RESUMO
Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Fúngicas/fisiologia , Complexos Multiproteicos/fisiologia , Nucleossomos/fisiologia , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Sistema Livre de Células , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/fisiologia , Pegada de DNA , DNA Bacteriano/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II , Histonas/genética , Histonas/metabolismo , Microscopia de Força Atômica , Nucleossomos/ultraestrutura , Plasmídeos/química , Proteínas de Ligação a RNA/fisiologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Xenopus laevis/genéticaRESUMO
Half of the human genome consists of repetitive DNA sequences. Recent studies in various organisms highlight the role of chromatin regulation of repetitive DNA in gene regulation as well as in maintainance of chromosomes and genome integrity. Hence, repetitive DNA sequences might be potential "sensors" for chromatin changes associated with pathogenesis. Therefore, we developed a new genomic tool called RepArray. RepArray is a repeat-specific microarray composed of a representative set of human repeated sequences including transposon-derived repeats, simple sequences repeats, tandemly repeated sequences such as centromeres and telomeres. We showed that combined to anti-methylcytosine immunoprecipitation assay, the RepArray can be used to generate repeat-specific methylation maps. Using cell lines impaired chemically or genetically for DNA methyltransferases activities, we were able to distinguish different epigenomes demonstrating that repeats can be used as markers of genome-wide methylation changes. Besides, using a well-documented system model, the thermal stress, we demonstrated that RepArray is also a fast and reliable tool to obtain an overview of overall transcriptional activity on whole repetitive compartment in a given cell type. Thus, the RepArray represents the first valuable tool for systematic and genome-wide analyses of the methylation and transcriptional status of the repetitive counterpart of the human genome.