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Epigenetics in Saccharomyces cerevisiae

Michael Grunstein1 and Susan M. Gasser2

1University of California, Los Angeles, Los Angeles, California 90095; 2Friedrich Miescher Institutefor Biomedical Research, 4058 Basel, Switzerland

Correspondence: [email protected] and [email protected]

SUMMARY

Saccharomyces cerevisiae provides awell-studied model system for heritable silent chromatin, in which anonhistone protein complex—the SIRcomplex—represses genes byspreading in a sequence-independentmanner, much like heterochromatin in higher eukaryotes. The ability to study mutations in histones and toscreen genome-wide for mutations that impair silencing has yielded an unparalleled depth of detail aboutthis system. Recent advances in the biochemistry and structural biology of the SIR-chromatin complexbring us much closer to a molecular understanding of how Sir3 selectively recognizes the deacetylatedhistone H4 tail and demethylated histone H3 core. The existence of appropriate mutants has also shownhow components of the silencing machinery affect physiological processes beyond transcriptionalrepression.

Outline

1 The genetic and molecular tools of yeast

2 The life cycle of yeast

3 Yeast heterochromatin is present at the silentHM mating loci and at telomeres

4 Sir protein structure and evolutionaryconservation

5 Silent chromatin is distinguished by a repressivestructure that spreads through the entiredomain

6 Distinct steps in heterochromatin assembly

7 The crucial role of histone H4K16 acetylationand its deacetylation by Sir2

8 Barrier functions: Histone modifications restrictSir complex spreading

9 A role for the H3 amino-terminal tail in higher-order chromatin structures

10 Trans-interaction of telomeres, and perinuclearattachment of heterochromatin

11 Telomere looping

12 Variable repression at natural subtelomericdomains

13 Inheritance of epigenetic states

14 Other functions of Sir proteins and silentchromatin

15 Summary

References

Editors: C. David Allis, Marie-Laure Caparros, Thomas Jenuwein, and Danny Reinberg

Additional Perspectives on Epigenetics available at www.cshperspectives.org

Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a017491

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1




OVERVIEW

The fraction of chromatin in a eukaryotic nucleus that bearsactive genes is termed euchromatin. This chromatin condens-es in mitosis to allow chromosomal segregation and decon-denses in interphase of the cell cycle to allow transcription tooccur. However, some chromosomal domains were observedby cytological criteria to remain condensed in interphase, andthis constitutively compacted chromatin was called hetero-chromatin. With the development of new techniques, molec-ular rather than cytological features have been used to definethis portion of the genome, and heterochromatin, which isoften found at centromeres and telomeres, was shown to con-tain many thousands of simple repeat sequences, particularlyin higher eukaryotic organisms. The repeat-rich genomicDNA tends to replicate late in S phase of the cell cycle, isfound clustered at the nuclear periphery or near the nucleo-lus, and is resistant to nuclease attack. Importantly, the char-acteristic chromatin structure that is formed on repeat DNAtends to spread and repress nearby genes. In the case of thefruit fly locus white, a gene that determines red eye color,epigenetic repression yields a red and white sectored eyethrough a phenomenon called position effect variegation(PEV). Mechanistically, PEV in flies reflects the recognitionof methylated histone H3K9 by heterochromatin protein 1(HP1), which can spread along the chromosomal arm. InSaccharomyces cerevisiae, also known as budding yeast, adistinct mechanism of heterochromatin formation hasevolved, yet it achieves a very similar result.

S. cerevisiae is a microorganism commonly used for mak-ing beer and baking bread. However, unlike bacteria, it is aeukaryote. The chromosomes of budding yeast, like those ofmore complex eukaryotes, are bound by histones, enclosed ina nucleus and replicated from multiple origins during S phase.

Still, the yeast genome is tiny with only 14 megabase pairs ofgenomic DNA divided among its 16 chromosomes, some notmuch larger than a bacteriophage genome. There are approx-imately 6000 genes in the yeast genome, closely packed alongchromosomal arms, generally with less than 2 kb spacing be-tween them. The vast majority of yeast genes are in an openchromatin state, meaning that they are either actively tran-scribed or can be rapidly induced. This, coupled with a verylimited amount of simple repeat DNA, makes the detection ofheterochromatin by cytological techniques very difficult inyeast.

