Thesis
Reference
In vitro reconstitution and biochemical characterization of yeast silent chromatin
MARTINO, Fabrizio
Abstract
Aux niveaux des télomères et du locus HM chez "Saccharomyces cerevisiae", la chromatine organisée en structure d'ordre supérieur conduit à une répression transcriptionnelle dans ces régions. Cette répression nécessite Sir2 ainsi que Sir3 et Sir4. Nous avons mis au point un système in vitro qui permet l'assemblage du complexe Sir2-3-4 sur un fragment d'ADN nucléosomique. L'hétéro-trimère Sir2-3-4 purifié s'associe de façon coopérative au fragment d'ADN produisant un complexe stable de masse moléculaire homogène. Alors que l'association de Sir3 aux nucléosomes est sensible à la présence de la queue de l'histone H4, celle de Sir2-4 ne l'est pas, en raison d'une forte interaction entre Sir4 et l'ADN. La méthylation de la lysine 79 de l'histone H3 par Dot1 réduit l'association de Sir3 ainsi que du complexe entier. Par contre, le produit de la réaction engendrée par Sir2,
"O"-acetyl-ADP-ribose, stimule fortement l'association de Sir3 ou du complexe Sir2-3-4 aux nucléosomes.
MARTINO, Fabrizio. In vitro reconstitution and biochemical characterization of yeast silent chromatin. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 4002
URN : urn:nbn:ch:unige-6251
DOI : 10.13097/archive-ouverte/unige:625
Available at:
http://archive-ouverte.unige.ch/unige:625
Disclaimer: layout of this document may differ from the published version.
Novartis research foundation
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES
Département de biologie moléculaire Professeur David Shore _____________________________________________________________________
In Vitro Reconstitution And Biochemical Characterization Of Yeast Silent Chromatin
THÈSE
Présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie
par
Fabrizio Martino De
Acqui Terme (Italy)
Thèse N° 4002
GENÈVE
Atelier de reproduction de la section de physique 2008
Résumé en français……….…...1
Summary of presented work……….3
Introduction Euchromatin and Heterochromatin………..7
Heterochromatin in yeast……….7
Silencing at Homothallic Mating-type loci Left and Right (HML and HMR)………..…8
The HMR……….9
The HML………...…12
Silencing at subtelomeric regions………..14
Silencing at the ribosomal DNA region……….…17
Histone modifications at silent chromatin………..20
Limiting or enhancing the spreading of silent chromatin…………..……….22
Anchoring to nuclear periphery and silencing...26
Differences between silencing at subtelomeric regions, HM loci and rDNA array………..27
Functional domains of Sir proteins………...……….30
Sir complexes……….32
The deacetylation reaction of Sir2: mechanism of catalysis, production, and importance of O-acetyl-ADP-ribose……….……….36
Mechanisms of transcriptional silencing in yeast………...…...38
Aims of this work……….…41
Results………..45
A homotrimer-heterotrimer switch in Sir2 structure differentiate rDNA and telomeric silencing………...………..…45
Manuscript and figures……….………47
Supplemental data………...59
Supplemental references………...…62
Supplemental figures...……….…63
In vitro reconstitution of yeast silent chromatin: multiple contacts favor Sir loading onto nucleosomes...…...67
Manuscript………...69
Conclusions and discussion………..…127
The complexes formed by the Sir proteins……….127
In vitro reconstitution of yeast silent chromatin reveals the mechanism of binding of the Sir2-3-4 complex to chromatin……….……..……130
Analysis of Sir4 domains revealed their importance in the formation of silenced chromatin in yeast………132
Importance of the deacetylation reaction of Sir2………...133
Model of recruitment and spreading of silencing………..…134
References...137
Résumé en français
La levure S. cerevisiae présente trois régions constituées d’hétéro-chromatine : Les régions chromosomiques adjacentes des télomères (Gottschling et al. 1990), les deux cassettes contenant les séquences du type sexuel (HML et HMR) (Haber 1998) ; ainsi que l’ADN condant pour les ARN ribosomiques (ADNr) (Smith and Boeke 1997).
Les gènes, transcrits par l’ARN polymerase II, positionnés à proximité de ces régions sont transcriptionnellement inactivés, on dit qu’ils sont soumis au phénomène de variégation ou PEV (position effect variegation) (Rine and Herskowitz 1987;
Gottschling et al. 1990; Smith and Boeke 1997). Cette répression transcriptionnelle est induite dans des régions bien précises sous le control de séquences d’ADN cis tel que les silencers ou encore les répétitions télomériques (TG1-3) (Abraham et al. 1984;
Feldman et al. 1984; Brand et al. 1985; Kyrion et al. 1993; Strahl-Bolsinger et al.
1997) : Les silencers contiennent des séquences qui sont spécifiquement reconnues par des facteurs comme le complexe ORC, Abf1 et Rap1 (Brand et al. 1987; Buchman et al. 1988a; Bell et al. 1993; Foss et al. 1993). Les répétitions télomériques TG1-3
sont quant à elles reconnues par Rap1 (Conrad et al. 1990). Les facteurs reconnaissant les séquences d’ADN cis ont pour fonction de recruter les protéines de la famille Sir (pour Silent Information Regulator proteins : Sir1, Sir2, Sir3 et Sir4). La suppression du gène Sir2, Sir3 ou Sir4 est suffisante pour induire la transcription au niveau des loci HM ou des régions télomériques (Haber and George 1979; Klar et al. 1979; Rine and Herskowitz 1987). La suppression du gène Sir1 quant à elle n’a pas d’effet sur les régions télomériques et réduit fortement l’efficacité du recrutement des facteurs induisant la répression transcriptionnelle au niveau du HMLα (Pillus and Rine 1989;
Gottschling et al. 1990). Sir4 est lui nécessaire au recrutement de Sir3 et Sir2 au niveau des télomères (Hecht et al. 1996; Strahl-Bolsinger et al. 1997; Luo et al. 2002) et fait sans doute parti intégrante avec Sir3 de la structure induisant la répression transcriptionnelle au niveau de la chromatine (Gasser and Cockell 2001). Au niveau de l’ADN ribosomique, la répression est dépendante uniquement de Sir2 qui est recrutée dans cette région par Net1 (Shou et al. 1999; Straight et al. 1999). Net1 forme avec Sir2 et Cdc14 un complexe appelé RENT (Regulator of Nucleolar silencing and Telophase exit) (Straight et al. 1999; Ghidelli et al. 2001). Sir2 est une histone déacétylase dépendante du NAD dont la fonction essentielle est de déacétyler la queue des histones présente au niveau des régions de chromatine réprimée chez la levure ainsi que chez les eucharyotes supérieurs (Sauve et al. 2006). Sir2, Sir3 et Sir4 interagissent pour former un complexe que l’on pense être l’unité de base induisant la répression transcriptionnelle chez la levure S. crevisiae (Liou et al. 2005; Cubizolles et al. 2006). Bien que la chromatine réprimée ait été étudiée abondamment chez la levure par une approche génétique, son analyse biochimique ainsi que sa structure ne l’ont été que de façon très limitée. Les caractéristiques biochimiques des protéines Sir ont également été très peu étudiées. Afin de répondre à ces manquements, nous avons mis au point un essai in vitro avec lequel il est possible d’assembler le complexe Sir2- 3-4 au tour d’une structure nucléosomique. Nous avons exprimé et purifié les protéines Sir seules ou sous forme de complexe à l’aide du système baculovirus.
