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Mechanisms of silencing in Saccharomyces cerevisiae
233
Mechanisms of silencing in Saccharomyces cerevisiae Arthur J Lustig In the yeast Saccharomycescerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.
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Addresses Department of Biochemistry, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA; e-maih alustig@ mailhost.tcs.tulane.edu
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Current Opinion in Genetics & Development 1998, 8:233-239
HMR silencer
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Abbreviations Abflp ARS-binding factor 1 CAF-1 chromatin assembly factor-1 ORC origin recognition complex Raplp repressor activator protein 1
Introduction q\vo mechanistically related forms of transcriptional silencing are present in yeast: first, tclomeric silencing, the metastable transcriptional repression that occurs when a Pol II or Pol I[I transcript is positioned adjacent to tclomeric poly(TGl_3) tracts; and second, the stable repression of MATcz information at the HML locus, located 12kb from the left telomere of chromosome III, and of MATcz transcription at the HMR locus, located 20kb from the right arm of the same chromosome [1,2"]. In each case, c/s-acting silencer elements are responsible for the repression of transcription in adjacent sequences (Figure 1). At the telomerc, the silcncer is composed of muhiplc binding sites for repressor activator protein 1 (Raplp) embedded within the tclomeric tract. At HML and HMR, the silencers consist of specific arrays of binding sites for ARS-binding factor 1 (Abflp), the origin recognition complex (ORC), and Raplp. Although silencers at the three regions differ in their degree of functional redundancy, they share a dependence on the silent information regulators Sir2p, Sir3p, Sir4p, as well as thc amino termini of histones H3 and H4. Common to all three classes of silencers is the formation of an adjacent 'closed' chromatin statc, inaccessiblc to exogenous probes both in vivo and in vitro. T h e characteristics of silenced regions, transcriptional quiescence, chromatin compaction, and late replication suggest that thcse position effects are the yeast equivalent of higher eukaryotic heterochromatin. Tclomeric silencing exhibits the lowest lcvel of functional redundancy, making it most amcnable to analysis. I will thcrcfore focus on this form of silencing, drawing
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Raplp-binding site Abfl p-binding site D-binding site
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Current Opinion in Genetics & Development
Structure of the telomeric and HM silencers in yeast. The telomeric silencer contains multiple sites for the telomere-binding factor Raplp, present at a density of one in every twenty base pairs of telomere tract. The structure of the HM loci, as depicted (HML), are more complex and involve the participation of silencers that flank the repressed gene. The identify of the factor that binds to the D region remains unknown. A greater level of redundancy is present at the HM loci than at telomeres. In a chromosomal context, elimination of either HMLE or HMLI is insufficient to eliminate silencing. Similarly, in the absence of HMLI, mutation in any one binding site of HMLE leads to only partial derepression. A similar situation is present at the HMR locus: elimination of HMRI has a small effect on silencing, whereas elimination of HMRE eliminates silencing completely. In addition, at HMRE, two out of three binding sites must normally be mutated to derepress silencing. All three silencers are capable of repressing transcription of both Pol II and Pol Ill genes [1].
comparisons to HML and HMR silencing when data is available. In this revicw, I emphasize recent studies providing mechanistic information on the nucleation and spreading of represscd chromatin and on the involvemcnt of silencing factors in both DNA repair and aging.
The nucleation of silencing Nucleation refers to the set of events acting at the silenccr that lead to transcriptional repression in adjacent sequences. Multiple lincs of evidence suggest that the nucleation of telomeric silencing is the consequence of recruitment of Sir3p and Sir4p to the carboxyl termini of multiple Raplp molecules bound to telomeric tracts (Figure 2). First, Raplp is capable of direct interactions with Sir3p, and, most likely, Sir4p [3-5]. Second, Sir3 and Sir4 fusion proteins, when artifically tethered to a site at a tclomeric/subtelomeric junction, overcomc the abrogation of telomeric silcncing in rapl mutants lacking
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Chromosomesand expression mechanisms
the carboxyl terminus [6,7]. Third, Raplp, Sir3p, and Sir4p co-immunolocalize to telomeric and/or subtelomeric domains at the nuclear periphery in a mutually dependent fashion [8]. Sir3p phosphorylation most likely serves as an additional regulatory step, as phosphorylated Sir3p enhances silencing [9]. Interestingly, two antagonists of silencing, Riflp and Rif2p, appear to compete with Sir3p and Sir4p for association with Rap l p [10,11,12°°]. This competition may explain the limiting function of Sir3p and Sir4p at the telomere, despite the large number of Raplp-binding sites. Consistent with this notion, artificial tethering of Sir3p or Sir4p to a telomeric site I w h i c h would be expected to overcome such competition--enhances silencing even in wild-type cells [6,7]. Sir3p and Sir4p appear to be capable of homo- and hetero-dimerization, although it is not known whether these associations take place within the telomeric tract [3,4,13°,14]. Nonetheless, these interactions provide a means of forming a higher-order structure mediated through association of Sir3p and Sir4p bound at adjacent Rapl molecules ([15]; Figure 2). Consistent with the viewpoint that these associations may stabilize the telomere as a silencer, long internal tracts of telomeric DNA can confer silencing [16]. An analogous process may take place at the HM silencers since both Raplp and Abflp can associate directly with Sir3p ([3]; P Moretti, D Shore, personal communication). Although ORC does not appear to interact with Sir3p directl-y; it may perform a similar function through the formation of Sirlp/Orclp and Sirlp/Sir4p interactions, both of which have been demonstrated experimentally [14,17]. Although the physical steps in recruitment are becoming clearer, their mechanistic consequence is uncertain. In some fashion, recruitment to silencers must initiate a cascade of events leading to a unidirectional and cooperative alteration in nucleosome structure (Figure 2). Recent investigations implicate two elements in this process. First, several studies indicate a requirement for a microenvironment containing a high concentration of Sir3p, Sir4p, and other silencing factors: one, Sir3p and Sir4p can associate with the amino termini of histories H3 and H4 in vitro and in vivo ([18]; S Gasser, personal communication); two, Raplp, Sir3p, Sir4p, and part of the cellular pool of Sir2p immunolocalize in vivo with clustered telomeric and/or subtelomeric regions [8,19"]; three, the introduction of elongated telomeres into a wild-type background diminishes HM silencing, suggesting titration of a limiting amount of Sir3p and Sir4p by the telomeric silencer [5]; four, silencing conferred by long internal tracts of telomeric sequence or by the HML silencer is enhanced by proximity to the telomere, even though silencing does not emanate from the telomerie tract in
either case [16,20"]; and five, the introduction of multiple Raplp binding sites adjacent to the MATc~ locus results in silencing of this normally active locus [21]. Second, although a high concentration of silencing factors may induce the cooperative formation of repressed chromatin, it is unclear how this process would result in the observed directionality of silencing. One possible means is through an initial 'bridge' between the silencer and adjacent histones (Figure 2). At the telomere, such a bridge might be provided by interaction of either Sir3p or Sir4p with both Raplp and specific forms of acetylated histones H3 and H4. This model would help to explain why tethering of Sir3p or Sir4p to the telomere confers silencing in rapl mutants unable to recruit either factor [6,7]. T h e nucleation of Hill silencing involves at least one additional level of complexity. A recent examination of the HMLE and HML1 silencing of a lacZ tester gene demonstrated that individual silencer binding sites--incapable of conferring silencing a l o n e - - c a n interact with silencers on the opposing side of thc lacZ gene, suggesting that protein-protein interactions flanking the gene, even if transient, facilitate the nucleation of silencing [22]. T h e ' s p r e a d i n g ' of r e p r e s s e d c h r o m a t i n Interaction of Sir3p and Sir4p with the histone amino termini The events that follow nucleation are somewhat clearer. As noted, direct interactions of the carboxyl terminus of Sir3p (and the amino terminus of Sir4p) with the amino termini of histones H3 and H4 have been demonstrated in vivo and in vitro ([18]; S Gasser, personal communication). T h e genomic sites of these interactions have, until recently, remained elusive. A reversible in vivo formaldehyde crosslinking method, however, has recently been used to demonstrate that Sir3p, Sir4p, Sir2p, and each core histone co-associate in subtelomeric DNA [23,24"]. The association of the Sir factors decrease with distance from the telomerc. Raplp also associates with these complexes, raising the possibility that a fold-back structure is formed between telomeric and subtelomeric regions [24"]. This method has also revealed that overproduction of Sir3p results in the physical presence of Sir3p at sites further from the telomere, consistent with the phenotypic spreading of silencing after overproduction of Sir3p [23,25]. Curiously, under these conditions, Sir2p and Sir4p association decreases significantly at these same distal sites. These data suggest that there are two forms of telomeric heterochromatin: a 'core' state composed of Sir2p, Sir3p and Sir4p, and an 'extended' region containing Sir3p but only limiting quantities of other Sir factors {24"°1.
These data also provide convincing evidence for the formation of condensed chromatin at bona fide telomeres. Both core and extended heterochromatin are found at naturally-occurring telomeres [23,24°°]. Further proof for
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
the existence of telomeric silencing at natural chromosomes comes from the finding that transcripts emanating from Ty5 elements, which insert preferentially into subtelomeric sites, are silenced in a SIR3-dependent fashion [26-28,29"°].