Nonetheless, budding yeast has distinct heterochromatin-like regions adjacent to all 32 telomeres and at two silentmating loci on chromosome III (Chr III), shown using molec-ular tools. Transcriptional repression at telomeres and the si-lent mating loci can spread into adjacent DNA and repressionof the silent mating loci is essential for maintaining a mating-competent haploid state. Both the subtelomeric regions andthe silent mating type loci repress integrated reporter genes ina position-dependent, epigenetic manner; they replicate latein S phase and are present at the nuclear periphery. Thus, theseloci bear most of the functional characteristics of heterochro-matin, without having cytologically visible condensation ininterphase. Byexploiting the advantages afforded by the smallgenome of yeast and its powerful genetic and biochemicaltools, many basic principles of chromatin-mediated repres-sion that are relevant to heterochromatin in more complexorganisms have been discovered. Nonetheless, silent chroma-tin in budding yeast is dependent on a unique set of nonhis-tone proteins that do not deposit nor recognize histone H3lysine 9 methylation.

M. Grunstein and S.M. Gasser

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1 THE GENETIC AND MOLECULAR TOOLSOF YEAST

Yeast provides a flexible and rapid genetic system for study-ing cellular events. With an approximate generation time of90 min, colonies containing millions of cells are producedafter just 2 d of growth. In addition, yeast can propagate inboth haploid and diploid forms, greatly facilitating geneticanalysis. Like bacteria, haploid yeast cells can be mutated toproduce specific nutritional requirements or auxotrophicgenetic phenotypes, and recessive lethal mutations can ei-ther be maintained in haploids as conditional lethal alleles(e.g., temperature-sensitive mutants), or in heterozygoticdiploids, which carry both wild-type and mutant alleles.

Extremely useful is the efficient homologous recombi-nation system of budding yeast, which allows the alterationof any chosen chromosomal sequence at will. In addition,portions of chromosomes can be manipulated and reintro-duced on plasmids that are stably maintained through celldivision, thanks to short sequences that provide centromereand replication origin function. Large linear plasmids, orminichromosomes, which carry telomeric repeats to captheir ends, also propagate stably in yeast.

Yeast also has a unique advantage in the genetic analysisof histones and their roles in gene regulation: Unlike mam-malian cells, which have as many as 60–70 copies of thecore histone coding genes (H2A, H2B, H3, and H4), yeastcontains only two copies of each of these genes. Becausethese two copies are functionally redundant, this has en-abled the production of cells containing single histone genecopies. Deletion analysis or mutation of defined histoneamino acids in these genes has uncovered specific rolesfor histone residues in heterochromatin and other cellularfunctions. By searching for suppressors of these mutantphenotypes it has also been possible to identify heterochro-matin proteins that interact with the histone sites in ques-tion. The ease of generating histone mutants in yeast hasalso led to the systematic mutagenesis of most amino acidresidues in each of the core histones (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2666297/) and analysisof their effects on genome function (Huang et al. 2009).

Budding yeast also provides powerful cellular readoutsfor epigenetic gene regulation, conceptually similar to PEVin flies, in which the white gene provides a visible screen forvariegated gene expression (see Elgin and Reuter 2013for more detail). A parallel phenomenon called telomereposition effect (TPE) occurs near yeast telomeres. Thestudy of TPE has been aided analogously by the use ofthe URA3 and ADE2 reporter genes (Fig. 1). The URA3gene product is necessary for pyrimidine biosynthesis, andcells that do not express the Ura3 protein cannot grow onsynthetic media lacking uracil. In addition, URA3 allows

for a counter-selection against expression in the presence of5-fluoroorotic acid (5-FOA), because the Ura3 gene prod-uct converts 5-FOA to 5-fluorouracil, an inhibitor of DNAsynthesis that causes cell death. Thus, when URA3 is inte-grated near heterochromatin and the gene is repressed insome but not all cells, only the cells that silence URA3 areable to grow in the presence of 5-FOA. Conversely, if thestrain lacks the strong activator of URA3, Ppr1, positiveselection is possible; only URA3-expressing cells can growon uracil-deficient plates. Thus the efficiency of URA3 re-pression/expression can be scored accurately in serial dilu-tion assays (over a 1–106-fold range) on plates that eithercontain 5-FOA, or lack uracil (Fig. 1A). Because 5-FOA ismutagenic and puts strong pressure on cells to repress thereporter gene, some conditions or yeast backgrounds favorthe use of uracil-drop out plates over counter-selection on5-FOA.