L’analyse de ces protéines purifiées a montré que Sir2, Sir3 et Sir4 forment un complexe stable dont la stœchiométrie est de 1:1:1. Sir2 est également capable de s’associer uniquement à Sir4 pour former un heterodimère ou de former seule un homotrimère. Cette étude nous a permis de conclure que la protéines Sir2 agit sous la forme d’un homotrimère au niveau de l’ADN ribosomique alors qu’elle agit au niveau des télomères sous la forme d’un hétérotrimère en s’associant avec Sir3 et Sir4
(Cubizolles et al. 2006). Nous avons également pu montrer que la protéine Sir2 seule présente une activité déacétylase très faible qui est fortement stimulée par la présence de Sir4 dans la réaction. Cette étude des protéines Sir a permis d’apporter un nouvel éclairage sur la façon dont elles peuvent agir de façon différente au niveau des différentes régions de chromatine réprimée. Ces protéines purifiées ont ensuite été utilisées pour assembler in vitro une structure de chromatine réprimée. La structure nucléosomique a été obtenue en utilisant des histones recombinantes de X. laevis ainsi que la séquence d’ADN répétitive « widom 601 ». L’assemblage séquentiel des protéines Sir sur cette structure a été analysé à l’aide de la technique de gel retard. La structure obtenue après assemblage des complexes Sir sur les nucléosomes est apparue homogène ainsi que sa masse moléculaire. Nous avons pu montrer que l’association de Sir3 était dépendante de la présence de la queue de l’histone H4, confirmant ainsi l’importance de cette queue observée in vivo. De plus la méthylation de la lysine 79 sur l’histone H3 réduit fortement l’association du complexe Sir2-3-4.
Comme cette réduction est également observée pour l’association de Sir3 seule, l’interaction du complexe Sir2-3-4 au niveau de la lysine 79 se fait par l’intermédiaire de Sir3. Nous avons également observé une forte affinité du complexe Sir2-3-4 pour l’ADN nu. Le domaine de Sir4 responsable de cette association a été caractérisé. Sir2 quant à elle ne présente pas d’affinité pour l’ADN nu ou la chromatine, cela suggère que sa fonction principale dans l’établissement de la répression transcriptionnelle est de déacétyler la queue des histones. Ainsi le produit de la réaction de déacétylation O- AADPR, augmente l’affinité du complexe Sir2-3-4 pour la chromatine.
Notre travail a permis de proposer le modèle suivant : le complexe Sir est recruité au niveau des répétitions télomériques via l’interaction entre Sir4, Rap1 et Ku70/Ku80 (Boulton and Jackson 1996; Hecht et al. 1996; Strahl-Bolsinger et al. 1997; Laroche et al. 1998; Mishra and Shore 1999). La grande stabilité du complexe Sir2-4 ainsi que sa forte activité de déacétylation des histones suggèrent que ce complexe est très probablement l’événement les plus en amont. Le complexe Sir2-4 déacétyle les histones au niveau des nucléosomes présents à proximité des télomères afin de recruter Sir3 (Braunstein et al. 1996; Tanny et al. 1999; Hoppe et al. 2002; Kimura et al. 2002; Suka et al. 2002). Puisque l’interaction entre Sir3 et le complexe Sir2-4 a été observée même en absence d’activité de déacétylation de la part de Sir2, elle est sans soute stabilisée en raison de la forte concentration de protéines ainsi obtenue au niveau des télomères (Rudner et al. 2005; Cubizolles et al. 2006). Une étude a proposé que le composé O-AADPR, obtenu lors de la réaction de déacétylation, induisait ainsi la multimérisation du complexe en se liant au domaine AAA+ de Sir3 (Liou et al. 2005). La queue de l’histone H4 ainsi que la région entourant la lysine 79 de l’histone H3 interagissent avec le complexe Sir2-3-4 via Sir3. La présence localement d’une forte concentration de complexe Sir2-3-4 favorise la formation de multimère de ce complexe par l’intermédiaire du domaime « coiled coil » de Sir4.
Cela peut expliquer l’association coopérative que nous avons observée ainsi que la propagation de la répression transcriptionnelle depuis l’extrémité des télémorères. Si l’on imagine qu’un complexe Sir2-3-4 est associé sur chaque surface de nucléosome, on peut penser que la compaction de la chromatine soit due à l’interaction des différents complexes Sir2-3-4 entre eux rendu possible par leur promiscuité ainsi obtenue. Bien sûr de multiples conformations peuvent être imaginées, seule l’étude structurale permettra de résoudre la conformation réelle. On peut également imaginer que le domaine amino-terminal de Sir4, nécessaire à son association avec l’ADN nu, joue un rôle important afin de stabiliser la conformation de la chromatine obtenue.
Summary of presented work
In vitro reconstitution and biochemical characterization of yeast silent chromatin
Budding yeast has three heterochromatic-like regions: the portion of the chromosomes adjacent to telomeres (Gottschling et al. 1990); the cryptic homothallic mating type loci HML and HMR (Rine and Herskowitz 1987); and the rRNA-encoding DNA (rDNA) (Smith and Boeke 1997). Polymerase II-transcribed genes located in these regions can be transcriptionally silenced due to a phenomenon called position effect (Rine and Herskowitz 1987; Gottschling et al. 1990; Smith and Boeke 1997). Silent chromatin is nucleated at specific locations characterized by special cis-acting DNA sequences: the silencers and the telomeric TG1-3 repeat tract (Abraham et al. 1984;
Feldman et al. 1984; Brand et al. 1985; Kyrion et al. 1993; Strahl-Bolsinger et al.
1997). The silencers contain binding sites for nucleating factors such as the ORC complex, Abf1, and Rap1 (Brand et al. 1987; Buchman et al. 1988a; Bell et al. 1993;
Foss et al. 1993), whereas TG1-3 repeat is bound by Rap1 (Conrad et al. 1990). This nucleation machinery has the role of recruiting the Silent Information Regulator proteins (Sir1, Sir2, Sir3, and Sir4).
Deletion of either SIR2, SIR3, or SIR4 disrupts silencing at HM loci and telomeres (Haber and George 1979; Klar et al. 1979; Rine and Herskowitz 1987; Gottschling et al. 1990; Aparicio et al. 1991), while deletion of SIR1 does not affect silencing at the telomeres and strongly weakens the nucleating efficiency of silencing at the HMLα. This generates a meta-stable silencing state represented by a mixed population of cells stably transmitting either a silent or an active HMLα (Pillus and Rine 1989;
Gottschling et al. 1990). The meta-stable repression can be explained by the important role played by Sir1 in recruiting and stabilizing Sir4 at the HM loci. In contrast, Sir4 is necessary for the recruitment of Sir3 and Sir2 at the telomeres (Hecht et al. 1996;
Strahl-Bolsinger et al. 1997; Luo et al. 2002). Sir2, Sir3, and Sir4 interact together to form a Sir2-3-4 complex, believed to be the basic unit of silent chromatin in budding yeast (Gasser and Cockell 2001; Liou et al. 2005; Cubizolles et al. 2006).
Silencing at the rDNA is different in that is dependent on Sir2, but independent on Sir3 and Sir4 (Bryk et al. 1997; Fritze et al. 1997; Smith and Boeke 1997). Sir2 is recruited to the rDNA by Net1 (Shou et al. 1999; Straight et al. 1999). Net1, together
with Sir2 and Cdc14, form a complex called the regulator of nucleolar silencing and telophase exit (RENT complex) (Straight et al. 1999; Ghidelli et al. 2001). The RENT complex, together with a complex formed by Fob1/Tof2/Lrs4/Csm1, is required, in a Sir2-dependent manner, for suppressing unequal exchange between rDNA repeats (Straight et al. 1999; Huang et al. 2006). Sir2 is a conserved NAD-dependent histone deacetylase with an essential role in deacetylating the histone tails at silent regions in yeast and higher eukaryotes (Sauve et al. 2006).