Figure 2
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Current Opinion in Genetics & Development
A speculative model for the steps involved in the formation of telomeric silencing, as described in the text, are depicted in this figure. In the first step, Rap1 p bound to telomeric consensus sites recruit the silencing factors Sir3p and Sir4p and the silencing-antagonistic factors Riflp and Rif2p. Subnuclear clustering of telomeres create a microenvironment high in concentration of silencing factors leading to a shift in the equilibrium to a preference for association with silencing factors. The association of Sir3p and Sir4p with themselves and with each other may help to stabilize the telomere as a silencer. This stabilized structure is subsequently able either to recruit a specifically modified set of histones H3 and H4 or to assist in the specific modification of these histones within the first nucleosome. Once Sir3p is associated with the initial amino-terminal tails, forming a 'histone bridge', subsequent Sir3p associations would similarly act on the adjacent nucleosomes. Raplp is shown as a dimer on the basis of recent biochemical studies (A Cassidy-Stone, SC Schultz, personal communication).
The role of histone acetylation and deacetylation
Ahhough transcriptionally inactive regions--such as subtelomeric and Hill loci [ 3 0 ] - - t e n d to contain hypoacetyfated histones H3 and H4, recent studies suggest a more complex relationship between histone acetylation and transcription. Indeed, it appears that acetylation of historic H4 lysine 12 is as critical for silencing as is deacetylation of lysine 16 [31]. It is therefore likely that repressed chromatin requires a specific set of partially acetylated histones [32]. T h e recent elucidation of the crystal structure of the nucleosome [33] will provide additional testable clues as to the role of this histone modification in silencing. Two histone deacetylases that may be linked to silencing have been identified recently. T h e first, Rpd3p, acts as a repressor of silencing. Intriguingl3q Rpd3p specifically deacetylates lysine 5 and 12 in histone H4 in vitro [34,35",36], lending support to the notion that acetylation of one or both lysines is critical for the silenced state. A second deacetylase, Hdalp, has a more uniform site preference in deacetylation and also represses silencing [36,37]. Numerous putative acetyltransferases with roles in silencing have also been identified. Two of these, Sas2p and Sas3p - - highly conserved among e u k a r y o t e s - - a r e required for telomeric and complete HM silencing [38,39"]. Although activity has not been demonstrated in vitro, both Sas2p and Sas3p display acetyltransferase consensus sequences. Despite the connection of these activities to silencing, it is not known whether histones H3 and H4 are the in vivo substrates for any of these enzymes and, if so, whether they confer similar acetylation patterns. An additional potential complication is presented by the large number of acetyltransferases and deacetylases associated with the plethora of recently identified chromatin remodeling machines (see [32]). Further studies will be required to ascertain whether these factors act in silencing through a direct alteration in chromatin structure or in the regulation of silencing factors. At what stage does selective acetylation participate in silencing? One viewpoint is that the spreading of repressed chromatin involves cooperative deposition of specifically acetylated histones in a unidirectional fashion. This might be mediated by a preference of Sir3p for association with a specific subset of deacetylated histones, leading to the recruitment and deposition of specifically modified histones H3 and H4 to adjacent nucleosomes either during nucleosome remodeling or onto pre-existing nucleosomes. An alternative possibility is that the observed acetylation
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Chromosomes and expression mechanisms
pattern is the product of deposition by a chromatin assembly machinery unique to repressed regions. A candidate for one such activity is the replication-dependent yeast chromatin assembly factor-1 (CAF-1), mutations in which derepress telomeric silencing [40"%41"], The vertebrate CAF-1 deposits cytoplasmically synthesized histones H3 and H4 (which, in the case of histone H4, is acetylated only at lysine 12) onto newly replicated DNA (see [32] and Waltrath review, this issue, pp 147-153). T h e specific deposition of these histones could very well account for the observed acetvlation pattern.
The role of replication in silencing Passage through S phase is an absolute requirement for the establishment of silencing [42] but the factors responsible for this S-phase requirement and their relationship to replication have remained elusive. One obvious candidate is ORe, binding sites for which are present at HM silencers and in subtelomeric regions. While providing an initially tantalizing link between replication and silencing, the identification of ore5 separation-of-function alleles has demonstrated that the function of O R e in silencing and in replication may be distinct [43,44,45"]. These data are more consistent with a role for O R e in Sir protein recruitment. Another candidate is CAF-1. All three subunits of CAF-1 have been purified and the corresponding genes (CACI, CAC2, and CAC3) cloned [41"]. One of these subunits, Caclp, was identified independently as R!f2p, a protein necessary for Raplp localization to telomeric loci [40"] and as a factor that associates with the Piflp helicase [46"]. Mutations in any one of the three subunits eliminate telomeric silencing, while having lesser effects on H,4I silencing. Ahhough CAF-1 activity at the telomere may explain the specific acetylation pattern in repressed regions, investigations into the function of this complex are still in their infancy.
Interrelationship between establishment, maintenance, and heritability Previous investigations have separated silencing into three distinct steps: establishment (formation of repressed chromatin from derepressed chromatin), maintenance (the stability of the repressed chromatin within a cell cycle), and inheritability (the stability of repression from generation to generation) [1]. These processes, although phenotypically distinct, may overlap mechanistically with each other and with nucleation and spreading as defined above. The primary indication of this is that Hell silencers are involved in both establishment and heritability [47]. This conclusion was drawn by following the characteristics of the repressed HAlL locus after physical elimination of the silencer by site-specific recombination. Cells that were in the repressed state did not require the silencer to maintain HAlL repression within that cell cycle. In subsequent cell cycles, however, repression was lost rapidly, indicating the requirement for the silencers in inheritability. These data raise the possibility that
silencing is erased transiently in every cell cycle and requires a re-establishment step either through facilitation of factor recruitment and/or association of the silencer directly with repressed chromatin. Although the processes are linked, several proteins, including Sirlp, Orc2p, and Sas2p, act predominantly in establishment [38,44,48], whereas the CAF-1 complex has a predominant role in heritability or maintenance ([46"]; J Berman, personal communication).
Sir4p ligands reveal links between silencing, ubiquitination and DNA repair The complexity of Sir-mediated interactions (Table 1) has been best illustrated by the sheer number of putative Sir4p ligands. Previous investigations have demonstrated that Sir4p can self-associate and associate with histoncs H3 and H4, Raplp, Sir3p, and Sirlp ([3,4,13",14,18]; S Gasser, personal communication). More recently, associations have been identified between Sir4p and the de-ubiquitinating enzyme Ubfp3 [49]--an inhibitor of telomeric silencing--providing the first link between the ubiquitination pathway and silencing. Consistent with this, mutations in Rad6p/Ubc2p, a ubiquitin-conjugating enzyme, reduces telomeric and HM silencing [2"]. Sir4p has also been shown to interact in a two-hybrid assay with Hdflp [50"], the yeast homologue of the 70kDa subunit of the mammalian Ku factor, a protein involved in non-homologous double strand DNA repair. It has been proposed that Ku recruits Sir4p to double-strand breaks, where it may induce a heterochromatin-like state important for DNA end-joining. Sir4p also associates in a two-hybrid assay with two distinct WD40 proteins of unknown function that interfere with silencing: Dislp and Sif2p ([51]; S Gasser, personal communication). Table 1 Evidence for the presence of multiple Sir4p ligands. Ligand
Method
IP Sir1 p Sir2p Sir3p Sir4p Rap 1 p Histone H3 Histone H4 Sif2p Ubf3p Hdfl p Dis 1 p
+ + + +
+ + -
Function in silencing
Two hybrid
GST m vivo
in vitro
GST
+ + + + + + + + + +
+ + + -
+ + + + -
Positive Positive Positive Positive Regulatory Regulatory Regulatory Negative Negative Unknown Positive
IP, interaction demonstrated by co-immunoprecipitation; G S T in vivo, interaction demonstrated by western blot analysis of G S T hybrid proteins partially purified after expression in yeast; G S T in vitro, demonstration of direct interaction b e t w e e n a G S T fusion protein and a s e c o n d protein, usually p r o d u c e d as a labeled translation product. Function in silencing refers to the role inferred from genetic studies. A regulatory role refers to the ability of the factor to act in opposing directions d e p e n d i n g on its state of modification or its associated ligands.
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
There are several caveats, however, influencing the interpretation of these associations: first, the domains involved in these associations may be unavailable or regulated in their normal context [13°]; second, associations that rest, It in opposing phenotypes may be regulated at specific cell-cycle windows; and third, many associations were identified by use of the two-hybrid method alone, raising the possibility that complex formation may be mediated
could not bc cited because of space limitations. I would like to thank Lucianne Morin for library research and Mary Ann Oslcy (SIoan-Kettering Institute), l.inda Hyman (Tulane University), Titia de l.ange (Rockefeller Univcrsity), and EB Hoffman for critical comments. Wnrk in my laboratory was supported by National Science l'oundation grant MCB 9604194.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
by a third factor.
