RESUMO
The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5(CA) mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5(CA) phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1delta cac2delta msi1delta (caf1delta) cells expressing apc5(CA) exhibit a phenotype more severe than that of apc5(CA) or caf1delta. The Ts- phenotypes observed in apc5(CA) and apc5(CA) caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts- defects. Synthetic genetic interactions were also observed in apc5(CA) asf1delta cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5(CA) Ts- defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5(CA) phenotypes. We discuss the implications of our observations.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação Fúngica da Expressão Gênica , Mitose , Ribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica , Histonas/metabolismo , Chaperonas Moleculares , Mutação/genética , Proteínas Nucleares/farmacologia , Fenótipo , Subunidades Proteicas , Proteínas Repressoras/farmacologia , Ribonucleases/genética , Ribonucleases/farmacologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/farmacologia , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Defects in anaphase-promoting complex (APC) activity, which regulates mitotic progression and chromatin assembly, results in genomic instability, a hallmark of premature aging and cancer. We investigated whether APC-dependent genomic stability affects aging and life span in yeast. Utilizing replicative and chronological aging assays, the APC was shown to promote longevity. Multicopy expression of genes encoding Snf1p (MIG1) and PKA (PDE2) aging-pathway components suppressed apc5CA phenotypes, suggesting their involvement in APC-dependent longevity. While it is known that PKA inhibits APC activity and reduces life span, a link between the Snf1p-inhibited Mig1p transcriptional modulator and the APC is novel. Our mutant analysis supports a model in which Snf1p promotes extended life span by inhibiting the negative influence of Mig1p on the APC. Consistent with this, we found that increased MIG1 expression reduced replicative life span, whereas mig1Delta mutations suppressed the apc5CA chronological aging defect. Furthermore, Mig1p and Mig2p activate APC gene transcription, particularly on glycerol, and mig2Delta, but not mig1Delta, confers a prolonged replicative life span in both APC5 and acp5CA cells. However, glucose repression of APC genes was Mig1p and Mig2p independent, indicating the presence of an uncharacterized factor. Therefore, we propose that APC-dependent genomic stability is linked to prolonged longevity by the antagonistic regulation of the PKA and Snf1p pathways.
Assuntos
Envelhecimento/fisiologia , Instabilidade Genômica , Longevidade , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Subunidade Apc5 do Ciclossomo-Complexo Promotor de Anáfase , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Fenótipo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements, whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model system Saccharomyces cerevisiae. Modifications to an in vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding two ubiquitin protein ligases (E3s): RSP5, and a subunit of the Anaphase Promoting Complex (APC), APC5. Additional modifications are described that allow for a rapid analysis and an in vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that the in vitro and invivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different molecular networks.