Although yeast silent chromatin has been thoroughly studied using genetic approaches, its biochemical analysis has been quite limited. We know little about the biochemical characteristics of the Sir proteins and of their complexes. Furthermore, little is known about the structure of silent chromatin or about the molecular mechanism behind the position effect mediated by silenced regions. To address these questions, we have set up an in vitro system in which we load the Sir2-3-4 complex on in vitro reconstituted nucleosome arrays. We expressed, purified, and characterize each Sir protein singly and in different combinations in Baculovirus. Purification and sedimentation analysis of the purified proteins and complexes showed that Sir2, Sir3, and Sir4 form a stable 1:1:1 heterotrimeric complex; Sir4p and Sir2p form a 1:1 heterodimer; while Sir2 alone forms a homotrimer. We concluded that Sir2 acts as a homotrimer at the rDNA and as a heterotrimer, with Sir3 and Sir4, at the telomeres and HM loci (Cubizolles et al. 2006). We could also show that Sir2 alone has a very low histone deacetylase activity, while Sir4, in the Sir2-4 complex, increases the activity of Sir2. Our biochemical characterization of the different Sir complexes gives a novel contribution towards understanding how different Sir complexes function at different silent locations.
The purified Sir proteins and Sir complexes were used to assemble in vitro yeast silent chromatin. Nucleosome arrays were reconstituted on tandem repeats of the "Widom 601" DNA sequence and X. laevis recombinant histones. The loading of Sir proteins and complexes, singularly and sequentially, has been monitored by band shift analysis.
The Sir complexes bind the chromatin resulting in Sir/nucleosome complexes that are homogeneous in molecular weight and, apparently, in structure. We can show that the binding of Sir3 to chromatin is sensitive to the lack of the H4 tail, confirming the H4 tail to be an important binding site for Sir3. Moreover, we demonstrate that methylation at H3K79 strongly reduces the binding of the Sir2-3-4 complex to chromatin. Since a similar effect is seen for Sir3 alone, we deduced that the binding of
the Sir2-3-4 complex to the region surrounding H3K79 is due to Sir3. We noticed that the Sir2-3-4 complex has a high binding affinity for naked DNA. We isolated the Sir4 domain responsible for this binding and compared it with other functional domains of Sir4 and with the full length protein. Sir2 does not show any affinity for either naked DNA or chromatin, suggesting that its main role in the establishment and maintenance of silent chromatin is to deacetylate the histone tails. In this regard we showed that the O-AADPR, the by-product of the deacetylation reaction of Sir2, increases the binding affinity of the Sir2-3-4 complex for chromatin.
Our work allows us to propose a model of recruitment and spreading of silencing at subtelomeric regions. The Sir complex is recruited at the telomeric TG1-3 repeat by the interaction of Sir4 with Rap1 and Ku70/Ku80 (Boulton and Jackson 1996; Hecht et al.
1996; Strahl-Bolsinger et al. 1997; Laroche et al. 1998; Mishra and Shore 1999). The high stability of the Sir2-4 complex, and its high histone deacetylase activity, suggests that the Sir2-4 complex is probably the first recruited at telomeres. The Sir2-4 complex deacetylates the histones on the nucleosomes adjacent to the telomeres and recruits Sir3 at the telomeres (Braunstein et al. 1996; Tanny et al. 1999; Hoppe et al.
2002; Kimura et al. 2002; Suka et al. 2002). The interaction between Sir3 and the Sir2-4 complex is probably stabilized by the high local concentrations of Sir proteins, since we and others have shown that the interaction between Sir2, Sir3, and Sir4 exists even without the Sir2 deacetylation reaction (Rudner et al. 2005; Cubizolles et al. 2006). The NAD+-dependent deacetylation reaction mediated by Sir2 produces O- AADPR that is thought to bind the AAA+ module of Sir3 and to induce its multimerization (Liou et al. 2005). The deacetylated tail of histone H4 and the region surrounding H3K79 are bound by the Sir2-3-4 complex through Sir3. The presence of high local concentrations of the Sir2-3-4 complex favors the formation of “dimers of Sir2-3-4 complexes” through the coiled coil domain of Sir4. This could explain the co-operative binding we have seen in our experiments, and could be a means of explaining the spreading of silencing inward along the chromosome. If we suppose that one Sir2-3-4 complex is bound to each surface of every nucleosome, we could imagine that the compaction of chromatin is mediated by interactions between Sir2-3- 4 complexes brought into close proximity. Of course, many possible folding patterns could be imagined, and future structural studies will answer this question. The binding of the N-terminal portion of Sir4 to naked DNA might be important for stabilizing the folding of chromatin into high-order structures. We still do not know where Sir4
contacts the DNA on the chromatin fiber. It is possible that Sir4 binds the linker DNA or the DNA at the dyad, thus promoting the folding of the chromatin fiber, or even the DNA wrapped around the nucleosome, thus stabilizing the binding of the Sir2-3-4 complex onto chromatin. We cannot exclude any of these possibilities. Furthermore, the O-AADPR is thought to promote interactions between separate arrays of nucleosomes bound by Sir proteins (Onishi et al. 2007). We showed that the O- AADPR increases the affinity of the Sir2-3-4 complex for binding to chromatin.
Whether the O-AADPR influences the structure of the silenced chromatin or acts only on the binding properties of the Sir2-3-4 complex remains to be determined.
Introduction
Euchromatin and Heterochromatin
Eukaryotic chromosomal DNA is compacted into a chromatin fiber with a hierarchical folding scheme. The basic unit of the chromatin fiber is the nucleosome, which is formed by 147 bp of DNA wrapped around a histone octamer (Noll 1974;
Luger et al. 1997a). Light-microscopy studies in the 1930s distinguished two types of chromatin in the interphase nuclei of many eukaryotic cells: a highly condensed form, associated to transcriptionally silent genes, called heterochromatin and a less condensed one, associated with transcribed genes, called euchromatin.
Heterochromatin is present in all eukaryotes, from yeast to mammals, and can organize up to 95% of the genome of highly differentiated cells of higher eukaryotes (reviewed in (Allis et al. 2007)). Heterochromatin can be divided into constitutive and facultative types (reviewed in (Grewal and Moazed 2003)). Constitutive heterochromatin is associated with repetitive sequences of the genomes, such as telomeres and centromeres, while facultative heterochromatin is associated with developmentally regulated genes. In all eukaryotes, heterochromatin shares some characteristics (reviewed in (Grewal and Moazed 2003)). First of all, the chromatin fiber at heterochromatin sites is compacted into higher-order structures that restrict accessibility of enzymes, such as endonucleases and RNA polymerase II. Because of its restricted accessibility, heterochromatin favors transcriptional gene silencing and low recombination rates. Importantly, heterochromatin spreads from nucleation sites into neighboring regions of the chromosome, as far as few kilo bases, thus exerting a position effect on genes placed in its proximity. The silent heterochromatic state is stably inherited through cells divisions by epigenetic marks such as histone hypoacetylation, histone and DNA methylation. Finally heterochromatic regions of the genome are late replicating and define functional domains of the nucleus.
Heterochromatin in yeast
Yeast has been one of the most studied organisms in the definition of the molecular mechanism of functioning of heterochromatin. Budding yeast has three
heterochromatic-like regions, also referred as regions of silent chromatin because of their transcriptionally repressed state: the regions adjacent to telomeres, (referred as subtelomeric regions) (Fig. 3) (Gottschling et al. 1990); the cryptic homothallic mating-type loci HML and HMR (Fig. 1 and 2) (Rine and Herskowitz 1987), and the rRNA-encoding DNA (rDNA) (Fig. 4) (Smith and Boeke 1997).
Silent chromatin is nucleated at specific locations characterized by special cis-acting DNA sequences: the silencers and the TG1-3 repeat tract (Abraham et al. 1984;
Feldman et al. 1984; Brand et al. 1985). The silencers contain binding sites for nucleating factors such as the ORC complex, Abf1, and Rap1 (Brand et al. 1987;
Buchman et al. 1988a; Bell et al. 1993; Foss et al. 1993), whereas the telomeric TG1-3
repeat tract is bound by Rap1 (Conrad et al. 1990). The role of this nucleation machinery is the recruitment of the Silent Information Regulator proteins (Sir1, Sir2, Sir3, and Sir4). Deletion of SIR2, SIR3, or SIR4 disrupts silencing at HM loci and telomeres (Haber and George 1979; Klar et al. 1979; Rine and Herskowitz 1987), while deletion of SIR1 strongly weakens the nucleating efficiency of silencing at HMLα, but does not affect silencing at the telomeres. Sir4 is necessary for the recruitment of Sir3 and Sir2 at telomeres and HM loci (Hecht et al. 1996; Strahl- Bolsinger et al. 1997; Luo et al. 2002). Sir2 is a conserved NAD-dependent histone deacetylase with an essential role in deacetylating the histone tails at silent regions in both yeast and higher eukaryotes (Sauve et al. 2006). Finally, Sir2, Sir3, and Sir4 interact together to form a Sir2-Sir3-Sir4 complex, which is believed to be the basic unit of silent chromatin in budding yeast (Gasser and Cockell 2001; Liou et al. 2005;
Cubizolles et al. 2006).