From telomeres to the nucleolus: the aging connection Sir2p-dependcnt transcriptional silencing can also take place at T3'I elements inserted into the rDNA array [52%53°]. Interestingly, this form of silencing is not dependent on either Sir3p or Sir4p. Consistent with the physiological significance of these data, Sir2p immunolocalizes to both telomeric loci and the nucleolus [19**]. Recent studies have suggested a novel physiological interrelationship between telomeric silencing and the nucleolus in aging, as defined by the capacity of mother cells to form viable daughter cells. Mutations in sit~2, sirs and sir4 decrease lifespan whereas cells carrying a novel gain-of-function allele, S I R 4 - 4 2 - - t h a t obliterates telomeric silencing--extends it [54"']. Remarkably, aging yeast cells and sir mutant cells share three phenotypes: loss of telomeric and Hilt silencing [55-57], localization of Sir2p, Sir3p, and Sir4p to the nucleolus [54•*], and nucleolar fragmentation [58°]. Although the mechanistic steps of this process remain a challenge, this provides the first example of a programmed function for telomeric silencing and strengthens the argument that telomeres are physiologically regulated repositories for silencing factors [5,(,,S8"].
• •. 1.
Acknowledgements I would like to thank members of the research community for sharing data prior to publication. I also apologize to those individuals whose work
of special interest of outstanding interest Leo S, Rine J: Silencing and heritable domains of gene expression. Annu Rev Cell Bio/1995, 11:519-548.
2. •
Huang H, Kahana A, Gottschling DE, Prakash L, Liebman SW: The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae. Mol Cell Bio/1997, 17:6693-6699. Ubc2p, a ubiquitin-conjugating enzyme, was shown to be required for telomeric and HM silencing. Mutational analysis demonstrated that this function is distinct from the role of ubiquitin in the N-end rule pathway of protein degradation. This paper also provides the first demonstration that Pol III genes are also subject to SIR-regulated telomeric silencing. 3.
Moretti P, Freeman K, Coodly L, Shore D: Evidence that a complex of SIR proteins interacts with the silencer and telomere binding protein RAP1. Genes Dev 1994, 8:2257-2269.
4.
Ceckell M, Palladino F, Laroche T, Kyrion G, Liu C, Lustig AJ, Gasser SM: The carboxy termini of Sir4p and Raplp affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing. J Cell Biol 1995, 129:909-924.
5.
Buck S, Shore D: Action of a RAP1 carboxy-terminal silencing domain reveals an underlying competition between HMR and telomeres in yeast. Genes Dev 1995, 9:3?0-384.
6.
Lustig AJ, Liu C, Zhang C, Hanish J: Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae. Mol Ceil Biol 1996, 16:2483-2495.
7.
Marcand S, Buck SW, Moretti P, Gilson E, Shore D: Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap1 protein. Genes Dev 1996, 10:1297-1309.
8.
Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM: The clustering of telomeres and co-localization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 1996, 134:1349-1363.
9.
Stone EM, Pillus L: Activation of an MAP kinase cascade leads to Sir3p hyperphosphorylation and strengthens transcriptional silencing. J Cell Biol 1996, 135:571-583.
10.
Hardy C, Sussel L, Shore D: A RAPl-interacting protein involved in transcriptional silencing and telomere length regulation. Genes Dev 1992, 6:801-814.
11.
Wotton D, Shore D: A novel Raplp-interacting factor, Rif2p, cooperates with Rifl p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 1997, 11:748-760.
Conclusions Investigations over the past year have helped to elucidate the steps and factors involved in telomeric and Hll/ silencing and have identified links between silencing and other processes. These include the identification and characterization of a replication-dependent chromatin assembly factor involved in silencing and the identification of a putative developmental program for aging dependent upon the silencing process. Three areas of study will probably be pursued during the coming year: first, the mechanism by which a silencer imposes directionality on repressed chromatin; second, the dissection of CAF-1 function in the heritability of silencing, and third, a molecular characterization of the factors that mobilize silencing proteins to different nuclear compartments. T h e recent identification of higher eukaryotic homologues of novel yeast silencing factors [35",38,44,58",59,60] will further catalyze investigations into the conserved functions of the silencing process.
237
12. *,,
Marcand S, Gilson E, Shore D: A protein-counting mechanism for telomere length regulation in yeast. Science 1997, 275:986990. To test whether the number of Raplp carboxyl termini are counted by the telomere-sizing machinery, multiple sites for the Gal4-binding domain were introduced adjacent to the telomeric tract. Introduction of fusions between the Gal4-binding domain and the Rap1 p carboxyl terminus led to a shortening of telomere tract length in cis. Tethering of Sir4p or Sir3p fusion proteins to the telemere resulted in elongated telomeres, whereas mutations in SIR4 and SIR3 normally lead to a reduction in telomere tract length. These data suggest the presence of a protein-counting mechanism and further imply a competition between silencing factors and regulators of telomere size for association with the carbexyl terminus of Rap1 p. 13. •
Moazed D, Kistler A, Axelrod A, Rine J, Johnson AD: Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3. Proc/Vat/ Acad Sci USA 1997, 94:2186-2191. GST fusion proteins with both full-length Sir4p and domains of Sir4p were tested for their ability to associate with Sir2p and Sir3p by a combination of
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Chromosomes and expression mechanisms
affinity chromatography and western blot analysis. Both Sir2p and Sir3p interacted with intact Sir4p and a Sir4p carboxy-terminal domain. Sir2p/Sir4p interactions were detected both by in vitro GST-based techniques and by co-immunoprecipitation of Sir2p with epitope-tagged Sir4p. Further domain structure analysis revealed a specific Sir4p region that abrogated interaction with Sir3p. These data raise the possibility that Sir3p/Sir4p interaction is regulated in vivo through the association of other Sir4p-interacting factors. 14.
Chien C-T, Barrel P, Sternglanz R, Fields S: The two hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Nat/Acad Sci USA 1991,88:9578-9582.
15.
Liu C, Lustig A J: Genetic analysis of Raplp/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae. Genetics 1996, 143:81-93.
16.
Stavenhagen J, Zakian VA: Internal tracts of telomeric DNA acts as silencers in Saccharomyces cerevisiae, Genes Dev 1994, 8:1411-1422.
1 "Z
TrioloT, Sternglanz R: Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing. Nature 1996, 381:251-253.
18.
Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M: Histone H3 and H4 N-termini interact with the silent information regulators Sir3 and Sir4: a molecular model for the formation of heterochromatin in yeast. Ceil 1995, 80:583-592.
19. ••
Gotta M, StrahI-Bolsinger S, Renauld H, Laroche T, Kennedy BK, Grunstein M, Gasser SM: Localization of Sir2p: the nucleolus as a compartment for silent information regulators. EMBO J 1997, 16:3243-3255. Sir2p was demonstrated immunocytochemically to be localized to two major regions of the nucleus: clustered telomeric loci and the nucleolus. Physical localization of Sir2p to the nucleolar rDNA was confirmed by the reversible formaldehyde crosslinking method. Mutations in SIR4 resulted in the relocalization of Sir3p from telomeric foci to the nucleolus in a process dependent upon both Sir2p a n d - a protein involved in mother cell aging- Uth4p. 20. ••
Maillet L, Boscheron C, Gotta M, Marcand S, Gilson E, Gasser SM: Evidence for silencing compartments within the yeast nucleus: a role for tel•mere proximity and Sir protein concentration in silencer-mediated repression. Genes Dev 1996, 10:1796-1811. A cassette containing the HMLE and HMLI silencers flanking a lacZ reporter gene was utilized to determine the effects of chromosomal position on HM silencing. Placement of the cassette at internal sites severely diminished silencing. High rates of silencing were regained, however, when a tel•mere was artificially positioned adjacent to the silencer. Silencing, in this case, did not propagate from the tel•mere itself; rather, the telomere appeared to promote repression of lacZ expression from the HML silencer. A stimulatory effect was also achieved when multiple Raplp-binding sites were placed adjacent to an internal silencer. Overexpression of Sir1 p, Sir3p and Sir4p also enhanced silencing when the cassette was present at an internal chromosomal position. These data argue that a microenvironment containing a high concentration of silencing factors and/or the relocalization of the internal silencer to a specific nuclear subcompartment stimulates the internal silencer. 21.
Shei G-J, Broach JR: Yeast silencers can act as orientationdependent gone inactivation centers that respond to environmental signals. Mol Cell Biol 1996, 15:3496-3506.
22.
Boscheron C, Maillet L, Marcand S, Tsai-Plugfelder M, Gasser SM, Gilson E: Cooperation at a distance between silencers and proto-silencers at the yeast HML locus. EMBO J 1996, 15:2184-2195.
23.
Hecht A, StrahI-Belsinger, Grunstein M: Spreading of transcriptional represser SIR3 from telomeric heterochromatin. Nature 1996, 383:92-96.
