RESUMO
Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.
Assuntos
Proteínas de Ciclo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Reparo do DNA/genética , Histonas/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA , Proteínas de Ligação a DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Recombinação Homóloga , Humanos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Sítio de Iniciação de Transcrição , CoesinasRESUMO
The International Knockout Mouse Consortium (IKMC) aims to mutate all protein-coding genes in the mouse using a combination of gene targeting and gene trapping in mouse embryonic stem (ES) cells and to make the generated resources readily available to the research community. The IKMC database and web portal (www.knockoutmouse.org) serves as the central public web site for IKMC data and facilitates the coordination and prioritization of work within the consortium. Researchers can access up-to-date information on IKMC knockout vectors, ES cells and mice for specific genes, and follow links to the respective repositories from which corresponding IKMC products can be ordered. Researchers can also use the web site to nominate genes for targeting, or to indicate that targeting of a gene should receive high priority. The IKMC database provides data to, and features extensive interconnections with, other community databases.
Assuntos
Bases de Dados Genéticas , Camundongos Knockout , Alelos , Animais , Marcação de Genes , Vetores Genéticos , Genômica , Internet , Camundongos , Anotação de Sequência Molecular , Interface Usuário-ComputadorRESUMO
Non-coding RNAs (ncRNAs) play major roles in proper chromatin organization and function. Senescence, a strong anti-proliferative process and a major anticancer barrier, is associated with dramatic chromatin reorganization in heterochromatin foci. Here we analyze strand-specific transcriptome changes during oncogene-induced human senescence. Strikingly, while differentially expressed RNAs are mostly repressed during senescence, ncRNAs belonging to the recently described vlincRNA (very long intergenic ncRNA) class are mainly activated. We show that VAD, a novel antisense vlincRNA strongly induced during senescence, is required for the maintenance of senescence features. VAD modulates chromatin structure in cis and activates gene expression in trans at the INK4 locus, which encodes cell cycle inhibitors important for senescence-associated cell proliferation arrest. Importantly, VAD inhibits the incorporation of the repressive histone variant H2A.Z at INK4 gene promoters in senescent cells. Our data underline the importance of vlincRNAs as sensors of cellular environment changes and as mediators of the correct transcriptional response.
Assuntos
Senescência Celular/fisiologia , RNA não Traduzido/genética , Linhagem Celular , Senescência Celular/genética , Cromatina/genética , Heterocromatina/genética , HumanosRESUMO
Although both homologous recombination (HR) and nonhomologous end joining can repair DNA double-strand breaks (DSBs), the mechanisms by which one of these pathways is chosen over the other remain unclear. Here we show that transcriptionally active chromatin is preferentially repaired by HR. Using chromatin immunoprecipitation-sequencing (ChIP-seq) to analyze repair of multiple DSBs induced throughout the human genome, we identify an HR-prone subset of DSBs that recruit the HR protein RAD51, undergo resection and rely on RAD51 for efficient repair. These DSBs are located in actively transcribed genes and are targeted to HR repair via the transcription elongation-associated mark trimethylated histone H3 K36. Concordantly, depletion of SETD2, the main H3 K36 trimethyltransferase, severely impedes HR at such DSBs. Our study thereby demonstrates a primary role in DSB repair of the chromatin context in which a break occurs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Linhagem Celular , Cromatina/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Rad51 Recombinase/metabolismo , Transcrição GênicaRESUMO
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.