Silencing at Homothallic Mating-type loci Left and Right (HML and HMR).
The mating type of Saccharomyces cerevisiae, either a or α, is determined by the genetic information present and expressed at the MAT locus, localized near the centromere of chromosome III. The complete copies of either a or α genes are stored, in a transcriptionally silent manner, in two other loci, named HML and HMR (Fig. 1 and 2), which are localized on the left and right arm of chromosome III respectively.
A haploid yeast cell carrying a wild type (WT) copy of the homothallic (HO) gene
can switch mating type by replacing the genetic information present at the MAT locus with the information stored at either the HML or HMR, via a gene conversion process.
The switching process is very important for yeast, since two opposite mating-type cells can mate and give rise to an a/α diploid which can then undergo meiotic division and generate haploid spores (reviewed in (Haber 1998)). When genes at both the HML and HMR are expressed, the haploid cell becomes sterile. To avoid this problem, yeast has developed a sophisticated mechanism of silencing the mating-type cassettes.
Silencing at the HML and HMR is mediated by cis-acting elements called silencers (Abraham et al. 1984; Feldman et al. 1984) which bind ORC, Rap1, and Abf1 (Shore and Nasmyth 1987; Buchman et al. 1988a; Kimmerly et al. 1988) which then recruit the Sir proteins (Haber and George 1979; Klar et al. 1979; Rine and Herskowitz 1987) (Fig. 1 and 2). Silencers on the left of HML and HMR are both essential, and they are called HML-E and HMR-E. Silencers on the right side are important for full repression and are called HML-I and HMR-I. The silencers are able to induce the silencing of both RNA polymerase II and RNA polymerase III promoters (Brand et al.
1985; Schnell and Rine 1986), and they are not gene-specific. Moreover, silencers are able to act bi-directionally at a distance of several hundred base pairs (as far as 2.7 kb) and they can function on ectopic chromosomal location (Brand et al. 1985).
The HMR locus
Silencers, like enhancers, are formed by short consensus elements that are recognized and bound by specific factors. The HMR-E silencer is formed by three small sequences originally called A, E, and B (Fig. 1) (Brand et al. 1985). The A element is an origin of replication: it is able to confer autonomous replication on plasmids (called ARS, Autonomous Replication Sequences); (Brand et al. 1987) and is bound by the Origin Recognition Complex (ORC) (Diffley and Cocker 1992; Bell et al. 1993;
Foss et al. 1993). Indeed HMR-E and HMR-I have been shown to be chromosomal origins of replication (Rivier and Rine 1992; Rivier et al. 1999). Interestingly, it has been shown that silenced regions of the yeast genome are late replicated, suggesting an influence of silencing on the timing of DNA replication (Ferguson and Fangman 1992). The B element also has ARS activity, but a minimal fragment that has silencing and no replicating activity has been isolated (Brand et al. 1987).
ACS
a2 a1 I
Abf1
Rap1 Abf1 ACS
tRNA
1 4
2 3 4
2 4
2 3 3 4
2 3 4
2 4
2 3 3
4 2
3 4
2 4
2 3 3 4
2 3 4
2 4
2 3
3 4
2 3 4
2 4
2 3 3 4
2 3 4
2 4
2 3 3 4
2 3 4
2 4
2 3 3
E
HMR
nucl
Fig.1) Organization of the HMR.
This fragment contains a consensus binding site for the transcription activator Abf1 (Brand et al. 1987; Shore and Nasmyth 1987; Shore et al. 1987; Buchman et al. 1988a;
Buchman et al. 1988b). The E element contains the consensus sequence for another transcription activator called Rap1 (Repressor Activator Protein 1) (Brand et al. 1987;
Shore and Nasmyth 1987; Shore et al. 1987; Buchman et al. 1988a; Buchman et al.
1988b). This discovery revealed the interesting property of certain sequences and factors that can act, depending on their chromosomal context, as either inhibitors or activators of transcription. Indeed, consensus sites from either the E or the B element are able to activate transcription when placed out of the context of a silencer (Brand et al. 1987). These observations suggest that not only the DNA sequence, but also the chromosomal context where a silencer is located, influences its mode of functioning.
The HMR-E silencer is also able to confer mitotic stability on plasmids (Kimmerly and Rine 1987) through Rap1, Abf1, and Sir4 (Kimmerly et al. 1988; Ansari and Gartenberg 1997). The silencers have the important role of recruiting Sir proteins, and thus silencing, at the HM loci. Mutations in SIR2, SIR3, and SIR4 genes completely abolished silencing at the HM loci (Ivy et al. 1986; Rine and Herskowitz 1987).
Interestingly, sir1 mutation confers a weakened control over the transcription state of HML, allowing it to switch from a repressed to a derepressed state and vice versa (Pillus and Rine 1989). Interestingly, in sir1 mutants, the transcriptional state of the cells, either repressed or derepressed, is mitotically inherited between generations (Pillus and Rine 1989), suggesting that Sir1 is required for the establishment of silencing, and not for its maintenance (Pillus and Rine 1989).
The Shore and Sternglanz laboratories have shown that Sir1 can nucleate silencing when artificially targeted to HMR-E silencers truncated of their elements (Chien et al.
1993). From their experiments and from literature, the authors could deduce that Sir1 is mainly recruited by ORC, while Rap1 and Abf1 are probably interacting directly with Sir3 and Sir4 (Chien et al. 1993; Moretti et al. 1994). Sternglanz’s laboratory finally proved that Sir1 binds Sir4 and Orc1, one of the subunits of the ORC complex (Triolo and Sternglanz 1996). They demonstrated that Sir1 acts downstream of ORC in recruiting silencing (Triolo and Sternglanz 1996). Importantly, Chien and co- workers showed that Sir2, Sir3, or Sir4 are not sufficient to nucleate silencing in the absence of either Sir1 or a functional E silencer (Chien et al. 1993), suggesting that Sir2, Sir3, and Sir4 have to be recruited at the silencers in a coordinated and tightly regulated manner that is ensured by ORC, Rap1, Abf1, and Sir1. Thus, A, B, and E elements work together to establish a tight, but reversible, transcriptional gene silencing.
The silencers have inherent redundancy in their organization. It has been shown that the presence of any two of these binding sites within the silencer is sufficient for its function (Brand et al. 1987). This suggests that nucleation of silencing requires the recruitment of a minimal number of Sir proteins. Interestingly, four Rap1 C-terminal fragments, shown to be required for silencing (Hardy et al. 1992a), were sufficient to promote silencing at an HMR-E with all three elements deleted (Hardy et al. 1992a).
When either the A or the B element was present, only two molecules of Rap1 were required. The targeting property of the C-terminal fragment of Rap1 was completely lost in the absence of Sir2, Sir3, or Sir4 but not of Sir1, suggesting that Sir1 recruitment at the silencer is not dependent on Rap1 (Hardy et al. 1992a). The redundancy of the A, B, and E elements may reflect the importance of silencing at the HM loci. In order to test if the three elements of the HMR-E silencer are indeed sufficient to promote silencing, Rine’s laboratory replaced the E element with a synthetic silencer containing an ARS consensus sequence, a Rap1 binding site present in the MATα UAS and a synthetic Abf1 binding site (McNally and Rine 1991). Even though the synthetic silencer could replace the wild-type HMR-E for silencing, a few differences were noticed. First of all, the artificial silencer required all three elements while a natural one works with just two (McNally and Rine 1991). Moreover, the artificial silencer required the presence of HMR-I and showed a complete dependence
on the SIR1 gene (McNally and Rine 1991). In contrast, the natural HMR-E was alone sufficient to promote silencing (Brand et al. 1985) and sir1 mutant strains still maintain substantial mating ability with a natural silencer (Rine and Herskowitz 1987).
