Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.
Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.
Lustig
A J
AJ
Graduate Program in Molecular Biology, Cornell University Graduate School of Medical Sciences, New York, NY 10021, USA.
Liu
C
C
Zhang
C
C
Hanish
J P
JP
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism; D001665:Binding Sites; D015825:Chromosomes, Fungal; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005838:Genotype; D008961:Models, Structural; D016296:Mutagenesis; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000502:physiology*; D012697:Serine Endopeptidases; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D013489:Suppression, Genetic; D016615:Telomere; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D020668:rap GTP-Binding Proteins; D000099160:LexA Repressor Protein
D001426:Bacterial Proteins; D005656:Fungal Proteins; D019204:GTP-Binding Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D015534:Trans-Activators; D020668:rap GTP-Binding Proteins; D000099160:LexA Repressor Protein; C084122:SIR3 protein, S cerevisiae