The maintenance of transcriptional silencing at HM mating-type loci and telomeres in yeast requires the SIR2, SIR3, and SIR4 proteins, none of which appear to be DNA-binding proteins. Here we show that SIR3 and SIR4 interact with a carboxy-terminal domain of the silencer, telomere, and UAS-binding protein RAP1. We identified SIR3 and SIR4 in a two-hybrid screen for RAPl-interacting factors and showed that SIR3 interacts both with itself and with SIR4. The interaction between RAP1 and SIR3 can be observed in vitro in the absence of other yeast proteins. Consistent with the notion that native SIR proteins interact with the RAP1 carboxyl terminus, we show that mutation of the endogenous SIR3 and SIR4 genes increases transcriptional activation by LexA/RAP1 hybrids. To test the importance of the RAP1-SIR3 interaction for silencing, we identified mutations in the RAP1 carboxyl terminus that either diminish or abolish this interaction. When introduced into the native RAP1 protein, these mutations cause corresponding defects in silencing at both HMR and telomeres. We propose that RAP1 acts in the initiation of transcriptional silencing by recruiting a complex of SIR proteins to the chromosome via protein-protein interactions. These data are consistent with a model in which SIR3 and SIR4 play a structural role in the maintenance of silent chromatin and indicate that their action is initiated at the silencer itself.

The yeast RAPl protein is a sequence-specific DNA-binding protein that functions as both a repressor and an activator of transcription. RAPl is also involved in the regulation of telomere structure, where its binding sites are found within the terminal poly(Ci_3A) sequences. Previous studies have indicated that the regulatory function of RAPl is determined by the context of its binding site and, presumably, its interactions with other factors. Using the two-hybrid system, a genetic screen for the identification of protein-protein interactions, we have isolated a gene encoding a RAPl-interacting factor (RIFl). Strains carrying gene disruptions of RIFl grow normally but are defective in transcriptional silencing and telomere length regulation, two phenotypes strikingly similar to those of silencing-defective rapl" mutants. Furthermore, hybrid proteins containing rapl^ missense mutations are defective in an interaction with RIFl in the two-hybrid system. Taken together, these data support the idea that the rapT phenotypes are attributable to a failure to recruit RIFl to silencers and telomeres and suggest that RIFl is a cofactor or mediator for RAPl in the establishment of a repressed chromatin state at these loci. By use of the two-hybrid system, we have isolated a mutation in RIFl that partially restores the interaction with rapl** mutant proteins.

The Saccharomyces cerevisiae Rapl protein binds with high affinity to sites within the poly(Ci_3A) tracts at telomeres, where it plays a role in both telomere length regulation and the initiation of telomeric silencing. Raplp initiates silencing at telomeres by interacting through its carboxy-terminal domain with Sir3p and Sir4p, both of which are required for repression. This same domain of Raplp also negatively regulates telomere elongation, through an unknown mechanism. We have identified a new Rapl-interacting factor (Rif2p) that plays a role in telomere length regulation. RifZp has considerable functional similarities with a Raplp-interacting factor (Riflp) identified previously. Mutations in RIFl or RJF2 (unlike mutations in the silencing genes SIRS and SIR4) result in moderate telomere elongation and improved telomeric silencing. However, deletion of both RIFl and RIF2 in the same cell results in a dramatic increase in telomere length, similar to that seen with a carboxy-terminal truncation of Raplp. In addition, overexpression of either jRIFl or RIF2 decreases telomere length, and co-overexpression of these proteins can reverse the telomere elongation effect of overexpression of the Raplp carboxyl terminus. Finally, we show that Riflp and Rif2p can interact with each other in vivo. These results suggest that telomere length regulation is mediated by a protein complex consisting of Riflp and Rif2p, each of which has distinct regulatory functions. One role of Raplp in telomere length regulation is to recruit these proteins to the telomeres.

In the yeast Saccharomyces cerevisiae, telomere elongation is negatively regulated by the telomere repeat-binding protein Rap1p, such that a narrow length distribution of telomere repeat tracts is observed. This length regulation was shown to function independently of the orientation of the telomere repeats. The number of repeats at an individual telomere was reduced when hybrid proteins containing the Rap1p carboxyl terminus were targeted there by a heterologous DNA-binding domain. The extent of this telomere tract shortening was proportional to the number of targeted molecules, consistent with a feedback mechanism of telomere length regulation that can discriminate the precise number of Rap1p molecules bound to the chromosome end.