RESUMO
The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.
Assuntos
Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Telômero/genética , Compartimento Celular , Núcleo Celular/metabolismo , Imunofluorescência , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Substâncias Macromoleculares , Proteínas Nucleares/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Solubilidade , Relação Estrutura-Atividade , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismoRESUMO
To investigate the role of the yeast telomere-, silencing-, and UAS-binding protein RAP1 in telomere position effects, we have characterized two sets of mutant cells: (1) a set of rap1 alleles (termed the rap1t alleles) that produce truncated RAP1 proteins missing the carboxy-terminal 144-165 amino acids; and (2) null mutants of the RIF1 gene, encoding a protein capable of interaction with the carboxyl terminus of RAP1. The data presented here indicate that loss of the carboxyl terminus of RAP1 abolishes position effects at yeast telomeres and diminishes silencing at the HML locus. Elimination of position effects in these cells is associated with increased accessibility to the Escherichia coli dam methylase in vivo. Thus, the carboxy-terminal domain of RAP1 is required for telomere position effects. In contrast, rif1 deletion alleles increase the frequency of repressed cells. Using the rap1t alleles to generate wild-type cells differing only in telomere tract lengths, we also show that telomere position effects are highly sensitive to changes in the size (or structure) of the telomeric tract. Longer poly(G1-3T) tracts can increase the frequency of transcriptional repression at the telomere, suggesting that telomeric poly(G1-3T) tracts play an active role in the formation or stability of subtelomeric transcriptional states.
Assuntos
Cromossomos Fúngicos , Proteínas Fúngicas/genética , Proteínas de Ligação ao GTP/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica) , Proteínas de Ligação a Telômeros , Telômero/química , Cromatina/química , DNA Fúngico/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Escherichia coli/enzimologia , Proteínas de Escherichia coli , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Metiltransferases/metabolismo , Proteínas Repressoras/genética , Deleção de Sequência , Supressão Genética , Transcrição Gênica , Proteínas rap de Ligação ao GTPRESUMO
The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.