24. ••
StrahI-Bolsinger S, Hecht A, Luo K, Grunstein M: SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 1997, 11:83-93. The reversible formaldehyde crosslinking method was used to determine the presence of Sir2p, Sir3p, and Sir4p as a function of distance from telomeric tracts in wild-type cells. All three factors were most concentrated at DNA proximal to the telemere, with their abundance decreasing with distance from the tel•mere. Raplp was also found to be associated with DNA regions close to the telomere. Upon overproduction of Sir3p-which extends the region of DNA associated with Sir3p-Sir2p and Sir4p were found in far lower abundance relative to Sir3p than under normal conditions. In addition, Rap1 p was not present in these tel•mere-distal regions. The authors propose two regions of heterochromatin: a core region composed of Sir2p, Sir3p, and Sir4p interacting with nucleosomal histones and forming a feldback structure with telomeric chromatin, and an extended chromatin region, made up pre-
dominantly of Sir3p associating with histones and not involving interactions with telomeric chromatin. 25.
Renauld H, Aparicio O, Zierath P, Billington B, Chhablani S, Gottschling DE: Silent domains are assembled continuously from the tel•mere and are defined by promoter distance and strength, and SIR3 dosage, Genes Dev 1993, 7:1133-1145.
26.
Vega-Palas MA, Venditt S, Di Mauro E: Telomeric transcriptional silencing in a natural context. Nat Genet 1997, 15:231-233.
27.
Zou S, Kim JM, Voytas DF: The Saccharomyces retrotransposon Ty5 influences the organization of chromosome ends. Nucleic Acids Res 1996, 24:4825-4831.
28.
Zeu S, Ke N, Kim JM, Voytas DF: The Saccharomyces retrotransposon Ty5 integrates preferentially into regions of silent chromatin at the tel•meres and mating loci. Genes Dev 1996, 10:634-645.
29. ee
Zou S, Voytas DF: Silent chromatin determines target preference of the Saccharomyces retrotransposon Ty5. Prec Nat/Acad Sci USA 1997, 94:7412-7416. An assay was established for induction of Ty5 transposition that was capable of distinguishing recombination from transposition. Previous data from this group had demonstrated that Ty5 preferentially integrates near silenced regions. In this study, the authors demonstrate that mutations in any one of the three binding sites present at HMRE partially reduced targeting of Ty5 to HMR, whereas elimination of any two sites abrogated preferential integration. These data parallel the effects of these same cis-acting mutations in silencing and suggest that the formation of a S/R-dependent heterochromatic region is essential for Ty5 targeting specificity. 30.
Braunstein M, Rose A, Holmes S, AIlis C, Broach J: Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev 1993, 7:592-604.
31.
Braunstein M, Sobel RE, Allis CD, Turner BM, Broach, JR: Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol 1996, 16:4349-4356.
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Grunstein M: Histone acetylation in chrematin structure and transcription. Nature 1997, 389:349-352.
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Luger K, Mader AW, Richmon RK, Sargent DF, Richmond T.I: Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 1997, 389:251-260.
34.
Vannier D, Balderes D, Shore D: Evidence that the transcriptional regulators SIN3 and RPD3 and a novel gene (SDS3) with similar functions are involved in transcriptional silencing in S. cerevisiae. Genetics 1996 144:1343-1353.
35. •
De Rubertis F, Kadosh D, Henchoz S, Pauli D, Reuter G, Struhl K, Spierer P: The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 1996, 384:589-591. An insertional mutation that enhances Drosophila position effect variegation was identified and demonstrated to be the homologue of the yeast RPD3 gene, encoding a histone deacetylase. Mutations in the yeast RPD3 gene enhanced telomeric silencing, consistent with a common function of the deacetylase in transcriptional repression in the two organisms. 36.
Rundlett SE, Carmen AA, Kobayashi R, Bavykin S, Turner BM, Grunstein M: HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proc Nat/Acad Sci USA 1996, 93:14503-14508.
37.
Carmen AA, Rundlett SE, Grunstein M: HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex. J Biol Chem 1996, 271:15837-15844.
38.
Reifsnyder C, Lowell J, Clarke A, Pillus L: Yeast SAS silencing genes and human genes associated with AML and HIV-1 Tat interactions are homologous with acetyltransferases. Nat Genet 1996, 14:42-49.
39. •
Ehrenhofer-Murray AE, Rivier DH, Rine J: The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function. Genetics 1997, 145:923-934. Second site-suppressors of cis-acting mutations in the HMRE silencer were identified. One of the suppressors, SAS2, reduced silencing at HML, while enhancing silencing at HMR in an Orc2p- and Orc5p-dependent fashion. Sequencing of the gene revealed significant homology to known acetyltransferases. 40. ••
Enomoto S, McCune-Zierath PD, Gerami-Nejad M, Sanders MA, Berman J: RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo. Genes Dev 1997, 11:358-370. Previous studies from this group identified a series of mutations defective in Rap1 p localization to discrete peripheral loci. In this study, one of these genes, RLF2, was demonstrated to be identical to CAC1, encoding a sub-
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
unit of chrematin assembly factor-I. Mutations in RLF2 reduced the efficiency of telomeric silencing, while not influencing telomere size. Although Raplp clustering was eliminated, telomeres remained clustered, suggesting that telomeres can cluster in the absence of high concentrations of Rap1 p. The authors propose that CAF-1 rapidly deposits a specific subclass of acetylated histones to form a repressed chromatin state. 41. •.
Kaufman PD, Kobayashi R, Stillman B: Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I. Genes Dev 199"7, 11:345-357. A replication-dependent chromatin assembly factor was identified in yeast by biochemical fractionation and shown to consist of three subunits, denoted Cacl p, Cac2p, and Cac3p. Peptides from each of the subunits were sequenced, and the wild-type genes were isolated. Each subunit displayed weak, but significant, homology to its human counterpart. Mutations in any of the subunits lead to UV hypersensitivity and a partial loss of telomeric silencing. 42.
Miller A, Nasmyth K: Role of DNA replication in the repression of silent mating type loci in yeast. Nature 1984, 312:247-251.
43.
Fox CA, Leo S, Dillin A, Rine J: The origin recognition complex has essential functions in transcriptional silencing and chromosomal replication. Genes Dev 1995, 9:911-924.
44.
Ehrenhofer-Murray AE, Gossen M, Pak DTS, Botchan MR, Rine J: Separation of origin recognition complex functions by crossspecies complementation. Science 1995, 270:1671-1674.
45. Dillon A, Rine .J: Separable functions of ORC5 in replication •* initiation and silencing. Genetics 1997, 147:1053-1062. Spontaneous revertants of the orc5-1 allele resulted in the identification of novel orc5 alleles that were specifically defective in either replication or in HMR silencing. Mutations that abrogated silencing were identified that were not defective in replication at the HMRE origin of replication, as determined by two-dimensional gel etectrophoresis. The presence of both classes of alleles in an orc5-1 mutant strain led to trans-complementation of both defects, further indicating that the role of ORC in silencing and replication may be functionally distinct. Monson EK, de Bruin D, Zakian VA: The yeast Cacl protein is required for the stable inheritance of transcriptionally repressed chromatin at telomeres. Proc Nat/Acad Sci USA 1997, 94:13081-13086. The authors use a single-cell assay to demonstrate that cacl mutant cells that were initially repressed were incapable of maintaining growth. These data suggest a defect in the inheritability of the repressed state. In contrast, no defect was found in the establishment of telomeric silencing.
51.
Holmes SG, Broach JR: Silencers are required for inheritance of the repressed state in yeast. Genes Dev 1996, 10:1021-1032.
48.
Pillus L, Rine J: Epigenetic inheritance of transcription states in S. cerevisiae. Cell 1989, 59:637-647.
49.
Moazed D, Johnson AD: A deubiquinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Ce//1996, 86:667-677.
50. •
Tsukamoto Y, Kate J-i, Ikeda, H: Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 199"7, 388:900-903. Two-hybrid analysis identified Sir4p as a protein capable of interacting with the 70 kDa subunit of the yeast Ku heterodimer, Hdfl p. Like half1 mutants, mutations in SIR2, SIR3, and SIR4 were defective in end-to-end joining and in combination with the rad52 mutation were defective in double-strand DNA repair. Epistasis analysis revealed that the Sir proteins and Hdfl p were acting in the same pathway. The authors propose that Hdfl p interacts with Sir4p at double-strand breaks to promote a heterochromatic state amenable to DNA repair.
Zhang Z, Buchman AR: Identification of a member of a DNAdependent ATPase family that causes interference with silencing. Mol Cell Biol 1997, 17:5461-54?2.
