RESUMEN
The telomere repeat-binding factor 1 (TERF1, referred to hereafter as TRF1) is a component of mammalian telomeres whose role in telomere biology and disease has remained elusive. Here, we report on cells and mice conditionally deleted for TRF1. TRF1-deleted mouse embryonic fibroblasts (MEFs) show rapid induction of senescence, which is concomitant with abundant telomeric gamma-H2AX foci and activation of the ATM/ATR downstream checkpoint kinases CHK1 and CHK2. DNA damage foci are rescued by both ATM and ATM/ATR inhibitors, further indicating that both signaling pathways are activated upon TRF1 deletion. Abrogation of the p53 and RB pathways bypasses senescence but leads to chromosomal instability including sister chromatid fusions, chromosome concatenation, and occurrence of multitelomeric signals (MTS). MTS are also elevated in ATR-deficient MEFs or upon treatment with aphidicolin, two conditions known to induce breakage at fragile sites, suggesting that TRF1-depleted telomeres are prone to breakage. To address the impact of these molecular defects in the organism, we deleted TRF1 in stratified epithelia of TRF1(Delta/Delta)K5-Cre mice. These mice die perinatally and show skin hyperpigmentation and epithelial dysplasia, which are associated with induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. p53 deficiency rescues mouse survival but leads to development of squamous cell carcinomas, demonstrating that TRF1 suppresses tumorigenesis. Together, these results demonstrate that dysfunction of a telomere-binding protein is sufficient to produce severe telomeric damage in the absence of telomere shortening, resulting in premature tissue degeneration and development of neoplastic lesions.
Asunto(s)
Fragilidad Cromosómica , Deficiencia de Proteína/complicaciones , Enfermedades de la Piel/etiología , Neoplasias Cutáneas/etiología , Telómero/genética , Proteína 1 de Unión a Repeticiones Teloméricas/deficiencia , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Envejecimiento/metabolismo , Animales , Ciclo Celular/fisiología , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/genética , Factor de Transcripción E2F1/metabolismo , Células Epidérmicas , Epidermis/patología , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Hiperpigmentación/etiología , Hiperpigmentación/genética , Ratones , Ratones Noqueados , Mutación/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Enfermedades de la Piel/genética , Neoplasias Cutáneas/genética , Células Madre/patología , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Zn²(+)-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn²(+)-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.
Asunto(s)
Proteínas Portadoras/fisiología , Reparación del ADN/fisiología , Pulmón/embriología , Proteínas Nucleares/fisiología , Organogénesis/fisiología , Animales , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Senescencia Celular , Daño del ADN , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Genotipo , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oxidantes/farmacología , Factores de Tiempo , Tráquea/embriología , Factores de Transcripción , Rayos UltravioletaRESUMEN
Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are present in variable number in most human cell types and have been linked to various cellular functions, including roles as depots for DNA repair proteins. Here, we show that treatment of human cells with DNA methylating agents leads to redistribution of PML from NBs to a diffuse nuclear localization. Biochemically, this correlates with a specific reduction of PML levels in the nuclear matrix fraction without affecting total PML levels. Similar results were obtained for the other major PML NB component, the Sp100 protein, indicating that DNA methylating agents lead to a general disassembly of PML NBs. Similar to the dispersal of PML NBs in response to some viral infections, PML redistribution after DNA damage was inhibited by the proteasome inhibitor MG132. We propose that the regulated dispersal of PML NBs may facilitate the enhanced release of DNA repair proteins from NB depots in order to respond adequately to extensive DNA damage.
Asunto(s)
Núcleo Celular/metabolismo , Cisteína Endopeptidasas/fisiología , Daño del ADN , Metilación de ADN , Complejos Multienzimáticos/fisiología , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Factores de Transcripción/metabolismo , Humanos , Proteína de la Leucemia Promielocítica , Complejo de la Endopetidasa Proteasomal , Proteínas Supresoras de TumorRESUMEN
Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética , Telomerasa/metabolismo , Telómero/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Células Cultivadas , Daño del ADN , Reparación del ADN , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Endogámicos , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/genética , Tasa de SupervivenciaRESUMEN
Abnormal regulation of progression from G(1) to S phase of the cell cycle by altered activity of cyclin-dependent kinases (CDKs) is a hallmark of cancer. However, inhibition of CDKs, particularly CDK2, has not shown selective activity against most cancer cells because the kinase seems to be redundant in control of cell cycle progression. Here, we show a novel role in the DNA damage response and application of CDK inhibitors in checkpoint-deficient cells. CDK2(-/-) mouse fibroblasts and small interfering RNA--mediated or small-molecule--mediated CDK2 inhibition in MCF7 or U2OS cells lead to delayed damage signaling through Chk1, p53, and Rad51. This coincided with reduced DNA repair using the single-cell comet assay and defects observed in both homologous recombination and nonhomologous end-joining in cell-based assays. Furthermore, tumor cells lacking cancer predisposition genes BRCA1 or ATM are 2- to 4-fold more sensitive to CDK inhibitors. These data suggest that inhibitors of CDK2 can be applied to selectively enhance responses of cancer cells to DNA-damaging agents, such as cytotoxic chemotherapy and radiotherapy. Moreover, inhibitors of CDKs may be useful therapeutics in cancers with defects in DNA repair, such as mutations in the familial breast cancer gene BRCA1.
Asunto(s)
Proteína BRCA1/deficiencia , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Quinasa 2 Dependiente de la Ciclina/deficiencia , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Reparación del ADN , Animales , Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Línea Celular Tumoral , Daño del ADN , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Fibroblastos/enzimología , Fibroblastos/fisiología , Humanos , Ratones , Ratones Noqueados , Purinas/farmacología , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , RoscovitinaRESUMEN
Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.
Asunto(s)
Apoptosis , Daño del ADN , Metilación de ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/fisiología , Proteínas Adaptadoras Transductoras de Señales , Disparidad de Par Base , Proteínas Portadoras , Línea Celular Tumoral , Supervivencia Celular , Humanos , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/fisiología , Estructura Terciaria de Proteína , Recombinasa Rad51 , Transducción de SeñalRESUMEN
Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.