUMEN

Canonical telomere repeats at chromosome termini can be maintained by a telomerase-independent pathway termed alternative lengthening of telomeres (ALT). Human cancers that survive via ALT can exhibit long and heterogeneous telomeres, although many telomerase-negative tumors possess telomeres of normal length. Here, we report that Caenorhabditis elegans telomerase mutants that survived via ALT possessed either long or normal telomere lengths. Most ALT strains displayed end-to-end chromosome fusions, suggesting that critical telomere shortening occurred before or concomitant with ALT. ALT required the 9-1-1 DNA damage response complex and its clamp loader, HPR-17. Deficiency for the POT-2 telomere binding protein promoted ALT in telomerase mutants, overcame the requirement for the 9-1-1 complex in ALT, and promoted ALT with normal telomere lengths. We propose that telomerase-deficient human tumors with normal telomere lengths could represent a mode of ALT that is facilitated by telomere capping protein dysfunction.


Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Homeostasis del Telómero/fisiología , Proteínas de Unión a Telómeros/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Indoles , Mutación/genética , Polimorfismo de Longitud del Fragmento de Restricción , Telomerasa/genética , Proteínas de Unión a Telómeros/deficiencia
9.
Nature ; 440(7085): 824-8, 2006 Apr 06.
Artículo en Inglés | MEDLINE | ID: mdl-16598261

RESUMEN

Telomere replication is achieved through the combined action of the conventional DNA replication machinery and the reverse transcriptase, telomerase. Telomere-binding proteins have crucial roles in controlling telomerase activity; however, little is known about their role in controlling semi-conservative replication, which synthesizes the bulk of telomeric DNA. Telomere repeats in the fission yeast Schizosaccharomyces pombe are bound by Taz1, a regulator of diverse telomere functions. It is generally assumed that telomere-binding proteins impede replication fork progression. Here we show that, on the contrary, Taz1 is crucial for efficient replication fork progression through the telomere. Using two-dimensional gel electrophoresis, we find that loss of Taz1 leads to stalled replication forks at telomeres and internally placed telomere sequences, regardless of whether the telomeric G-rich strand is replicated by leading- or lagging-strand synthesis. In contrast, the Taz1-interacting protein Rap1 is dispensable for efficient telomeric fork progression. Upon loss of telomerase, taz1Delta telomeres are lost precipitously, suggesting that maintenance of taz1Delta telomere repeats cannot be sustained through semi-conservative replication. As the human telomere proteins TRF1 and TRF2 are Taz1 orthologues, we predict that one or both of the human TRFs may orchestrate fork passage through human telomeres. Stalled forks at dysfunctional human telomeres are likely to accelerate the genomic instability that drives tumorigenesis.


Asunto(s)
Replicación del ADN , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Cinética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Telomerasa/deficiencia , Telomerasa/genética , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Moldes Genéticos
10.
EMBO J ; 26(22): 4709-19, 2007 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-17948054

RESUMEN

The POT1 (protection of telomeres) protein binds the single-stranded G-rich overhang and is essential for both telomere end protection and telomere length regulation. Telomeric binding of POT1 is enhanced by its interaction with TPP1. In this study, we demonstrate that mouse Tpp1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres. Knockdown of Tpp1 elicits a p53-dependent growth arrest and an ATM-dependent DNA damage response at telomeres. In contrast to depletion of Trf2, which activates ATM, removal of Pot1a and Pot1b from telomeres initiates an ATR-dependent DNA damage response (DDR). Finally, we show that telomere dysfunction as a result of Tpp1 depletion promotes chromosomal instability and tumorigenesis in the absence of an ATM-dependent DDR. Our results uncover a novel ATR-dependent DDR at telomeres that is normally shielded by POT1 binding to the single-stranded G-overhang. In addition, our results suggest that loss of ATM can cooperate with dysfunctional telomeres to promote cellular transformation and tumor formation in vivo.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Ratones , Neoplasias/genética , Neoplasias/metabolismo , ARN Mensajero/metabolismo , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo
11.
Genetics ; 219(2)2021 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-34849883

RESUMEN

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich's ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Expansión de Repetición de Trinucleótido , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismo
12.
Ophthalmic Genet ; 41(6): 656-658, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-32940098

RESUMEN

INTRODUCTION: Recently, You, Hoover-Fong, and colleagues described a disease caused by a deficiency of the telomere maintenance 2 gene (TELO2) function. The clinical spectrum includes early-onset global delay, dysmorphic facial features, auditory disorder, and reduced vision. MATERIALS AND METHODS: We report two siblings, diagnosed with You-Hoover-Fong syndrome at the age of 28 and 14 months. Both were genetically studied to find the cause of their developmental delay and microcephaly. RESULTS: The identical compound heterozygous missense mutations in the TELO2gene were found in each. Ophthalmologically, both siblings were diagnosed with progressive congenital bilateral nuclear-lamellar cataracts. CONCLUSIONS: We report nuclear-lamellar cataracts in two siblings diagnosed with You-Hoover-Fong syndrome.