52. •
Smith JS, Boeke JD: An unusual form of transcriptional silencing in yeast ribosomal DNA. Genes Dev 1997, 11:241254. Genes cloned into a Tyl element, when transposed into the rDNA array, are transcriptionally repressed. This effect was independent of the gene tested but completely dependent on its presence in the rDNA array. A single repeat of rDNA containing the transposon placed elsewhere in the genome was not subject to silencing. This process was dependent on Sir2p but not on other Sir proteins. Psoralen is a reagent that photoreacts with accessible DNA upon UV treatment. Psoralen crosslinking analysis of silenced regions was consistent with a more condensed chromatin state dependent on Sir2p. 53. •
Bryk M, Banerjee M, Murphy M, Knudsen KE, Garfinkel DJ, Curcio MJ: Transcriptional silencing of Tyl elements in the RDN1 locus of yeast. Genes Dev 1997, 11:255-269. Transposition of a HIS3-marked Tyl element into the rDNA array led to SIR2-dependent silencing of the HIS3 gene. Silencing was dependent on Rad6p/Ubc2p, a ubiquitin-conjugating activity. The domains of Rad6p involved suggested, however, that this process was not mediated by the N-end rule protein degradation pathway. Depletion of the histenes H2A and H2B also led to a loss of silencing, linking alterations in chromatin structure with the rDNA-silencing process. 54. ••
Kennedy BK, Gotta M, Sinclair DA, Mills K, McNabb DS, Murthy M, Pak SM, Laroche T, Gasser SM, Guarente L: Redistribution of silencing proteins from telomeres to the nucleolus is associated with extension of life span in S. cerevisiae. Ceil 1997, 89:381-391. The SIR4-42 allele suppresses uth4 truncation alleles, resulting in extended mother cell lifespan. Interestingly, these cells also display a relocalization of both Sir3p and Sir4-42p to the nucleolus. Using a magnetic-sorting technique that separates young from old mother cells on the basis of the degree of biotin incorporation in the cell surface (previously developed in this lab), the authors demonstrated that Sir3p relocalizes from the telomere to the nucleolus during the aging process. 55.
Smeal T, Claus J, Kennedy B, Cole F, Guarente L: Loss of transcriptional silencing causes sterility in old mother cells of S. cerevisiae. Cell 1996, 84:633-642.
56.
Kim S, Viileponteau B, Jazwinski SM: Effect of replicative age on transcriptional silencing near telomeres in Saccharomyces cerevisiae. Biochem Biophys Res Comm 1996, 219:370-376.
57.
Austriaco NR, Guarente LP: Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae. Proc Nail Acad Sci USA 1997, 94:9768-9772.
46. •
4?.
239
58. •
Sinclair DA, Mills K, Guarente L: Accelerated aging and nucleolar fragmentation in yeast sgsl mutants. Science 1997, 277:1313-1316. The yeast SGS1 gene is homologous to the human gene mutated in Werner's syndrome, one of the symptoms of which is premature aging. To test the role of S g s l p in yeast aging, the number of generations a mother cell could produce a viable daughter was assayed. Mutants in Sgsl p, which localize to the nucleolus, exhibited a severely truncated lifespan, with a concomitant loss of HM silencing, relocalization of Sir3p to the nucleolus, and enlargement and fragmentation of the nucleoli. 59.
Laible G, Wolf A, Dorn R, Reuter G, Nislow C, Lebersorger A, Popkin D, Pillus L, Jenuwein T: Mammalian homologues of the Polycomb-group gene Enhancer ofzeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. EMBO J 1997, 11:3219-3232.
60.
Nislow C, Ray E, Pillus L: SET1, a yeast member of the Trithorax family, functions in transcriptional silencing and diverse cellular processes. Mol Biol Ceil 1998, 18:2421-2436.
Mechanisms of silencing in Saccharomyces cerevisiae Arthur J Lustig In the yeast Saccharomycescerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.
Figure 1 Telomeric silencer
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Addresses Department of Biochemistry, Tulane University Medical Center, 1430 Tulane Avenue, New Orleans, Louisiana 70112, USA; e-maih alustig@ mailhost.tcs.tulane.edu
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Current Opinion in Genetics & Development 1998, 8:233-239
HMR silencer
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Abbreviations Abflp ARS-binding factor 1 CAF-1 chromatin assembly factor-1 ORC origin recognition complex Raplp repressor activator protein 1
Introduction q\vo mechanistically related forms of transcriptional silencing are present in yeast: first, tclomeric silencing, the metastable transcriptional repression that occurs when a Pol II or Pol I[I transcript is positioned adjacent to tclomeric poly(TGl_3) tracts; and second, the stable repression of MATcz information at the HML locus, located 12kb from the left telomere of chromosome III, and of MATcz transcription at the HMR locus, located 20kb from the right arm of the same chromosome [1,2"]. In each case, c/s-acting silencer elements are responsible for the repression of transcription in adjacent sequences (Figure 1). At the telomerc, the silcncer is composed of muhiplc binding sites for repressor activator protein 1 (Raplp) embedded within the tclomeric tract. At HML and HMR, the silencers consist of specific arrays of binding sites for ARS-binding factor 1 (Abflp), the origin recognition complex (ORC), and Raplp. Although silencers at the three regions differ in their degree of functional redundancy, they share a dependence on the silent information regulators Sir2p, Sir3p, Sir4p, as well as thc amino termini of histones H3 and H4. Common to all three classes of silencers is the formation of an adjacent 'closed' chromatin statc, inaccessiblc to exogenous probes both in vivo and in vitro. T h e characteristics of silenced regions, transcriptional quiescence, chromatin compaction, and late replication suggest that thcse position effects are the yeast equivalent of higher eukaryotic heterochromatin. Tclomeric silencing exhibits the lowest lcvel of functional redundancy, making it most amcnable to analysis. I will thcrcfore focus on this form of silencing, drawing
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Current Opinion in Genetics & Development
Structure of the telomeric and HM silencers in yeast. The telomeric silencer contains multiple sites for the telomere-binding factor Raplp, present at a density of one in every twenty base pairs of telomere tract. The structure of the HM loci, as depicted (HML), are more complex and involve the participation of silencers that flank the repressed gene. The identify of the factor that binds to the D region remains unknown. A greater level of redundancy is present at the HM loci than at telomeres. In a chromosomal context, elimination of either HMLE or HMLI is insufficient to eliminate silencing. Similarly, in the absence of HMLI, mutation in any one binding site of HMLE leads to only partial derepression. A similar situation is present at the HMR locus: elimination of HMRI has a small effect on silencing, whereas elimination of HMRE eliminates silencing completely. In addition, at HMRE, two out of three binding sites must normally be mutated to derepress silencing. All three silencers are capable of repressing transcription of both Pol II and Pol Ill genes [1].
comparisons to HML and HMR silencing when data is available. In this revicw, I emphasize recent studies providing mechanistic information on the nucleation and spreading of represscd chromatin and on the involvemcnt of silencing factors in both DNA repair and aging.
The nucleation of silencing Nucleation refers to the set of events acting at the silenccr that lead to transcriptional repression in adjacent sequences. Multiple lincs of evidence suggest that the nucleation of telomeric silencing is the consequence of recruitment of Sir3p and Sir4p to the carboxyl termini of multiple Raplp molecules bound to telomeric tracts (Figure 2). First, Raplp is capable of direct interactions with Sir3p, and, most likely, Sir4p [3-5]. Second, Sir3 and Sir4 fusion proteins, when artifically tethered to a site at a tclomeric/subtelomeric junction, overcomc the abrogation of telomeric silcncing in rapl mutants lacking
234
Chromosomesand expression mechanisms
the carboxyl terminus [6,7]. Third, Raplp, Sir3p, and Sir4p co-immunolocalize to telomeric and/or subtelomeric domains at the nuclear periphery in a mutually dependent fashion [8]. Sir3p phosphorylation most likely serves as an additional regulatory step, as phosphorylated Sir3p enhances silencing [9]. Interestingly, two antagonists of silencing, Riflp and Rif2p, appear to compete with Sir3p and Sir4p for association with Rap l p [10,11,12°°]. This competition may explain the limiting function of Sir3p and Sir4p at the telomere, despite the large number of Raplp-binding sites. Consistent with this notion, artificial tethering of Sir3p or Sir4p to a telomeric site I w h i c h would be expected to overcome such competition--enhances silencing even in wild-type cells [6,7]. Sir3p and Sir4p appear to be capable of homo- and hetero-dimerization, although it is not known whether these associations take place within the telomeric tract [3,4,13°,14]. Nonetheless, these interactions provide a means of forming a higher-order structure mediated through association of Sir3p and Sir4p bound at adjacent Rapl molecules ([15]; Figure 2). Consistent with the viewpoint that these associations may stabilize the telomere as a silencer, long internal tracts of telomeric DNA can confer silencing [16]. An analogous process may take place at the HM silencers since both Raplp and Abflp can associate directly with Sir3p ([3]; P Moretti, D Shore, personal communication). Although ORC does not appear to interact with Sir3p directl-y; it may perform a similar function through the formation of Sirlp/Orclp and Sirlp/Sir4p interactions, both of which have been demonstrated experimentally [14,17]. Although the physical steps in recruitment are becoming clearer, their mechanistic consequence is uncertain. In some fashion, recruitment to silencers must initiate a cascade of events leading to a unidirectional and cooperative alteration in nucleosome structure (Figure 2). Recent investigations implicate two elements in this process. First, several studies indicate a requirement for a microenvironment containing a high concentration of Sir3p, Sir4p, and other silencing factors: one, Sir3p and Sir4p can associate with the amino termini of histories H3 and H4 in vitro and in vivo ([18]; S Gasser, personal communication); two, Raplp, Sir3p, Sir4p, and part of the cellular pool of Sir2p immunolocalize in vivo with clustered telomeric and/or subtelomeric regions [8,19"]; three, the introduction of elongated telomeres into a wild-type background diminishes HM silencing, suggesting titration of a limiting amount of Sir3p and Sir4p by the telomeric silencer [5]; four, silencing conferred by long internal tracts of telomeric sequence or by the HML silencer is enhanced by proximity to the telomere, even though silencing does not emanate from the telomerie tract in
