No abstract

RAP1 is a sequence-specific DNA-binding protein in yeast that can either repress or activate transcription. Previous studies have demonstrated a direct role for RAP1 in silencing at HM mating-type loci and telomeres. Here, we show that a small carboxy-terminal domain of RAP1 is sufficient to establish repression when fused to the GAL4 DNA-binding domain (GBr,) and targeted to mutated HMR silencers containing GAL4 DNA-binding sites. Silencing by GBD/RAP1 hybrids, like normal silencing at HMR, requires the trans-acting factors SIR2, SIR3, and SIR4. However, GBD/RAPl-mediated silencing is independent of SIR1, whose product is normally required for the establishment of repression at HMR. Targeted silencing also displays an unusual response to silencing-defective rap1 ~ mutations. The incorporation of a rapl s missense mutation into GBD/RAP1 hybrids can improve targeted silencing, yet wild-type GBI~/RAP1 hybrids fail to establish repression in strains in which the endogenous RAP1 locus carries a rapF mutation. In addition, we find that telomeric silencing is increased in rapF strains. We propose that the rapF mutation creates an HMR-specific silencing defect by shifting a balance between silencing at HMR and telomeres in favor of telomeric silencing. This balance is regulated by telomere length and by interactions between the RAP1 carboxyl terminus and both RIF1 and SIR4 proteins. In support of this model, we show that abnormally long telomeres antagonize silencing at HMR and a rapl ~ hybrid protein displays a strengthened interaction with SIR4 in a two-hybrid assay.

The ribosomal DNA (rDNA) tandem array in Saccharomyces cerevisiae induces transcriptional silencing of RNA polymerase II-transcribed genes. This SIR2-dependent form of repression (rDNA silencing) also functions to limit rDNA recombination and is involved in life span control. In this report, we demonstrate that rDNA silencing spreads into the centromere-proximal unique sequence located downstream of RNA polymerase I (Pol I) transcription, but fails to enter the upstream telomere-proximal sequences. The spreading of silencing correlates with SIR2-dependent histone H3 and H4 deacetylation and can be extended by SIR2 overexpression. Surprisingly, rDNA silencing required transcription by RNA polymerase I and the direction of spreading was controlled by the direction of Pol I transcription.

Raplp binds to silencer elements and telomeric repeats in yeast, where it appears to initiate silencing by recruiting Sir3p and Sir4p to the chromosome through interactions with its carboxy-terminal domain. Sir3p and Sir4p interact in vitro with histones H3 and H4 and are likely to be structural components of silent chromatin. We show that targeting of these Sir proteins to the chromosome is sufficient to initiate stable silencing either at a silent mating-type locus lacking a functional silencer element or at a telomere in a strain in which the Raplp carboxy-terminal silencing domain has been deleted. Silencing by Sir protein targeting can also be initiated at a telomere-proximal site {ADH4), but is much weaker at an internal chromosomal locus {LYS2). Strikingly, deletion of the Raplp silencing domain, which abolishes telomeric silencing, improves targeted silencing at LYS2 by both SirSp and Sir4p, while weakening the silencing activity of these proteins at or near a telomere. This effect may result from the release of Sir proteins from the telomeres, thus increasing their effective concentration at other chromosomal sites. We suggest that telomeres and Raplp serve a regulatory role in sequestering Sir proteins at telomeres, controlling silencing at other loci in trans and preventing indiscriminate gene silencing throughout the genome. The yeast Saccharomyces cerevisiae uses a form of po sition-effect regulation, commonly referred to as tran scriptional silencing (Brand et al. 1985), to control the expression of genes that determine mating type (for re view, see Laurenson and Rine 1992). A very similar but less stable form of repression is also observed when genes are placed near telomeres in yeast (Gottschling et al. 1990;Aparicio et al. 1991), suggesting that silencing may play a more general role in regulating gene expres sion in this organism (for review, see Shore 1995). Many of the properties of silencing in yeast are reminiscent of position-effect variegation in Drosophila, and recent molecular studies (Hecht et al. 1995) suggest that silenc ing in yeast may be analogous to heterochromatin for mation in more complex eukaryotes.A number of cis-and trans-acting elements involved in silencing have now been identified and characterized in some detail. The silencer elements that flank the two silent mating-type loci {HML and HMR] all contain an autonomously replicating sequence (ARS) consensus sePresent address:

The silent information regulator (Sir2) family of protein deacetylases (Sirtuins) are nicotinamide adenine dinucleotide (NAD)(+)-dependent enzymes that hydrolyze one molecule of NAD(+) for every lysine residue that is deacetylated. The Sirtuins are phylogenetically conserved in eukaryotes, prokaryotes, and Archeal species. Prokaryotic and Archeal species usually have one or two Sirtuin homologs, whereas eukaryotes typically have multiple versions. The founding member of this protein family is the Sir2 histone deacetylase of Saccharomyces cerevisiae, which is absolutely required for transcriptional silencing in this organism. Sirtuins in other organisms often have nonhistone substrates and in eukaryotes, are not always localized in the nucleus. The diversity of substrates is reflected in the various biological activities that Sirtuins function, including development, metabolism, apoptosis, and heterochromatin formation. This review emphasizes the great diversity in Sirtuin function and highlights its unusual catalytic properties.