To summarize, the synthetic silencer is weaker than the wild-type one, suggesting that some elements, not strictly required but important for the stability of the silencer, are missing in the synthetic silencer. These missing elements could be important for recruiting additional factors such as, for example, cohesins (Chang et al. 2005), or for the folding of the HMR locus into a high-order structure, as suggested by Kamakaka’s laboratory (Valenzuela et al. 2008). It is also possible that the spacing between the elements is important for the correct functioning of the silencer. Interestingly, Nasmyth’s laboratory has shown that the HMR-E silencer functions when inverted, but only at its natural location (Brand et al. 1985). An inverted HMR-E was not functional when located in place of HMR-I. These data, and the experiment discussed above, suggest that the chromosomal context and the complete sequence of each silencer are important for the nucleation of fully stable and functional silent chromatin.
The HML locus
The HML locus is flanked by an E silencer on its left and by an I silencer on its right (Fig. 2). The E silencer contains an ARS consensus sequence and a Rap1 binding site (Feldman et al. 1984; Buchman et al. 1988a; Boscheron et al. 1996). The I silencer has binding sites for Abf1, Rap1 and an ARS consensus sequence (Feldman et al.
1984; Buchman et al. 1988a; Boscheron et al. 1996). In a manner similar to HMR, HML silencers function bi-directionally and can induce silencing not only on the α genes but also on heterologous marker genes (Mahoney and Broach 1989). In contrast to HMR, each element, either HML-E or HML-I, is sufficient to promote silencing (Mahoney and Broach 1989). Moreover, each silencer is able to function completely with just the ARS or Rap1 (or Abf1) binding site (Mahoney and Broach 1989). In contrast, HMR-E is not fully functional without the Rap1 binding site (Kimmerly et al.
1988). Nevertheless a report from Gilson’s laboratory showed that an HML locus lacking one of the two silencers only partially silence a lacZ reporter gene (Boscheron et al. 1996). Interestingly they showed that individual elements of the silencers act asprotosilencers, since they are able to enhance the strength of a distant silencer (Boscheron et al. 1996).
α 2 α 1 I E
Abf1 ACS ACS
Rap1
HML
Rap1
Rap1
Fig.2) Organization of the HML.
Since the silencers at both the HMR and the HML contain similar sequences and recruit similar factors, and are both located near the telomeres, it is difficult to understand why they function differently. It is possible that the chromosomal context where they are placed influences their properties. Interestingly, Kamakaka’s laboratory has shown that RNA polymerase II and III promoters can act as barriers for the spreading of heterochromatin (Donze and Kamakaka 2001) and a tRNA gene, that serves as a heterochromatin barrier, has been mapped on the right side of HMR-I (Oki and Kamakaka 2005). Another possibility is that the factors responsible for silencing formation and maintenance are differentially regulated at HML and HMR.
One example for the different regulation is the inactivation of ARD1 or NAT1, the genes coding for the two subunits of an N-terminal acetyltransferase. This leads to complete derepression of HML but not of HMR (Whiteway and Szostak 1985;
Whiteway et al. 1987; Mullen et al. 1989). ORC and Sir3 have been described as targets of the Nat1/Ard1 acetyl transferase complex (Wang et al. 2004). Strains with mutations in SIR3 that prevent the N-terminal acetylation of the protein present a decreased silencing at HML and at the telomeres, but not at HMR. In addition, it has been shown that Sir1 over-expression can partially suppress deletions of either NAT1 or ARD1 genes and also certain mutations in the SIR3 and in HHF2 genes (coding for histone H4) (Stone et al. 1991). These observations suggest that the silencing defect of Ard1/Nat1 mutants is caused by a reduced targeting of Sir3 at the HML and telomeres. HMR is not affected by these mutations probably because of the strong nucleating properties of its silencers. Both HML and HMR silencers have ORC binding sites but only HMR-E and HMR-I present chromosomal origin of replication activity (Dubey et al. 1991; Rivier and Rine 1992; Rivier et al. 1999). It seems that the ARS elements at the HML silencers do not function as origins of replication, and
that replication of the HML is accomplished by the activity of replication origins outside of it (Dubey et al. 1991). It is not clear what inhibits the A sequence from functioning as an ARS, we could exclude that such a behavior is due to the interaction of ORC with the Sir proteins since the ARS at the HML do not fire in sir4Δ strains (Dubey et al. 1991).
Silencing at subtelomeric regions
In budding yeast, telomeres exert transcriptional silencing on genes located in their vicinity in a fashion that resembles the silencing at the HML and HMR loci (Gottschling et al. 1990; Aparicio et al. 1991). The repressed state conferred by the telomeres on nearby genes is mitotically inherited through cell generations. Repressed genes can escape from telomeric repression and switch to a state of active transcription (Gottschling et al. 1990) that is then stably transmitted between cell generations. Thus telomeres, in a similar way to centromeres in fruit flies, exert a position effect on nearby genes (Raffel and Muller 1940; Gottschling et al. 1990) known as the Telomere Position Effect (TPE).
Telomeric silencing presents many similarities and some differences with silencing at the HM loci. Mutations in SIR2, SIR3, or SIR4 genes, but not in SIR1, completely disrupt silencing at telomeres (Aparicio et al. 1991). Moreover, both deletions of the tails of histone H4 and point mutation of lysine 16 and arginine 17 to neutral amino acids was sufficient to disrupt silencing at the telomeres (Aparicio et al. 1991). Finally, deletion of either NAT1 or ARD1 genes had a similar, if not stronger, effect on telomeric silencing (Aparicio et al. 1991). On the other hand, mutations in the end binding factor Ku70/Ku80 derepresses telomeric but not HM silencing (Boulton and Jackson 1998; Laroche et al. 1998; Mishra and Shore 1999).
Silent chromatin is nucleated at subtelomeric regions by the binding of Rap1 to the TG1-3 repeat (Conrad et al. 1990; Gilson et al. 1993; Kyrion et al. 1993; Moretti et al.
1994) and by the Ku70/Ku80 heterodimer, which binds chromosomal ends of both telomeres and DNA double-strand breaks (Boulton and Jackson 1998; Laroche et al.
1998; Mishra and Shore 1999) (Fig. 3). Chromatin immuno-precipitation experiments have shown that localization of Sir2, Sir3, and Sir4 at the telomeres depends on the C- terminus of Rap1 and on Ku70/Ku80 (Hecht et al. 1996; Strahl-Bolsinger et al. 1997;
Martin et al. 1999). Yeast telomeres are formed by 250-630 bp of the TG1-3 repeat which is bound by Rap1 (Shore and Nasmyth 1987; Buchman et al. 1988b; Longtine et al. 1989). Rap1 binds telomeres in vivo (Conrad et al. 1990; Klein et al. 1992) and has a role in maintaining telomere length, since temperature-sensitive Rap1 mutants showed a telomere shortening phenotype (Conrad et al. 1990). One molecule of Rap1 binds approximately 20 bp of telomeric TG1-3 repeat (Gilson et al. 1993), and the number of molecules of Rap1 bound to the telomeres is used by the cell to sense the length of the telomeres (Marcand et al. 1997). The C-terminal portion of Rap1 (precisely the fragment between amino acids 655 and 827) binds Rif1 (Rap1 Interacting Factor 1) and Rif2, which form a complex and are negative regulators of telomere length (Hardy et al. 1992a; Hardy et al. 1992b; Wotton and Shore 1997).
