RESUMEN
Genomic instability can be a hallmark of both human genetic disease and cancer. We identify a deleterious UBQLN4 mutation in families with an autosomal recessive syndrome reminiscent of genome instability disorders. UBQLN4 deficiency leads to increased sensitivity to genotoxic stress and delayed DNA double-strand break (DSB) repair. The proteasomal shuttle factor UBQLN4 is phosphorylated by ATM and interacts with ubiquitylated MRE11 to mediate early steps of homologous recombination-mediated DSB repair (HRR). Loss of UBQLN4 leads to chromatin retention of MRE11, promoting non-physiological HRR activity in vitro and in vivo. Conversely, UBQLN4 overexpression represses HRR and favors non-homologous end joining. Moreover, we find UBQLN4 overexpressed in aggressive tumors. In line with an HRR defect in these tumors, UBQLN4 overexpression is associated with PARP1 inhibitor sensitivity. UBQLN4 therefore curtails HRR activity through removal of MRE11 from damaged chromatin and thus offers a therapeutic window for PARP1 inhibitor treatment in UBQLN4-overexpressing tumors.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Nucleares/genética , Proteínas Portadoras/metabolismo , Cromatina/metabolismo , ADN , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Femenino , Inestabilidad Genómica , Mutación de Línea Germinal , Recombinación Homóloga , Humanos , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Cultivo Primario de Células , Reparación del ADN por RecombinaciónRESUMEN
Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.
Asunto(s)
Roturas del ADN de Doble Cadena , Histona Demetilasas/fisiología , Proteínas de Homeodominio/fisiología , Reparación del ADN por Recombinación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Línea Celular , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Regulación de la Expresión Génica , Inestabilidad Genómica , Células HeLa , Histona Demetilasas/metabolismo , Proteínas de Homeodominio/metabolismo , HumanosRESUMEN
Cell fitness and survival upon exposure to DNA damage depends on the repair of DNA lesions. Interestingly, cellular identity does affect and finetunes such response, although the molecular basis of such differences between tissues and cell types is not well understood. Thus, a possibility is that DNA repair itself is controlled by the mechanisms that govern cell identity. Here we show that the KLF4, involved in cellular homeostasis, proliferation, cell reprogramming and cancer development, directly regulates resection and homologous recombination proficiency. Indeed, resection efficiency follows KLF4 protein levels, i.e. decreases upon KLF4 downregulation and increases when is overexpressed. Moreover, KLF4 role in resection requires its methylation by the methyl-transferase PRMT5. Thus, PRMT5 depletion not only mimics KLF4 downregulation, but also showed an epistatic genetic relationship. Our data support a model in which the methylation of KLF4 by PRMT5 is a priming event required to license DNA resection and homologous recombination.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Epistasis Genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Reparación del ADN por Recombinación , Línea Celular Tumoral , ADN/metabolismo , Roturas del ADN de Doble Cadena , Regulación de la Expresión Génica , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Metilación , Proteína-Arginina N-Metiltransferasas/genéticaRESUMEN
The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.
Asunto(s)
ADN de Cadena Simple/química , Colorantes Fluorescentes/química , Interacciones Hidrofóbicas e Hidrofílicas , Tensoactivos/química , Cationes , Etidio/química , Micelas , Estructura Molecular , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
Acquired genomic instability is one of the major concerns for the clinical use of induced pluripotent stem cells (iPSCs). All reprogramming methods are accompanied by the induction of DNA damage, of which double-strand breaks are the most cytotoxic and mutagenic. Consequently, DNA repair genes seem to be relevant for accurate reprogramming to minimize the impact of such DNA damage. Here, we reveal that reprogramming is associated with high levels of DNA end resection, a critical step in homologous recombination. Moreover, the resection factor CtIP is essential for cell reprogramming and establishment of iPSCs, probably to repair reprogramming-induced DNA damage. Our data reveal a new role for DNA end resection in maintaining genomic stability during cell reprogramming, allowing DNA repair fidelity to be retained in both human and mouse iPSCs. Moreover, we demonstrate that reprogramming in a resection-defective environment has long-term consequences on stem cell self-renewal and differentiation.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Supervivencia Celular/genética , Reprogramación Celular/genética , Aptitud Genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteínas Nucleares/genética , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Autorrenovación de las Células/genética , Daño del ADN , Endodesoxirribonucleasas , Inestabilidad Genómica , Humanos , Proteínas Nucleares/metabolismoRESUMEN
DNA breaks are complex DNA lesions that can be repaired by two alternative mechanisms: non-homologous end-joining and homologous recombination. The decision between them depends on the activation of the DNA resection machinery, which blocks non-homologous end-joining and stimulates recombination. On the other hand, post-translational modifications play a critical role in DNA repair. We have found that the SUMO E3 ligase CBX4 controls resection through the key factor CtIP. Indeed, CBX4 depletion impairs CtIP constitutive sumoylation and DNA end processing. Importantly, mutating lysine 896 in CtIP recapitulates the CBX4-depletion phenotype, blocks homologous recombination and increases genomic instability. Artificial fusion of CtIP and SUMO suppresses the effects of both the non-sumoylatable CtIP mutant and CBX4 depletion. Mechanistically, CtIP sumoylation is essential for its recruitment to damaged DNA. In summary, sumoylation of CtIP at lysine 896 defines a subpopulation of the protein that is involved in DNA resection and recombination.The choice between non-homologous end-joining and homologous recombination to repair a DNA double-strand break depends on activation of the end resection machinery. Here the authors show that SUMO E3 ligase CBX4 sumoylates subpopulation of CtIP to regulate recruitment to breaks and resection.
Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Ligasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Western Blotting , Proteínas Portadoras/genética , Línea Celular Tumoral , ADN/genética , ADN/metabolismo , Endodesoxirribonucleasas , Células HEK293 , Recombinación Homóloga , Humanos , Ligasas/genética , Microscopía Confocal , Proteínas Nucleares/genética , Proteínas del Grupo Polycomb/genética , Interferencia de ARN , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , SumoilaciónRESUMEN
Advanced ovarian cancer is an incurable disease. Thus, novel therapies are required. We wished to identify new therapeutic targets for ovarian cancer. ShRNA screen performed in 42 ovarian cancer cell lines identified the centriolar replication factor STIL as an essential gene for ovarian cancer cells. This was verified in-vivo in orthotopic human ovarian cancer mouse models. STIL depletion by administration of siRNA in neutral liposomes resulted in robust anti-tumor effect that was further enhanced in combination with cisplatin. Consistent with this finding, STIL depletion enhanced the extent of DNA double strand breaks caused by DNA damaging agents. This was associated with centrosomal depletion, ongoing genomic instability and enhanced formation of micronuclei. Interestingly, the ongoing DNA damage was not associated with reduced DNA repair. Indeed, we observed that depletion of STIL enhanced canonical homologous recombination repair and increased BRCA1 and RAD51 foci in response to DNA double strand breaks. Thus, inhibition of STIL significantly enhances the efficacy of DNA damaging chemotherapeutic drugs in treatment of ovarian cancer.