RESUMEN
Comprehensive analyses of the histone-GLibrary in previous studies showed that most mutants of modification sites in the histone core regions show phenotypes, whereas those with modifications in the histone N-terminal unstructured tail regions (N-tails) do not. One possible reason is that modifications in N-tails are linked to each other to form a scale-free network termed histone 'modification web'. In the network, the compensatory pathways are created to acquire the robustness against the any defects. Because of this robustness, it is difficult to determine the significance of the individual histone modifications in N-tails in vivo. To overcome this problem, we used a strategy using drugs coordinately to inhibit modification enzymes and observed the mutant phenotypes when the compensatory pathways are largely interrupted. We analyzed histone-GLibrary using inhibitors of histone deacetylases (HDACs) and identified novel phenotypic mutants. We also examined the phenotypic changes through the combined use of an HDAC inhibitor and an inhibitor of DNA-mediated reactions. Mutation of modifiable sites H3-K4 and H4-K16 in histone N-tails, which are presumed to be the 'hubs' of the network, resulted in identifiable phenotypes. The data obtained provide valuable information for speculation on novel relationships between histone modification in N-tails and biological function and for predicting unknown modification sites in core histones.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histonas/genética , Mutación Puntual , Acetilación/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/metabolismo , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Terciaria de ProteínaRESUMEN
Eukaryotic chromatin is regulated by chromatin factors such as histone modification enzymes, chromatin remodeling complexes and histone chaperones in a variety of DNA-dependent reactions. Among these reactions, transcription in the chromatin context is well studied. On the other hand, how other DNA-dependent reactions, including postreplicative homologous recombination, are regulated in the chromatin context remains elusive. Here, histone H3 Lys56 acetylation, mediated by the histone acetyltransferase Rtt109 and the histone chaperone Cia1/Asf1, is shown to be required for postreplicative sister chromatid recombination. This recombination did not occur in the cia1/asf1-V94R mutant, which lacks histone binding and histone chaperone activities and which cannot promote the histone acetyltransferase activity of Rtt109. A defect in another histone chaperone, CAF-1, led to an increase in acetylated H3-K56 (H3-K56-Ac)-dependent postreplicative recombination. Some DNA lesions recognized by the putative ubiquitin ligase complex Rtt101-Mms1-Mms22, which is reported to act downstream of the H3-K56-Ac signaling pathway, seem to be increased in CAF-1 defective cells. Taken together, these data provide the framework for a postreplicative recombination mechanism controlled by histone modifiers and histone chaperones in multiple ways.
Asunto(s)
Cromatina/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Recombinación Genética , Acetilación , Sitios de Unión/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Replicación del ADN , ADN de Hongos/genética , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Chaperonas de Histonas/genética , Histonas/química , Histonas/genética , Lisina/química , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Intercambio de Cromátides HermanasRESUMEN
Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.