either case [16,20"]; and five, the introduction of multiple Raplp binding sites adjacent to the MATc~ locus results in silencing of this normally active locus [21]. Second, although a high concentration of silencing factors may induce the cooperative formation of repressed chromatin, it is unclear how this process would result in the observed directionality of silencing. One possible means is through an initial 'bridge' between the silencer and adjacent histones (Figure 2). At the telomere, such a bridge might be provided by interaction of either Sir3p or Sir4p with both Raplp and specific forms of acetylated histones H3 and H4. This model would help to explain why tethering of Sir3p or Sir4p to the telomere confers silencing in rapl mutants unable to recruit either factor [6,7]. T h e nucleation of Hill silencing involves at least one additional level of complexity. A recent examination of the HMLE and HML1 silencing of a lacZ tester gene demonstrated that individual silencer binding sites--incapable of conferring silencing a l o n e - - c a n interact with silencers on the opposing side of thc lacZ gene, suggesting that protein-protein interactions flanking the gene, even if transient, facilitate the nucleation of silencing [22]. T h e ' s p r e a d i n g ' of r e p r e s s e d c h r o m a t i n Interaction of Sir3p and Sir4p with the histone amino termini The events that follow nucleation are somewhat clearer. As noted, direct interactions of the carboxyl terminus of Sir3p (and the amino terminus of Sir4p) with the amino termini of histones H3 and H4 have been demonstrated in vivo and in vitro ([18]; S Gasser, personal communication). T h e genomic sites of these interactions have, until recently, remained elusive. A reversible in vivo formaldehyde crosslinking method, however, has recently been used to demonstrate that Sir3p, Sir4p, Sir2p, and each core histone co-associate in subtelomeric DNA [23,24"]. The association of the Sir factors decrease with distance from the telomerc. Raplp also associates with these complexes, raising the possibility that a fold-back structure is formed between telomeric and subtelomeric regions [24"]. This method has also revealed that overproduction of Sir3p results in the physical presence of Sir3p at sites further from the telomere, consistent with the phenotypic spreading of silencing after overproduction of Sir3p [23,25]. Curiously, under these conditions, Sir2p and Sir4p association decreases significantly at these same distal sites. These data suggest that there are two forms of telomeric heterochromatin: a 'core' state composed of Sir2p, Sir3p and Sir4p, and an 'extended' region containing Sir3p but only limiting quantities of other Sir factors {24"°1.
These data also provide convincing evidence for the formation of condensed chromatin at bona fide telomeres. Both core and extended heterochromatin are found at naturally-occurring telomeres [23,24°°]. Further proof for
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
the existence of telomeric silencing at natural chromosomes comes from the finding that transcripts emanating from Ty5 elements, which insert preferentially into subtelomeric sites, are silenced in a SIR3-dependent fashion [26-28,29"°].
Figure 2
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Current Opinion in Genetics & Development
A speculative model for the steps involved in the formation of telomeric silencing, as described in the text, are depicted in this figure. In the first step, Rap1 p bound to telomeric consensus sites recruit the silencing factors Sir3p and Sir4p and the silencing-antagonistic factors Riflp and Rif2p. Subnuclear clustering of telomeres create a microenvironment high in concentration of silencing factors leading to a shift in the equilibrium to a preference for association with silencing factors. The association of Sir3p and Sir4p with themselves and with each other may help to stabilize the telomere as a silencer. This stabilized structure is subsequently able either to recruit a specifically modified set of histones H3 and H4 or to assist in the specific modification of these histones within the first nucleosome. Once Sir3p is associated with the initial amino-terminal tails, forming a 'histone bridge', subsequent Sir3p associations would similarly act on the adjacent nucleosomes. Raplp is shown as a dimer on the basis of recent biochemical studies (A Cassidy-Stone, SC Schultz, personal communication).
The role of histone acetylation and deacetylation
Ahhough transcriptionally inactive regions--such as subtelomeric and Hill loci [ 3 0 ] - - t e n d to contain hypoacetyfated histones H3 and H4, recent studies suggest a more complex relationship between histone acetylation and transcription. Indeed, it appears that acetylation of historic H4 lysine 12 is as critical for silencing as is deacetylation of lysine 16 [31]. It is therefore likely that repressed chromatin requires a specific set of partially acetylated histones [32]. T h e recent elucidation of the crystal structure of the nucleosome [33] will provide additional testable clues as to the role of this histone modification in silencing. Two histone deacetylases that may be linked to silencing have been identified recently. T h e first, Rpd3p, acts as a repressor of silencing. Intriguingl3q Rpd3p specifically deacetylates lysine 5 and 12 in histone H4 in vitro [34,35",36], lending support to the notion that acetylation of one or both lysines is critical for the silenced state. A second deacetylase, Hdalp, has a more uniform site preference in deacetylation and also represses silencing [36,37]. Numerous putative acetyltransferases with roles in silencing have also been identified. Two of these, Sas2p and Sas3p - - highly conserved among e u k a r y o t e s - - a r e required for telomeric and complete HM silencing [38,39"]. Although activity has not been demonstrated in vitro, both Sas2p and Sas3p display acetyltransferase consensus sequences. Despite the connection of these activities to silencing, it is not known whether histones H3 and H4 are the in vivo substrates for any of these enzymes and, if so, whether they confer similar acetylation patterns. An additional potential complication is presented by the large number of acetyltransferases and deacetylases associated with the plethora of recently identified chromatin remodeling machines (see [32]). Further studies will be required to ascertain whether these factors act in silencing through a direct alteration in chromatin structure or in the regulation of silencing factors. At what stage does selective acetylation participate in silencing? One viewpoint is that the spreading of repressed chromatin involves cooperative deposition of specifically acetylated histones in a unidirectional fashion. This might be mediated by a preference of Sir3p for association with a specific subset of deacetylated histones, leading to the recruitment and deposition of specifically modified histones H3 and H4 to adjacent nucleosomes either during nucleosome remodeling or onto pre-existing nucleosomes. An alternative possibility is that the observed acetylation
236
Chromosomes and expression mechanisms
pattern is the product of deposition by a chromatin assembly machinery unique to repressed regions. A candidate for one such activity is the replication-dependent yeast chromatin assembly factor-1 (CAF-1), mutations in which derepress telomeric silencing [40"%41"], The vertebrate CAF-1 deposits cytoplasmically synthesized histones H3 and H4 (which, in the case of histone H4, is acetylated only at lysine 12) onto newly replicated DNA (see [32] and Waltrath review, this issue, pp 147-153). T h e specific deposition of these histones could very well account for the observed acetvlation pattern.
The role of replication in silencing Passage through S phase is an absolute requirement for the establishment of silencing [42] but the factors responsible for this S-phase requirement and their relationship to replication have remained elusive. One obvious candidate is ORe, binding sites for which are present at HM silencers and in subtelomeric regions. While providing an initially tantalizing link between replication and silencing, the identification of ore5 separation-of-function alleles has demonstrated that the function of O R e in silencing and in replication may be distinct [43,44,45"]. These data are more consistent with a role for O R e in Sir protein recruitment. Another candidate is CAF-1. All three subunits of CAF-1 have been purified and the corresponding genes (CACI, CAC2, and CAC3) cloned [41"]. One of these subunits, Caclp, was identified independently as R!f2p, a protein necessary for Raplp localization to telomeric loci [40"] and as a factor that associates with the Piflp helicase [46"]. Mutations in any one of the three subunits eliminate telomeric silencing, while having lesser effects on H,4I silencing. Ahhough CAF-1 activity at the telomere may explain the specific acetylation pattern in repressed regions, investigations into the function of this complex are still in their infancy.
Interrelationship between establishment, maintenance, and heritability Previous investigations have separated silencing into three distinct steps: establishment (formation of repressed chromatin from derepressed chromatin), maintenance (the stability of the repressed chromatin within a cell cycle), and inheritability (the stability of repression from generation to generation) [1]. These processes, although phenotypically distinct, may overlap mechanistically with each other and with nucleation and spreading as defined above. The primary indication of this is that Hell silencers are involved in both establishment and heritability [47]. This conclusion was drawn by following the characteristics of the repressed HAlL locus after physical elimination of the silencer by site-specific recombination. Cells that were in the repressed state did not require the silencer to maintain HAlL repression within that cell cycle. In subsequent cell cycles, however, repression was lost rapidly, indicating the requirement for the silencers in inheritability. These data raise the possibility that
silencing is erased transiently in every cell cycle and requires a re-establishment step either through facilitation of factor recruitment and/or association of the silencer directly with repressed chromatin. Although the processes are linked, several proteins, including Sirlp, Orc2p, and Sas2p, act predominantly in establishment [38,44,48], whereas the CAF-1 complex has a predominant role in heritability or maintenance ([46"]; J Berman, personal communication).