Interestingly, the same domain of Rap1 has been shown to bind directly to Sir3 and Sir4 (Moretti et al. 1994; Buck and Shore 1995). It has been proposed that Sir4 and Rif1 compete for the binding of Rap1 at telomeres (Buck and Shore 1995). According to this model, deletion of RIF1 and RIF2 improves silencing at telomeres by releasing a subset of Rap1 molecules that would be normally bound to Rif1 and Rif2 (Buck and Shore 1995). Notably, improvement of telomeric silencing in strains deleted for RIF1 and RIF2 is also due to the elongated state of the TG1-3 repeat tract that accounts for an increased recruitment of Rap1, and thus of Sir4 compared to WT strains (Hardy et al. 1992b; Buck and Shore 1995). The increased recruitment of Sir4 at the telomeres generates derepression of silencing at the HM loci (Buck and Shore 1995). This observation suggested that the amount of Sir proteins is limiting in the cell and is shared between the telomeres and the HM loci (Buck and Shore 1995; Maillet et al.
1996; Cockell et al. 1998a). Nucleation of Sir4 occurs by Rap1 (Moretti et al. 1994;
Buck and Shore 1995; Luo et al. 2002). Indeed Sir4 could be detected at the TG1-3 repeat of telomeres of cells deleted for SIR2, SIR3, HDF1 and HDF2 (the genes coding for Ku70 and Ku80 respectively) (Luo et al. 2002). Nevertheless in those mutants, the recruitment of Sir4 at subtelomeric regions was reduced compared to WT and silencing was completely absent (Luo et al. 2002). yKu70/Ku80 directly binds Sir4 with high affinity, thus arguing that, like Rap1, it contributes to recruit Sir4 at subtelomeric regions (Boulton and Jackson 1998; Evans et al. 1998; Laroche et al.
1998; Mishra and Shore 1999; Roy et al. 2004; Taddei et al. 2004). Moreover, yKu70/Ku80 plays a crucial role is Sir4 recruitment at telomeres by helping Sir4 to counteract the competition with Rif1/Rif2 for binding Rap1 (Mishra and Shore 1999).
Ku 4
2 3 4
2 4 2 3 3
Rap1 4 4
Rif1Rif2 Rif1Rif2
Sir4
Ku70/Ku80 Sir2-3-4
Rap1 nucleosome
Rif1/Rif2
Subtelomeric regions
4 4 4 4 4 4
Rap1 Rap1 Rap1
3 4 2 3 4
2
Ku
4 2 4 3 2 4 2
3 4 3
2 3 4
2 4
2 3 3 4
2 3 4
2 4 2
3
3 4
2 3 4
2 4 2
3 4 3
2 3 4
2 4 2
3
3 4
2 4 3 2 4 2
3 4 3
2 4 3 2 4 2
33 4 2
3 4
2 4 2
3 3
nucleosomes
4 Rif1Rif2 4
Rif1Rif2
Fig.3) Silent chromatin truncated telomeres.
The reduced recruitment of Sir4 at the TG1-3 repeat, in sir2 and sir3 mutants, suggests that Sir2 and Sir3 play an important role in stabilizing the interaction of Sir4 with Rap1 and yKu70/Ku80 at the TG1-3 repeat. The importance of Ku70/Ku80 in telomeric silencing is reinforced by the observation that 350 bp of TG1-3 repeat tract alone cannot nucleate silencing when not present at the ends of chromosomes (Stavenhagen and Zakian 1994). For instance, silencing at telomeres, but not at an internal locus, could be nucleated by just 80bp of TG1-3 repeat (Gottschling et al.
1990). Moreover, tracts of 270 bp of TG1-3 repeats placed on an extrachromosomal plasmid were sufficient to de-repress silencing of an internally located TG1-3 tract but not of a telomeric one (Stavenhagen and Zakian 1994). These data support the idea that a telomere-bound factor other than Rap1 and Sir proteins positively influence silencing at telomeres and this factor is undoubtedly yKu70/Ku80 (Boulton and Jackson 1998; Laroche et al. 1998; Mishra and Shore 1999; Roy et al. 2004; Taddei et al. 2004).
The fact that genes located up to 2 kb from the end of the chromosome were silenced, in a Sir-dependent fashion, implied that the factors involved in silencing were able to spread inward along the chromosome arm. In support of this model, Sir2, Sir3, and Sir4 have been shown to localize as far as 2.8 kb from the telomeres (Strahl-Bolsinger et al. 1997). When Sir3 is over-expressed, silencing spreads to 7 kb from the telomere of V-R (Renauld et al. 1993), and Sir3 can be detected as far as 17.5 kb from the end of chromosomes V-R and VI-L, without the stoichiometric binding of Sir2 and Sir4 (Hecht et al. 1996; Strahl-Bolsinger et al. 1997). The spreading of silencing, from telomeres along the chromosome, depends on a balanced amount of functional Sir3
and Sir4 and on Sir2 that deacetylates the histone tails of the nucleosomes adjacent to the TG1-3 repeat (Renauld et al. 1993; Maillet et al. 1996; Tanny et al. 1999; Luo et al.
2002). The current model is that the Sir proteins spread by binding to the deacetylated histone tails (Hecht et al. 1995; Kimura et al. 2002; Suka et al. 2002). Recent data from Moazed’s laboratory suggest that the deacetylation reaction of Sir2 is important not only for creating Sir3 and Sir4 binding sites. They argue that the deacetylation reaction of Sir2 and, more specifically, O-acetyl ADP ribose, the byproduct of this reaction, are able to promote oligomerization of arrays in a Sir-dependent manner (Luo et al. 2002). Under those conditions, the oligo-nucleosomes formed 10-20 nm thick and 100 nm long filaments in vitro (Luo et al. 2002). The authors proposed that the deacetylation reaction and, consequently, the O-AADPR are the motors of the spreading of Sirs from nucleating sites inward along the chromosome. The lack of resolution and the lack of contrast of the images presented in that paper make it difficult to determine the structure of these filaments. The preparation of better and more homogeneous samples, in combination with a biochemical and biophysical analysis, will be needed for a first model of the structure and function of yeast silent chromatin. Finally, it is also possible that the spreading of telomeric silent chromatin is dependent of the looping of the end of the chromosome on itself, as proposed by several laboratories (Pryde and Louis 1999; de Bruin et al. 2001). We do not know if Ku70/Ku80 has a role in the formation of this looped structure.
Silencing at the ribosomal DNA region
In budding yeast, the ribosomal DNA, the portion of the genome coding for the ribosomal RNA, is organized into a tandem array of 9.1 kb units repeated 100-200 times (Petes and Botstein 1977; Philippsen et al. 1978) (Fig.4). The yeast rDNA is localized on chromosome XII and it is confined to the nucleolus, a membrane-less organelle occupying a distinctive half-moon crescent within the yeast nucleus (Petes and Botstein 1977; Philippsen et al. 1978; Melese and Xue 1995). Each rDNA repeat contains a 5S rRNA gene transcribed by RNA polymerase III (Pol III) and a 35S pre- rRNA gene transcribed by RNA polymerase I (Pol I). The rDNA repeats, like any repeated sequence of the genome, are naturally subject to recombination, and thus genomic instability (Reid and Rothstein 2004).
35S 5S ARS 35S
NTS1 NTS2
RFB
RENT RENT
rDNA array
9.1 kb repeat
Fig.4) Organization of the rDNA array.
Since the integrity and the correct functioning of rDNA genes are very important for the life of the cell, ribosomal DNA recombination is repressed. The suppression of recombination of the rDNA arrays depends on the SIR2 gene (Gottlieb and Esposito 1989). It has been proposed that Sir2 suppresses rDNA recombination by inducing compaction of the chromatin fiber into higher order structures (Fritze et al. 1997;
Smith and Boeke 1997).
