RESUMEN
Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28-1 mutant and to a lesser extent in a cdc7-1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.
Asunto(s)
Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Factor 1 de Ensamblaje de la Cromatina/química , Factor 1 de Ensamblaje de la Cromatina/genética , Ensamble y Desensamble de Cromatina , Replicación del ADN , Silenciador del Gen , Datos de Secuencia Molecular , Mutación , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telómero/genética , Telómero/metabolismoRESUMEN
Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in Δsir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.