Sir4p ligands reveal links between silencing, ubiquitination and DNA repair The complexity of Sir-mediated interactions (Table 1) has been best illustrated by the sheer number of putative Sir4p ligands. Previous investigations have demonstrated that Sir4p can self-associate and associate with histoncs H3 and H4, Raplp, Sir3p, and Sirlp ([3,4,13",14,18]; S Gasser, personal communication). More recently, associations have been identified between Sir4p and the de-ubiquitinating enzyme Ubfp3 [49]--an inhibitor of telomeric silencing--providing the first link between the ubiquitination pathway and silencing. Consistent with this, mutations in Rad6p/Ubc2p, a ubiquitin-conjugating enzyme, reduces telomeric and HM silencing [2"]. Sir4p has also been shown to interact in a two-hybrid assay with Hdflp [50"], the yeast homologue of the 70kDa subunit of the mammalian Ku factor, a protein involved in non-homologous double strand DNA repair. It has been proposed that Ku recruits Sir4p to double-strand breaks, where it may induce a heterochromatin-like state important for DNA end-joining. Sir4p also associates in a two-hybrid assay with two distinct WD40 proteins of unknown function that interfere with silencing: Dislp and Sif2p ([51]; S Gasser, personal communication). Table 1 Evidence for the presence of multiple Sir4p ligands. Ligand
Method
IP Sir1 p Sir2p Sir3p Sir4p Rap 1 p Histone H3 Histone H4 Sif2p Ubf3p Hdfl p Dis 1 p
+ + + +
+ + -
Function in silencing
Two hybrid
GST m vivo
in vitro
GST
+ + + + + + + + + +
+ + + -
+ + + + -
Positive Positive Positive Positive Regulatory Regulatory Regulatory Negative Negative Unknown Positive
IP, interaction demonstrated by co-immunoprecipitation; G S T in vivo, interaction demonstrated by western blot analysis of G S T hybrid proteins partially purified after expression in yeast; G S T in vitro, demonstration of direct interaction b e t w e e n a G S T fusion protein and a s e c o n d protein, usually p r o d u c e d as a labeled translation product. Function in silencing refers to the role inferred from genetic studies. A regulatory role refers to the ability of the factor to act in opposing directions d e p e n d i n g on its state of modification or its associated ligands.
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
There are several caveats, however, influencing the interpretation of these associations: first, the domains involved in these associations may be unavailable or regulated in their normal context [13°]; second, associations that rest, It in opposing phenotypes may be regulated at specific cell-cycle windows; and third, many associations were identified by use of the two-hybrid method alone, raising the possibility that complex formation may be mediated
could not bc cited because of space limitations. I would like to thank Lucianne Morin for library research and Mary Ann Oslcy (SIoan-Kettering Institute), l.inda Hyman (Tulane University), Titia de l.ange (Rockefeller Univcrsity), and EB Hoffman for critical comments. Wnrk in my laboratory was supported by National Science l'oundation grant MCB 9604194.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
by a third factor.
From telomeres to the nucleolus: the aging connection Sir2p-dependcnt transcriptional silencing can also take place at T3'I elements inserted into the rDNA array [52%53°]. Interestingly, this form of silencing is not dependent on either Sir3p or Sir4p. Consistent with the physiological significance of these data, Sir2p immunolocalizes to both telomeric loci and the nucleolus [19**]. Recent studies have suggested a novel physiological interrelationship between telomeric silencing and the nucleolus in aging, as defined by the capacity of mother cells to form viable daughter cells. Mutations in sit~2, sirs and sir4 decrease lifespan whereas cells carrying a novel gain-of-function allele, S I R 4 - 4 2 - - t h a t obliterates telomeric silencing--extends it [54"']. Remarkably, aging yeast cells and sir mutant cells share three phenotypes: loss of telomeric and Hilt silencing [55-57], localization of Sir2p, Sir3p, and Sir4p to the nucleolus [54•*], and nucleolar fragmentation [58°]. Although the mechanistic steps of this process remain a challenge, this provides the first example of a programmed function for telomeric silencing and strengthens the argument that telomeres are physiologically regulated repositories for silencing factors [5,(,,S8"].
• •. 1.
Acknowledgements I would like to thank members of the research community for sharing data prior to publication. I also apologize to those individuals whose work
of special interest of outstanding interest Leo S, Rine J: Silencing and heritable domains of gene expression. Annu Rev Cell Bio/1995, 11:519-548.
2. •
Huang H, Kahana A, Gottschling DE, Prakash L, Liebman SW: The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae. Mol Cell Bio/1997, 17:6693-6699. Ubc2p, a ubiquitin-conjugating enzyme, was shown to be required for telomeric and HM silencing. Mutational analysis demonstrated that this function is distinct from the role of ubiquitin in the N-end rule pathway of protein degradation. This paper also provides the first demonstration that Pol III genes are also subject to SIR-regulated telomeric silencing. 3.
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Gotta M, Laroche T, Formenton A, Maillet L, Scherthan H, Gasser SM: The clustering of telomeres and co-localization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J Cell Biol 1996, 134:1349-1363.
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Wotton D, Shore D: A novel Raplp-interacting factor, Rif2p, cooperates with Rifl p to regulate telomere length in Saccharomyces cerevisiae. Genes Dev 1997, 11:748-760.
Conclusions Investigations over the past year have helped to elucidate the steps and factors involved in telomeric and Hll/ silencing and have identified links between silencing and other processes. These include the identification and characterization of a replication-dependent chromatin assembly factor involved in silencing and the identification of a putative developmental program for aging dependent upon the silencing process. Three areas of study will probably be pursued during the coming year: first, the mechanism by which a silencer imposes directionality on repressed chromatin; second, the dissection of CAF-1 function in the heritability of silencing, and third, a molecular characterization of the factors that mobilize silencing proteins to different nuclear compartments. T h e recent identification of higher eukaryotic homologues of novel yeast silencing factors [35",38,44,58",59,60] will further catalyze investigations into the conserved functions of the silencing process.
237
12. *,,
Marcand S, Gilson E, Shore D: A protein-counting mechanism for telomere length regulation in yeast. Science 1997, 275:986990. To test whether the number of Raplp carboxyl termini are counted by the telomere-sizing machinery, multiple sites for the Gal4-binding domain were introduced adjacent to the telomeric tract. Introduction of fusions between the Gal4-binding domain and the Rap1 p carboxyl terminus led to a shortening of telomere tract length in cis. Tethering of Sir4p or Sir3p fusion proteins to the telemere resulted in elongated telomeres, whereas mutations in SIR4 and SIR3 normally lead to a reduction in telomere tract length. These data suggest the presence of a protein-counting mechanism and further imply a competition between silencing factors and regulators of telomere size for association with the carbexyl terminus of Rap1 p. 13. •
Moazed D, Kistler A, Axelrod A, Rine J, Johnson AD: Silent information regulator protein complexes in Saccharomyces cerevisiae: a SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3. Proc/Vat/ Acad Sci USA 1997, 94:2186-2191. GST fusion proteins with both full-length Sir4p and domains of Sir4p were tested for their ability to associate with Sir2p and Sir3p by a combination of
238
Chromosomes and expression mechanisms
affinity chromatography and western blot analysis. Both Sir2p and Sir3p interacted with intact Sir4p and a Sir4p carboxy-terminal domain. Sir2p/Sir4p interactions were detected both by in vitro GST-based techniques and by co-immunoprecipitation of Sir2p with epitope-tagged Sir4p. Further domain structure analysis revealed a specific Sir4p region that abrogated interaction with Sir3p. These data raise the possibility that Sir3p/Sir4p interaction is regulated in vivo through the association of other Sir4p-interacting factors. 14.
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1 "Z
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18.
Hecht A, Laroche T, Strahl-Bolsinger S, Gasser SM, Grunstein M: Histone H3 and H4 N-termini interact with the silent information regulators Sir3 and Sir4: a molecular model for the formation of heterochromatin in yeast. Ceil 1995, 80:583-592.
19. ••
Gotta M, StrahI-Bolsinger S, Renauld H, Laroche T, Kennedy BK, Grunstein M, Gasser SM: Localization of Sir2p: the nucleolus as a compartment for silent information regulators. EMBO J 1997, 16:3243-3255. Sir2p was demonstrated immunocytochemically to be localized to two major regions of the nucleus: clustered telomeric loci and the nucleolus. Physical localization of Sir2p to the nucleolar rDNA was confirmed by the reversible formaldehyde crosslinking method. Mutations in SIR4 resulted in the relocalization of Sir3p from telomeric foci to the nucleolus in a process dependent upon both Sir2p a n d - a protein involved in mother cell aging- Uth4p. 20. ••
Maillet L, Boscheron C, Gotta M, Marcand S, Gilson E, Gasser SM: Evidence for silencing compartments within the yeast nucleus: a role for tel•mere proximity and Sir protein concentration in silencer-mediated repression. Genes Dev 1996, 10:1796-1811. A cassette containing the HMLE and HMLI silencers flanking a lacZ reporter gene was utilized to determine the effects of chromosomal position on HM silencing. Placement of the cassette at internal sites severely diminished silencing. High rates of silencing were regained, however, when a tel•mere was artificially positioned adjacent to the silencer. Silencing, in this case, did not propagate from the tel•mere itself; rather, the telomere appeared to promote repression of lacZ expression from the HML silencer. A stimulatory effect was also achieved when multiple Raplp-binding sites were placed adjacent to an internal silencer. Overexpression of Sir1 p, Sir3p and Sir4p also enhanced silencing when the cassette was present at an internal chromosomal position. These data argue that a microenvironment containing a high concentration of silencing factors and/or the relocalization of the internal silencer to a specific nuclear subcompartment stimulates the internal silencer. 21.