As a side effect of the particular chromatin structure present at the rDNA repeat Pol II-transcribed genes artificially placed in the rDNA are subject to variegated repression (Bryk et al. 1997; Fritze et al. 1997; Smith and Boeke 1997). The sites of repression occurred preferentially in three regions: the non-transcribed region 1 (NTS1) downstream of the 5S gene which contains the replication fork barrier (RFB), a DNA element responsible for stimulating recombination (Kobayashi and Horiuchi 1996; Johzuka and Horiuchi 2002; Benguria et al. 2003) and for blocking DNA replication forks moving toward RNA Pol I (Brewer and Fangman 1988); the non- transcribed region 2 (NTS2) upstream of the 5S gene; and into the 35S gene, downstream of the Pol I promoter. Interestingly, silencing at the rDNA depends on Pol I activity and spreads only unidirectionally, downstream of a functional Pol I promoter (Buck et al. 2002; Cioci et al. 2003). Silencing at the rDNA is dependent on SIR2 but only at NTS1 and NTS2 and not in the transcribed region of the 35S gene, which is dependent on Pol I only (Bryk et al. 1997). On the other hand, Fritze and colleagues support a model, even though only indirectly, in which SIR2 influences silencing even in the transcribed region of the 35S gene (Fritze et al. 1997).
At the rDNA, Sir2 is part of a complex called the regulator of nucleolar silencing and telophase exit (RENT) formed by Net1 and Cdc14 (Shou et al. 1999; Straight et al.
1999). Net1 is required for silencing and for localization of Sir2 in the nucleolus and at the rDNA (Shou et al. 1999; Straight et al. 1999). Moreover, Net1 plays a role in maintaining nucleolar integrity and in regulating the exit from telophase by controlling the release of the phosphatase Cdc14 (Visintin et al. 1998; Straight et al.
1999; Visintin et al. 1999; Shou et al. 2001). The RENT complex, through its subunit Net1, is recruited at the RFB by Fob1 and at the 35S gene promoter by RNA Pol I (Shou et al. 2001; Huang and Moazed 2003). Interestingly, Fob1 is responsible for both inducing recombination within the rDNA, by a yet unknown mechanism, and repressing it, through the recruitment of the RENT complex (Kobayashi and Horiuchi 1996; Kobayashi et al. 1998; Johzuka and Horiuchi 2002). At NTS1, Fob1 recruits the RENT complex and a complex formed by Tof2, Lrs4, and Csm1 (Huang and Moazed 2006). This complex is required for silencing at NTS1 in a Sir2-independent manner, and acts synergistically with Sir2 in the suppression of unequal recombination at the rDNA (Huang et al. 2006). It has been proposed that the Fob1/Tof2/Lrs4/Csm1 complex binds the rDNA and the cohesin complex at the same time, thus fixing the position of two sister chromatids relative to each other, and thereby inhibiting unequal exchange between repeats (Huang and Moazed 2006). This model is consistent with the observation that Sir2 is required for maximal association of cohesin with the RFB (Kobayashi et al. 2004). Deletion of SIR2 increases the Fob1-dependent recombination rate at the rDNA, with consequent formation of extrachromosomal rDNA circles (ERCs) (Kaeberlein et al. 1999). The accumulation of ERCs leads to premature cell senescence in budding yeast (Sinclair and Guarente 1997). On the other hand, either increasing the dosage of SIR2 or deleting FOB1 suppresses recombination and increases lifespan (Kaeberlein et al. 1999).
At the chromatin level, both the H3 tail and H4K16 are important for silencing at the rDNA (Hoppe et al. 2002). The rDNA, the regions occupied by Sir2 at the rDNA present hypoacetylation of H3 tails and of H4K16 (Buck et al. 2002; Huang and Moazed 2003). Interestingly, the lowest chromatin acetylation state was initially mapped at the NTS1 region (Buck et al. 2002) even though, in a more recent report, the NTS1 and NTS2 regions have been shown to have the same hypoacetylated state (Huang and Moazed 2003). Even though hypoacetylation of H3 and H4K16 is a clear characteristic of the rDNA region, the papers referred to do not take into account the histone loss that can occur at the highly transcribed rDNA arrays, thus influencing the efficiency of signal recovery (Buck et al. 2002; Huang and Moazed 2003).
Interestingly, silencing at the rDNA is also dependent on RAD6 (also known as UBC2) (Bryk et al. 1997; Huang et al. 1997; Smith et al. 1999; Robzyk et al. 2000). Rad6 has been shown to ubiquitinate H2B at lysine 123 (Robzyk et al. 2000). Ubiquitinated H3K123 is in turn necessary for H3K79 methylation (Sun and Allis 2002), which is thought to act as a barrier for Sir proteins spreading at the telomeres (van Leeuwen et al. 2002). Thus, the Sir complex may spread inward on the chromosome in the absence of methylated H3K79 and thus recruit all the cellular Sir proteins there. Thus, Rad6 would have an indirect effect in rDNA silencing. Nevertheless, we cannot exclude a model with a more direct role of Rad6.
Histone modifications at silent chromatin
Histones play a crucial role in the formation and maintenance of silent chromatin at the HM loci and at the telomeres (Grunstein 1998). Two regions of histone H4 have been shown to be of particular importance for the correct recruitment of Sir proteins at silent regions, and thus for the establishment and maintenance of silencing (Kayne et al. 1988; Johnson et al. 1990; Park and Szostak 1990; Johnson et al. 1992). The first region is a basic domain spanning between glycine 14 and arginine 19 (Kayne et al.
1988). Any point mutation to a neutral amino acid of either lysine 16, arginine 17, histidine 18, or arginine 19 leads to a strong derepression of silencing at HMLα (Johnson et al. 1990; Park and Szostak 1990) and at the telomeres (Aparicio et al.
1991). In contrast, silencing was only slightly decreased when the charge of the single amino acid substitution was preserved (Johnson et al. 1990). Interestingly, insertions of even a single alanine between arginine 19 and lysine 20 affected silencing as much as any single point mutation of amino acids from position 16 to 19 (Johnson et al.
1992). This and the previous data suggest that the tail of H4, with its positively charged amino acids, presents a recognition motive for the binding of Sir proteins.
Interestingly, a search for suppressors of point mutations in the basic domain of the H4 tail led to the isolation of two alleles of SIR3: sir3R1, with a substitution of tryptophan 86 to arginine, and sir3R3 with a substitution of aspartic acid 205 to asparagine, revealing that the basic domain of H4 tail is bound by the N-terminal portion of Sir3 (Johnson et al. 1990). This region of Sir3 contains the BAH domain (Bromo-adjacent homology). The BAH domain has been identified in several other proteins such as Dnmt1, the major DNA methyltransferase in mouse and human cells,
Rsc1 and Rsc2, components of the RSC chromatin remodeling complex in S.
cerevisiae, and Orc1 the major subunit of the Origin Recognition Complex (Zhang et al. 2002; Connelly et al. 2006). Interestingly, sir3R1 and sir3R3 were suppressors of histone H4 mutations only at the HML locus, but not at telomeres. It is possible that the sir3R1and sir3R3 alleles still have a partial defect in silencing that could be masked by redundancy at HML, but not at the sub-telomeric regions, because the silencers are stronger nucleating elements than the TG1-3 repeat (Gottschling et al.
1990). Indeed, pull-down experiments with bacterially expressed H4 tails showed that in vitro translated Sir3 and Sir4 bind to the basic domain of the H4 tail lying between lysines 16 and 20 (Hecht et al. 1995). Interestingly, point mutation of lysine 16 to glutamine did not disrupt binding of either Sir3 or Sir4 to H4 N-terminal polypeptides, except when it was in combination with either deletions or single point mutations of the lysines in position 5, 8, and 12 (Hecht et al. 1995). The fact that a single point mutation at lysine 16 disrupts silencing in vivo (Johnson et al. 1990; Park and Szostak 1990), but does not compromise binding of Sir3 and Sir4 to polypeptides mimicking the tail of histone H4 (Hecht et al. 1995), suggests that the tail of H4 is more than just a binding site for Sir proteins. For instance, the deacetylation reaction of Sir2, acting on acetylated H4 tail, might be very important for the binding of Sir2-3-4 complex and for its spreading inward the chromosome arms.
