RESUMEN
Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.
Asunto(s)
Roturas del ADN de Doble Cadena , Recombinasa Rad51 , Cromatina , Reparación del ADN por Unión de Extremidades , Reparación del ADN , ADN de Cadena Simple/genética , Humanos , Recombinasa Rad51/metabolismoRESUMEN
Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired and rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited to nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry analysis revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveal that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability through its role in nucleolar caps formation.
Asunto(s)
Daño del ADN , Histona Demetilasas con Dominio de Jumonji/genética , ARN Ribosómico/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Unión Proteica , ARN Ribosómico/metabolismoRESUMEN
The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs. These data reveal a role for the cohesin complex in protecting against genome rearrangements arising from the ligation of distant DSEs in S/G2 phases (both long-range DSEs and those that are only a few kilobases apart), while keeping end joining fully active for close DSEs. Therefore, this role likely involves limitation of DSE motility specifically in S phase, rather than inhibition of the end-joining machinery itself.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular , Proteínas Cromosómicas no Histona/genética , Aberraciones Cromosómicas , Proteínas de Unión al ADN , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Reordenamiento Génico , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Interferencia de ARN , Puntos de Control de la Fase S del Ciclo Celular , Factores de Tiempo , Transfección , CohesinasRESUMEN
Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.
Asunto(s)
Adenosina Trifosfatasas/genética , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Poli(ADP-Ribosa) Polimerasas/genética , Cromatina/genética , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Inestabilidad Genómica/genética , Recombinación Homóloga/genética , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificaciónRESUMEN
Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN/fisiología , ADN/aislamiento & purificación , Endonucleasas/metabolismo , Secuencia de Bases , ADN/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , EstreptavidinaRESUMEN
In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Histonas/genética , Puntos de Control del Ciclo Celular/genética , Línea Celular Transformada , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Humanos , Rayos LáserRESUMEN
DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate-dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Ciclo Celular , Línea Celular , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Humanos , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Interferencia de ARN , Proteína de Replicación A/metabolismo , Transducción de SeñalRESUMEN
In proliferating myoblasts, muscle specific genes are silenced by epigenetic modifications at their promoters, including histone H3K9 methylation. Derepression of the promoter of the gene encoding the myogenic factor myogenin (Myog) is key for initiation of muscle differentiation. The mechanism of H3K9 demethylation at the Myog promoter is unclear, however. Here, we identify an isoform of the histone demethylase JMJD2A/KDM4A that lacks the N-terminal demethylase domain (ΔN-JMJD2A). The amount of ΔN-JMJD2A increases during differentiation of C2C12 myoblasts into myotubes. Genome-wide expression profiling and exon-specific siRNA knockdown indicate that, in contrast to the full-length protein, ΔN-JMJD2A is necessary for myotube formation and muscle-specific gene expression. Moreover, ΔN-JMJD2A promotes MyoD-induced conversion of NIH3T3 cells into muscle cells. ChIP-on-chip analysis indicates that ΔN-JMJD2A binds to genes mainly involved in transcriptional control and that this binding is linked to gene activation. ΔN-JMJD2A is recruited to the Myog promoter at the onset of differentiation. This binding is essential to promote the demethylation of H3K9me2 and H3K9me3. We conclude that induction of the ΔN-JMJD2A isoform is crucial for muscle differentiation: by directing the removal of repressive chromatin marks at the Myog promoter, it promotes transcriptional activation of the Myog gene and thus contributes to initiation of muscle-specific gene expression.