Shei G-J, Broach JR: Yeast silencers can act as orientationdependent gone inactivation centers that respond to environmental signals. Mol Cell Biol 1996, 15:3496-3506.
22.
Boscheron C, Maillet L, Marcand S, Tsai-Plugfelder M, Gasser SM, Gilson E: Cooperation at a distance between silencers and proto-silencers at the yeast HML locus. EMBO J 1996, 15:2184-2195.
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24. ••
StrahI-Bolsinger S, Hecht A, Luo K, Grunstein M: SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev 1997, 11:83-93. The reversible formaldehyde crosslinking method was used to determine the presence of Sir2p, Sir3p, and Sir4p as a function of distance from telomeric tracts in wild-type cells. All three factors were most concentrated at DNA proximal to the telemere, with their abundance decreasing with distance from the tel•mere. Raplp was also found to be associated with DNA regions close to the telomere. Upon overproduction of Sir3p-which extends the region of DNA associated with Sir3p-Sir2p and Sir4p were found in far lower abundance relative to Sir3p than under normal conditions. In addition, Rap1 p was not present in these tel•mere-distal regions. The authors propose two regions of heterochromatin: a core region composed of Sir2p, Sir3p, and Sir4p interacting with nucleosomal histones and forming a feldback structure with telomeric chromatin, and an extended chromatin region, made up pre-
dominantly of Sir3p associating with histones and not involving interactions with telomeric chromatin. 25.
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Zeu S, Ke N, Kim JM, Voytas DF: The Saccharomyces retrotransposon Ty5 integrates preferentially into regions of silent chromatin at the tel•meres and mating loci. Genes Dev 1996, 10:634-645.
29. ee
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Braunstein M, Rose A, Holmes S, AIlis C, Broach J: Transcriptional silencing in yeast is associated with reduced nucleosome acetylation. Genes Dev 1993, 7:592-604.
31.
Braunstein M, Sobel RE, Allis CD, Turner BM, Broach, JR: Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern. Mol Cell Biol 1996, 16:4349-4356.
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Luger K, Mader AW, Richmon RK, Sargent DF, Richmond T.I: Crystal structure of the nucleosome core particle at 2.8A resolution. Nature 1997, 389:251-260.
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Vannier D, Balderes D, Shore D: Evidence that the transcriptional regulators SIN3 and RPD3 and a novel gene (SDS3) with similar functions are involved in transcriptional silencing in S. cerevisiae. Genetics 1996 144:1343-1353.
35. •
De Rubertis F, Kadosh D, Henchoz S, Pauli D, Reuter G, Struhl K, Spierer P: The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 1996, 384:589-591. An insertional mutation that enhances Drosophila position effect variegation was identified and demonstrated to be the homologue of the yeast RPD3 gene, encoding a histone deacetylase. Mutations in the yeast RPD3 gene enhanced telomeric silencing, consistent with a common function of the deacetylase in transcriptional repression in the two organisms. 36.
Rundlett SE, Carmen AA, Kobayashi R, Bavykin S, Turner BM, Grunstein M: HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription. Proc Nat/Acad Sci USA 1996, 93:14503-14508.
37.
Carmen AA, Rundlett SE, Grunstein M: HDA1 and HDA3 are components of a yeast histone deacetylase (HDA) complex. J Biol Chem 1996, 271:15837-15844.
38.
Reifsnyder C, Lowell J, Clarke A, Pillus L: Yeast SAS silencing genes and human genes associated with AML and HIV-1 Tat interactions are homologous with acetyltransferases. Nat Genet 1996, 14:42-49.
39. •
Ehrenhofer-Murray AE, Rivier DH, Rine J: The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function. Genetics 1997, 145:923-934. Second site-suppressors of cis-acting mutations in the HMRE silencer were identified. One of the suppressors, SAS2, reduced silencing at HML, while enhancing silencing at HMR in an Orc2p- and Orc5p-dependent fashion. Sequencing of the gene revealed significant homology to known acetyltransferases. 40. ••
Enomoto S, McCune-Zierath PD, Gerami-Nejad M, Sanders MA, Berman J: RLF2, a subunit of yeast chromatin assembly factor-I, is required for telomeric chromatin function in vivo. Genes Dev 1997, 11:358-370. Previous studies from this group identified a series of mutations defective in Rap1 p localization to discrete peripheral loci. In this study, one of these genes, RLF2, was demonstrated to be identical to CAC1, encoding a sub-
Mechanisms of silencing in Saccharomyces cerevisiae Lustig
unit of chrematin assembly factor-I. Mutations in RLF2 reduced the efficiency of telomeric silencing, while not influencing telomere size. Although Raplp clustering was eliminated, telomeres remained clustered, suggesting that telomeres can cluster in the absence of high concentrations of Rap1 p. The authors propose that CAF-1 rapidly deposits a specific subclass of acetylated histones to form a repressed chromatin state. 41. •.
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Fox CA, Leo S, Dillin A, Rine J: The origin recognition complex has essential functions in transcriptional silencing and chromosomal replication. Genes Dev 1995, 9:911-924.
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Ehrenhofer-Murray AE, Gossen M, Pak DTS, Botchan MR, Rine J: Separation of origin recognition complex functions by crossspecies complementation. Science 1995, 270:1671-1674.
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Moazed D, Johnson AD: A deubiquinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Ce//1996, 86:667-677.
50. •
Tsukamoto Y, Kate J-i, Ikeda, H: Silencing factors participate in DNA repair and recombination in Saccharomyces cerevisiae. Nature 199"7, 388:900-903. Two-hybrid analysis identified Sir4p as a protein capable of interacting with the 70 kDa subunit of the yeast Ku heterodimer, Hdfl p. Like half1 mutants, mutations in SIR2, SIR3, and SIR4 were defective in end-to-end joining and in combination with the rad52 mutation were defective in double-strand DNA repair. Epistasis analysis revealed that the Sir proteins and Hdfl p were acting in the same pathway. The authors propose that Hdfl p interacts with Sir4p at double-strand breaks to promote a heterochromatic state amenable to DNA repair.
Zhang Z, Buchman AR: Identification of a member of a DNAdependent ATPase family that causes interference with silencing. Mol Cell Biol 1997, 17:5461-54?2.
52. •
Smith JS, Boeke JD: An unusual form of transcriptional silencing in yeast ribosomal DNA. Genes Dev 1997, 11:241254. Genes cloned into a Tyl element, when transposed into the rDNA array, are transcriptionally repressed. This effect was independent of the gene tested but completely dependent on its presence in the rDNA array. A single repeat of rDNA containing the transposon placed elsewhere in the genome was not subject to silencing. This process was dependent on Sir2p but not on other Sir proteins. Psoralen is a reagent that photoreacts with accessible DNA upon UV treatment. Psoralen crosslinking analysis of silenced regions was consistent with a more condensed chromatin state dependent on Sir2p. 53. •
Bryk M, Banerjee M, Murphy M, Knudsen KE, Garfinkel DJ, Curcio MJ: Transcriptional silencing of Tyl elements in the RDN1 locus of yeast. Genes Dev 1997, 11:255-269. Transposition of a HIS3-marked Tyl element into the rDNA array led to SIR2-dependent silencing of the HIS3 gene. Silencing was dependent on Rad6p/Ubc2p, a ubiquitin-conjugating activity. The domains of Rad6p involved suggested, however, that this process was not mediated by the N-end rule protein degradation pathway. Depletion of the histenes H2A and H2B also led to a loss of silencing, linking alterations in chromatin structure with the rDNA-silencing process. 54. ••
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Austriaco NR, Guarente LP: Changes of telomere length cause reciprocal changes in the lifespan of mother cells in Saccharomyces cerevisiae. Proc Nail Acad Sci USA 1997, 94:9768-9772.
46. •
4?.
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58. •
Sinclair DA, Mills K, Guarente L: Accelerated aging and nucleolar fragmentation in yeast sgsl mutants. Science 1997, 277:1313-1316. The yeast SGS1 gene is homologous to the human gene mutated in Werner's syndrome, one of the symptoms of which is premature aging. To test the role of S g s l p in yeast aging, the number of generations a mother cell could produce a viable daughter was assayed. Mutants in Sgsl p, which localize to the nucleolus, exhibited a severely truncated lifespan, with a concomitant loss of HM silencing, relocalization of Sir3p to the nucleolus, and enlargement and fragmentation of the nucleoli. 59.
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