RESUMO
Centromeres are essential for ensuring proper chromosome segregation in eukaryotes. Their definition relies on the presence of a centromere-specific H3 histone variant CenH3, known as CENP-A in mammals. Its overexpression in aggressive cancers raises questions concerning its effect on chromatin dynamics and contribution to tumorigenesis. We find that CenH3 overexpression in human cells leads to ectopic enrichment at sites of active histone turnover involving a heterotypic tetramer containing CenH3-H4 with H3.3-H4. Ectopic localization of this particle depends on the H3.3 chaperone DAXX rather than the dedicated CenH3 chaperone HJURP. This aberrant nucleosome occludes CTCF binding and has a minor effect on gene expression. Cells overexpressing CenH3 are more tolerant of DNA damage. Both the survival advantage and CTCF occlusion in these cells are dependent on DAXX. Our findings illustrate how changes in histone variant levels can disrupt chromatin dynamics and suggests a possible mechanism for cell resistance to anticancer treatments.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autoantígenos/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Proteína Centromérica A , Cromatina/metabolismo , Mapeamento Cromossômico , Proteínas Correpressoras , Dano ao DNA , Epitopos/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Espectrometria de Massas , Microscopia de Fluorescência , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.
Assuntos
Cromossomos Fúngicos/química , Saccharomyces cerevisiae/genética , Fosfatase Ácida/genética , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , Genômica/métodos , Histonas/metabolismo , Espectrometria de Massas , Nucleossomos/química , Regiões Promotoras Genéticas , Proteoma/isolamento & purificação , RNA Ribossômico 5S/química , RNA Ribossômico 5S/ultraestrutura , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Chromatin is unevenly distributed within the eukaryote nucleus and it contributes to the formation of morphologically and functionally distinct substructures, called chromatin domains and nuclear bodies. Here we describe an approach to assess specific chromatin features, the histone posttranslational modifications (PTMs), of the largest nuclear sub-compartment, the nucleolus. In this chapter, methods for the isolation of nucleolus-associated chromatin from native or formaldehyde-fixed cells and the effect of experimental procedures on the outcome of mass spectrometry analysis of histone PTMs are compared.