Asunto(s)
Diferenciación Celular , Histona Demetilasas/fisiología , Músculo Esquelético/citología , Animales , Células Cultivadas , Cromatina/metabolismo , Epigénesis Genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Ratones , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Células 3T3 NIH , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TransfecciónRESUMEN
Chromatin modifications and chromatin-modifying enzymes are believed to play a major role in the process of DNA repair. The histone acetyl transferase Tip60 is physically recruited to DNA DSBs (double-strand breaks) where it mediates histone acetylation. In the present study, we show, using a reporter system in mammalian cells, that Tip60 expression is required for homology-driven repair, strongly suggesting that Tip60 participates in DNA DSB repair through homologous recombination. Moreover, Tip60 depletion inhibits the formation of Rad50 foci following ionizing radiation, indicating that Tip60 expression is necessary for the recruitment of the DNA damage sensor MRN (Mre11-Rad50-Nbs1) complex to DNA DSBs. Moreover, we found that endogenous Tip60 physically interacts with endogenous MRN proteins in a complex which is distinct from the classical Tip60 complex. Taken together, our results describe a physical link between a DNA damage sensor and a histone-modifying enzyme, and provide important new insights into the role and mechanism of action of Tip60 in the process of DNA DSB repair.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Ácido Anhídrido Hidrolasas , Western Blotting , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de la radiación , Enzimas Reparadoras del ADN/genética , Proteínas de Unión al ADN/genética , Células HeLa , Histona Acetiltransferasas/genética , Histonas/genética , Histonas/metabolismo , Humanos , Inmunoprecipitación , Células Jurkat , Lisina Acetiltransferasa 5 , Proteína Homóloga de MRE11 , Proteínas Nucleares/genética , Unión Proteica , Interferencia de ARN , Radiación Ionizante , Recombinación GenéticaRESUMEN
Permanent silencing of E2F-dependent genes is a hallmark of the irreversible cell cycle exit that characterizes terminally differentiated and senescent cells. The determinant of this silencing during senescence has been proposed to be the binding of the retinoblastoma protein Rb and the consequent methylation of H3K9. During ex vivo skeletal muscle differentiation, while most cells terminally differentiate and form myotubes, a subset of myoblasts remains quiescent and can be reinduced by growth factor stimulation to enter the cell cycle. Thus, differentiating cells are composed of two different populations: one in which E2F-dependent genes are permanently repressed and the other not. We observed that, in a manner reminiscent to senescent cells, permanent silencing of the E2F-dependent cdc6, dhfr, and p107 promoters in myotubes was associated with a specific increase in H3K9 trimethylation. To investigate the role of Rb in this process, we developed a reliable method to detect Rb recruitment by chromatin immunoprecipitation. Surprisingly, we observed that Rb was recruited to these promoters more efficiently in quiescent cells than in myotubes. Thus, our data indicate that during muscle differentiation, permanent silencing and H3K9 trimethylation of some E2F-dependent genes are not directly specified by Rb binding, in contrast to what is proposed for senescence.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Músculo Esquelético/fisiología , Mioblastos/fisiología , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Animales , Ciclo Celular , Diferenciación Celular , Línea Celular , Senescencia Celular , Cromatina/metabolismo , Amplificación de Genes , Ratones , Músculo Esquelético/citología , Mioblastos/citología , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The histone variant H3.3 can be incorporated in chromatin independently of DNA synthesis. By imaging using green fluorescent protein-tagged histones, H3.3 deposition has been found to be linked with transcriptional activation. Here, we investigated H3.3 incorporation during G1 progression on cell-cycle-regulated E2F-dependent genes and on some control loci. We transiently transfected resting cells with an expression vector for tagged H3.3 and we analysed its presence by chromatin immunoprecipitation. We found that replication-independent H3.3 deposition occurred on actively transcribed genes, but not on silent loci, thereby confirming its link with transcription. Interestingly, we observed similar levels of H3.3 occupancy on promoters and on the coding regions of the corresponding genes, indicating that H3.3 deposition is not restricted to promoters. Finally, H3.3 occupancy correlated with the presence of transcription-competent RNA polymerase II. Taken together, our results support the hypothesis that H3.3 is incorporated after disruption of nucleosomes mediated by transcription elongation.
Asunto(s)
Factores de Transcripción E2F/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Transcripción Genética , Animales , Ciclo Celular/fisiología , Histonas/genética , Ratones , Células 3T3 NIH , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
We show here that the two antagonists ICI 182 780, a pure estrogen antagonist, and 4-hydroxy-tamoxifen, a selective estrogen receptor modulator (SERM) have distinct effects on TFF1 (formerly pS2) gene chromatin structure and transcription. Indeed, ICI 182 780 decreased both the intensity of the hormone-dependent DNase I hypersensitive site pS2 HS-1 and transcription of the pS2 gene whereas 4-hydroxy-tamoxifen (OH-Tam) increased the intensity of pS2-HS1 and had no effect on pS2 gene transcription. Interestingly, these differential effects are associated with different fates of ERalpha following the two treatments: The ERalpha-OH-Tam complex was retained in the nucleus more efficiently than the ERalpha-estradiol complex. In contrast, ICI 182 780 provoked a rapid relocation of ERalpha complex to an insoluble nuclear fraction, followed by its degradation. Taken together, these data suggest that regulating the amount of ERalpha in the nucleus is a major way of action of estrogen antagonists with respect to chromatin remodeling and transcriptional control.