The second important region of the H4 tail is represented by a hydrophobic patch found between isoleucines 21 and 29. A single point mutation of each of these residues or one alanine insertion between aspartate 24 and asparagine 25 and between glycine 28 and isoleucine 29 leads to derepression of both HML and HMR (Johnson et al. 1992). Interestingly, no mutation suppressor has ever been found in this hydrophobic domain of the H4 tail (Johnson et al. 1990; Thompson et al. 2003).
Moreover, in vitro translated Sir3 and Sir4 did not show any affinity for polypeptides representing only the hydrophobic region of the tail of H4 (Hecht et al. 1995). Finally, point mutations in this hydrophobic domain of the H4 tail did not compromise the binding of in vitro translated Sir3 and Sir4 to peptides representing the full length histone tails (amino acids 1-34). (Hecht et al. 1995). These data suggest that the hydrophobic region of the H4 tail is not a binding site for Sir3 or Sir4.
Not only the tail of histone H4, but also the core surface of the nucleosome, is important in the establishment and maintenance of silencing at HM loci, telomeres, and rDNA. For example, a recent report from Grunstein’s laboratory has suggested
that the deacetylation of H3K56 by Sir2, following DNA replication, is necessary for silencing (Xu et al. 2007). Based on predictions from the structure of the core nucleosome, Grunstein’s laboratory has suggested that the acetylation of H3K56 opens the nucleosome, preventing the binding of Sir proteins (Xu et al. 2007).
Another region of the nucleosome important for silencing is the surface surrounding H3K79. Interestingly, mutations at H3K79 affect all silent loci, whereas mutations at residues around H3K79 selectively affect repression at the telomeres, at HM loci, or at the rDNA (Park et al. 2002). H3K79 is methylated by Dot1; a methyltransferase whose over-expression or deletion leads to loss of silencing at telomeres (Singer et al.
1998; Ng et al. 2002; van Leeuwen et al. 2002). H3K79 methylation requires ubiquitination at H2B123 by Rad6/Bre1 (Sun and Allis 2002). It has been proposed that methylation at H3K79 together with Htz1, the variant of H2A enriched at actively transcribed regions, prevents the Sir2-3-4 complex from binding to chromatin (van Leeuwen and Gottschling 2002; Meneghini et al. 2003), although this model has not been proven conclusively. Interestingly, the sir3R1 allele is able to suppress both point mutations in the basic domain of the H4 tail and at H3 lysine 79 (Johnson et al.
1990; Thompson et al. 2003). From these data we could speculate that the positively charged region of the tail of H4 (formed by the first 20 amino acids) is involved in the first step of Sir3 recruitment, while the hydrophobic region would have a role in a second step, to direct Sir3 to bind to the region around lysine 79 of histone H3. This model requires biochemical and structural data for support.
Limiting or enhancing the spreading of silent chromatin
As we discussed above, in budding yeast, silencing is localized in the proximity of telomeres, at the HM loci, and at the rDNA array. In order to restrict silencing to only these three locations, and to avoid misregulation of important nearby genes, the strength of silencing has to be tightly controlled. A simple mechanism of such a control consists in limiting the amount of Sir proteins in the cell, and in tightly balancing their distribution between the three silent regions (Buck and Shore 1995;
Maillet et al. 1996; Marcand et al. 1996; Fritze et al. 1997). Telomeres artificially truncated of their subtelomeric sequences (called truncated telomeres) are particularly sensitive to any variation in the cellular amount of Sir proteins (Gottschling et al.
1990; Renauld et al. 1993). It seems that the spreading of silent chromatin at these
telomeres is mainly limited by the amount of Sirs and by the activity of chromatin modifying enzymes, such as Sas2 and Dot1, and chromatin remodeling activities, such as the SWR1 complex. These activities create zones of chromatin proficient for active transcription, thus presenting acetylation at H4K16, methylation at H3K79, and acetylated Htz1 in place of H2A (Kimura et al. 2002; Ng et al. 2002; Suka et al. 2002;
van Leeuwen et al. 2002; Meneghini et al. 2003).
In contrast, telomeres possessing subtelomeric elements (called native telomeres) present a more complex pattern of silencing (Fig. 5). Native yeast chromosome ends have a number of subtelomeric repeat elements, which vary between ends and strains.
The core X is found at all ends and contains an ARS consensus sequence (ACS) in all cases, and an Abf1 binding site at 31 out of 32 ends (Louis 1995).The Y’ element is found at some ends in up to four tandem copies, and contains two large open reading frames (ORFs) expressed during meiosis and an ACS sequence (Louis and Haber 1992; Louis 1995). The STR (SubTelomeric Repeats) regions consist of one or more
core X STR
TG1-3 TG1-3
Y’ ORF Y’-STAR
TG1-3 silencing
TG1-3
core X STR Y’ ORF Y’-STAR
silencing core X
TG1-3
silencing TG1-3
TG1-3 TG1-3 silencing
silencing core X
core X STR STR
STR
a)
f) e)
d) c) b)
g)
Fig.5) Silencing at native telomeres. a) Possible organization of subtelomeric sequences at native telomeres. b) Silencing at truncated telomeres. c) Silencing at native telomeres having all possible subtelomeric sequences. d) Silencing at native telomeres having either only the coreX or only the STR e). f) and g) Silencing at telomeres with both coreX and STR located in different positions.
(adapted from (Fourel et al. 1999)).
of several small elements designated STR-D, -C, -B, and -A which contain binding sites for Tbf1 and Reb1 (Chasman et al. 1990; Liu and Tye 1991; Brigati et al. 1993).
When Y’ elements are present, they are combined with a core X sequence on their centromeric side. The junctions between the core X and the Y’ element usually have variable numbers of the STR elements and can also contain internal telomeric repeats.
Gilson’s laboratory has dissected the role of each single subtelomeric sequence by testing their ability in either blocking or promoting silencing, coming from either telomeric repeats or HMR silencers, on a URA3 marker gene (Fourel et al. 1999).
They were able to show that the core X is able to enhance the effect of a distant silencer such as the E and I silencers, or the TG1-3 repeat. In contrast, the STR elements were able to block silencing, and were thus behaving as insulator elements.
Interestingly, the core X could recruit silencing even when the STRs were placed between the core X and the telomere, but not if the STRs were placed between the core X and the marker gene. This observation suggested that telomeres and the core X interact together through a long-range interaction. Work from the Louis laboratory has confirmed this hypothesis. They placed a URA3 marker gene at different locations of several ends of yeast chromosomes (Fig. 6) (Pryde and Louis 1999). First of all, they showed that silencing at native ends is less sensitive to increased expression of Sir3 than silencing at artificial truncated telomeres. They also showed that the Y’ element was refractory to silencing and that, in contrast, the core X was a region of silenced chromatin. They proposed that the telomere folds back on the chromosome, because of the interaction between the telomere-associated Sir2-3-4/Rap1 complexes and the proteins bound at the core X. In this model, Y’ would loop out, forming a transcriptionally active region. Also truncated telomeres are thought to fold back on themselves by mean of an interaction between the Sir2-3-4/Rap1 complexes bound to telomeres and the Sir2-3-4 complexes bound to nucleosomes inward the chromosome (Fig. 6) (de Bruin et al. 2000; de Bruin et al. 2001). A long range interaction has also been proposed for the HM loci. It was proposed that the HMR silencers interact together in a Sir-dependent manner, and cause the HM cassette to form a chromatin loop (Hofmann et al. 1989; Valenzuela et al. 2008). It is possible to imagine that the silencers of the HMR interact with one another and with telomeres to bring these silenced domains into a nuclear compartment that favors silencing. This model is supported by the experiments described above and by a few more observations. First of all the telomeres have been shown to cluster at the nuclear periphery (Gotta et al.
