RESUMO
At yeast telomeres and silent mating-type loci, chromatin assumes a higher-order structure that represses transcription by means of the histone deacetylase Sir2 and structural proteins Sir3 and Sir4. Here, we present a fully reconstituted system to analyze SIR holocomplex binding to nucleosomal arrays. Purified Sir2-3-4 heterotrimers bind chromatin, cooperatively yielding a stable complex of homogeneous molecular weight. Remarkably, Sir2-3-4 also binds naked DNA, reflecting the strong, albeit nonspecific, DNA-binding activity of Sir4. The binding of Sir3 to nucleosomes is sensitive to histone H4 N-terminal tail removal, while that of Sir2-4 is not. Dot1-mediated methylation of histone H3K79 reduces the binding of both Sir3 and Sir2-3-4. Additionally, a byproduct of Sir2-mediated NAD hydrolysis, O-acetyl-ADP-ribose, increases the efficiency with which Sir3 and Sir2-3-4 bind nucleosomes. Thus, in small cumulative steps, each Sir protein, unmodified histone domains, and contacts with DNA contribute to the stability of the silent chromatin complex.
Assuntos
Cromatina/metabolismo , Nucleossomos/metabolismo , O-Acetil-ADP-Ribose/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Histona Desacetilases/isolamento & purificação , Histona Desacetilases/metabolismo , Modelos Biológicos , Modelos Moleculares , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/isolamento & purificação , Sirtuína 2 , Sirtuínas/isolamento & purificação , Sirtuínas/metabolismoRESUMO
In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sequência de Aminoácidos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Teste de Complementação Genética , Dados de Sequência Molecular , Fenótipo , Pneumocystis carinii/química , Pneumocystis carinii/genética , Pneumocystis carinii/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/química , Saccharomycetales/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência de Aminoácidos , Telômero/química , Telômero/metabolismo , Fatores de TranscriçãoRESUMO
We investigated the role of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. We identified apparent null alleles of lis-1, which result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. We raised antibodies against LIS-1 and generated transgenic animals expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, we found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. We established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, we found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another.