Asunto(s)
Catarata/patología , Discapacidades del Desarrollo/patología , Discapacidad Intelectual/patología , Microcefalia/patología , Mutación Missense , Homeostasis del Telómero , Proteínas de Unión a Telómeros/deficiencia , Catarata/etiología , Preescolar , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Femenino , Humanos , Lactante , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/genética , Masculino , Microcefalia/complicaciones , Microcefalia/genética , Hermanos , Proteínas de Unión a Telómeros/metabolismo
13.
Mol Biol Cell ; 30(14): 1664-1675, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-31091167

RESUMEN

The cardiomyocyte cytoskeleton, including the sarcomeric contractile apparatus, forms a cohesive network with cellular adhesions at the plasma membrane and nuclear--cytoskeletal linkages (LINC complexes) at the nuclear envelope. Human cardiomyopathies are genetically linked to the LINC complex and A-type lamins, but a full understanding of disease etiology in these patients is lacking. Here we show that SUN2-null mice display cardiac hypertrophy coincident with enhanced AKT/MAPK signaling, as has been described previously for mice lacking A-type lamins. Surprisingly, in contrast to lamin A/C-null mice, SUN2-null mice fail to show coincident fibrosis or upregulation of pathological hypertrophy markers. Thus, cardiac hypertrophy is uncoupled from profibrotic signaling in this mouse model, which we tie to a requirement for the LINC complex in productive TGFß signaling. In the absence of SUN2, we detect elevated levels of the integral inner nuclear membrane protein MAN1, an established negative regulator of TGFß signaling, at the nuclear envelope. We suggest that A-type lamins and SUN2 play antagonistic roles in the modulation of profibrotic signaling through opposite effects on MAN1 levels at the nuclear lamina, suggesting a new perspective on disease etiology.


Asunto(s)
Cardiomegalia/metabolismo , Cardiomegalia/patología , Eliminación de Gen , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Animales , Adhesión Celular , Forma del Núcleo Celular , Proteínas de Unión al ADN/metabolismo , Fibrosis , Integrinas/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/deficiencia , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/metabolismo , Miocardio/patología , Miocardio/ultraestructura , Membrana Nuclear/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sarcómeros/metabolismo , Sarcómeros/ultraestructura , Proteínas de Unión a Telómeros/deficiencia , Factor de Crecimiento Transformador beta/metabolismo
14.
Cell Rep ; 29(11): 3708-3725.e5, 2019 12 10.
Artículo en Inglés | MEDLINE | ID: mdl-31825846

RESUMEN

Telomeres use shelterin to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), repressing ataxia-telangiectasia, mutated (ATM) and ATM and Rad3-related (ATR) dependent DNA damage checkpoint responses. The MRE11 nuclease is thought to be essential for the resection of the 5' C-strand to generate the microhomologies necessary for alternative non-homologous end joining (A-NHEJ) repair. In the present study, we uncover DNA damage signaling and repair pathways engaged by components of the replisome complex to repair dysfunctional telomeres. In cells lacking MRN, single-stranded telomeric overhangs devoid of POT1-TPP1 do not recruit replication protein A (RPA), ATR-interacting protein (ATRIP), and RAD 51. Rather, components of the replisome complex, including Claspin, Proliferating cell nuclear antigen (PCNA), and Downstream neighbor of SON (DONSON), initiate DNA-PKcs-mediated p-CHK1 activation and A-NHEJ repair. In addition, Claspin directly interacts with TRF2 and recruits EXO1 to newly replicated telomeres to promote 5' end resection. Our data indicate that MRN is dispensable for the repair of dysfunctional telomeres lacking POT1-TPP1 and highlight the contributions of the replisome in telomere repair.


Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/metabolismo , Telómero/metabolismo , Ácido Anhídrido Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aminopeptidasas/deficiencia , Aminopeptidasas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/deficiencia , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Humanos , Proteína Homóloga de MRE11/metabolismo , Ratones , Complejos Multienzimáticos/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Serina Proteasas/deficiencia , Serina Proteasas/metabolismo , Complejo Shelterina , Telómero/genética , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo
15.
J Genet Genomics ; 43(7): 451-65, 2016 07 20.
Artículo en Inglés | MEDLINE | ID: mdl-27477026

RESUMEN

In the budding yeast Saccharomyces cerevisiae, heterochromatin structure is found at three chromosome regions, which are homothallic mating-type loci, rDNA regions and telomeres. To address how telomere heterochromatin is assembled under physiological conditions, we employed a de novo telomere addition system, and analyzed the dynamic chromatin changes of the TRP1 reporter gene during telomere elongation. We found that integrating a 255-bp, but not an 81-bp telomeric sequence near the TRP1 promoter could trigger Sir2 recruitment, active chromatin mark(s)' removal, chromatin compaction and TRP1 gene silencing, indicating that the length of the telomeric sequence inserted in the internal region of a chromosome is critical for determining the chromatin state at the proximal region. Interestingly, Rif1 but not Rif2 or yKu is indispensable for the formation of intra-chromosomal silent chromatin initiated by telomeric sequence. When an internal short telomeric sequence (e.g., 81 bp) gets exposed to become a de novo telomere, the herterochromatin features, such as Sir recruitment, active chromatin mark(s)' removal and chromatin compaction, are detected within a few hours before the de novo telomere reaches a stable length. Our results recapitulate the molecular dynamics and reveal a coherent picture of telomere heterochromatin formation.


Asunto(s)
Heterocromatina/genética , Saccharomyces cerevisiae/genética , Telómero/genética , Isomerasas Aldosa-Cetosa/deficiencia , Isomerasas Aldosa-Cetosa/genética , Secuencia de Bases , Silenciador del Gen , Sitios Genéticos/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética
16.
Cell Rep ; 9(4): 1273-80, 2014 Nov 20.
Artículo en Inglés | MEDLINE | ID: mdl-25453752

RESUMEN

The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres.


Asunto(s)
Secuencia Conservada , Endonucleasas/metabolismo , Técnicas de Inactivación de Genes , Homeostasis del Telómero/genética , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Transactivadores/metabolismo , Animales , Proliferación Celular , Cromatina/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Complejo Shelterina , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/metabolismo , Transcripción Genética
17.
J Clin Invest ; 124(1): 353-66, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24316971

RESUMEN

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.


Asunto(s)
Células Madre Hematopoyéticas/fisiología , Mutación , Proteínas de Unión a Telómeros/genética , Animales , Apoptosis , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Células Cultivadas , Inestabilidad Cromosómica , Aberraciones Cromosómicas , Activación Enzimática , Puntos de Control de la Fase G2 del Ciclo Celular , Genes Letales , Trasplante de Células Madre Hematopoyéticas , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pancitopenia/genética , Acortamiento del Telómero , Proteínas de Unión a Telómeros/deficiencia
18.
Mol Cell Biol ; 34(7): 1349-62, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24469404

RESUMEN

The shelterin protein TIN2 is required for the telomeric accumulation of TPP1/POT1 heterodimers and for the protection of telomeres by the POT1 proteins (POT1a and POT1b in the mouse). TIN2 also binds to TRF1 and TRF2, improving the telomeric localization of TRF2 and its function. Here, we ask whether TIN2 needs to interact with both TRF1 and TRF2 to mediate the telomere protection afforded by TRF2 and POT1a/b. Using a TIN2 allele deficient in TRF1 binding (TIN2-L247E), we demonstrate that TRF1 is required for optimal recruitment of TIN2 to telomeres and document phenotypes associated with the TIN2-L247E allele that are explained by insufficient TIN2 loading onto telomeres. To bypass the requirement for TRF1-dependent recruitment, we fused TIN2-L247E to the TRF2-interacting (RCT) domain of Rap1. The RCT-TIN2-L247E fusion showed improved telomeric localization and was fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. These data indicate that when sufficient TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. We therefore conclude that shelterin can protect chromosome ends as a TRF2-tethered TIN2/TPP1/POT1 complex that lacks a physical connection to TRF1.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Células Cultivadas , Ratones , Mutagénesis Sitio-Dirigida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Complejo Shelterina , Telómero/genética , Homeostasis del Telómero/genética , Homeostasis del Telómero/fisiología , Proteínas de Unión a Telómeros/deficiencia , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/química , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/genética
20.
Mol Cell Biol ; 30(12): 2971-82, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20404094

RESUMEN

Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres--TPP1 and the protein that tethers TPP1 to the complex, TIN2--results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.


Asunto(s)
Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/enzimología , Inmunoprecipitación de Cromatina , Daño del ADN , Células HeLa , Humanos , Unión Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas , Complejo Shelterina , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/deficiencia
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