Another useful assay for epigenetic repression is basedon the insertion of the reporter gene ADE2 near hetero-chromatin. When ADE2 is repressed, a precursor in adeninebiosynthesis accumulates, turning the cell red. When ADE2is expressed, cells are white (Fig. 1B). The epigenetic natureof repression of a subtelomeric ADE2 gene is visible withina single colony of genetically identical cells because thevariegated expression of ADE2 generates white sectors ina red colony background, attributable to ADE2 repression(Fig. 1B). This reporter avoids the stress of selective pressureagainst cells that fail to repress ADE2, and thus the sectoredphenotype illustrates both the switching rate and inheri-tance of the epigenetic state through mitotic division, muchlike the sectored white phenotype in the Drosophila mela-nogaster eye.

Combined with these genetic approaches, biochemicaltechniques for mapping epigenetic modifiers are readilyapplied to yeast. Large yeast cultures can be grown eithersynchronously or asynchronously. A battery of moleculartools, which includes transcriptome analysis and chro-matin immunoprecipitation (ChIP), can be combinedwith multiplexed next generation sequencing for efficientwhole-genome coverage. Combining this with proteomicapproaches that map protein networks enables a compre-hensive and quantitative comparison of gene expression,transcription factor binding, histone modification, andprotein–protein interactions. Finally, a technology devel-oped in yeast, called chromosome conformation capture,scores protein-mediated contacts between unlinked chro-mosomal domains to monitor long-range chromatin in-teractions (for more detail, see Dekker and Misteli 2014).With this broad range of tools, scientists have explored themechanisms that regulate both the establishment of het-erochromatin and its physiological roles in budding yeastfor more than 30 years. Before describing these discoveries,

Epigenetics in S. cerevisiae

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YPD

+ 5-FOA

URA3-Tel VIIL

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Variegatedrepression

Telomere

TG repeats

Red and white sectors

ade2–

ADE2-TelVR

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ade2–

ade2–

ADE2

Figure 1. Silencing and TPE in yeast. (A) The URA3 gene, inserted near the telomeric simple TG-rich repeat at the leftarm of Chr VII, is silenced by telomeric heterochromatin in this yeast strain. In normal rich media (YPD) no growthdifference can be detected between wild-type (wt) cells that repress the subtelomeric URA3 gene, and silencingmutants that lose telomeric heterochromatin and express URA3. In media containing 5-FOA (middle panel), on theother hand, cells that repress URA3 (e.g., wt cells) can grow, whereas cells that express it (sir2D and yku70D) cannot,because the URA3 gene product converts 5-FOA to the toxic intermediate 5-flourouracil. The serial dilution/dropassay allows detection of silencing in as few as 1 in 106 cells. In cells deleted for the URA3 activator, Ppr1 ( ppr1D), onecan screen for repression by plating on synthetic dextrose (SD) medium, lacking uracil. In this case, silencing thegene inhibits colony growth. (B) Cells containing the wild-type ADE2 gene produce a colony that is white, whereasthose containing mutant ade2 appear red, because of the accumulation of a reddish intermediate in adeninebiosynthesis. When the ADE2 gene is inserted near the telomere at the right arm of Chr V it is silenced in anepigenetic manner. The silent ADE2 state and the active ADE2 state in genetically identical cells are both inheritedcreating red and white sectors in a colony.






however, we will first review the life cycle of yeast in moredetail.

2 THE LIFE CYCLE OF YEAST

S. cerevisiae multiplies through mitotic division in either ahaploid or a diploid state by producing a bud that enlargesand eventually separates from the mother cell (Fig. 2A).This is why it is called budding yeast. Haploid yeast cellscan mate with each other (i.e., conjugate) because they existin one of two mating types, termed a or a, reminiscent ofthe two sexes in mammals. Yeast cells of each mating typeproduce a distinct pheromone that attracts the cells of theopposite mating type: a cells produce a peptide of 12 aminoacids (aa) called a factor, which binds to a membrane span-ning a-factor receptor on the surface of an a cell. Converse-ly, a cells produce a 13 aa peptide (a-factor) that binds tothe a-factor-receptor on the surface of a cells. These in-teractions result in the arrest of the two cell types in mid-to-late G1 phase of the cell cycle. The arrested cells assume“shmoo”-like shapes (named after the pear-shaped AlCapp cartoon character; Fig. 2B). Shmoos of opposite mat-ing type fuse at their tips to produce an a/a diploid cell.

In diploid cells the mating response is repressed, andcells propagate vegetatively (i.e., by mitotic division) unlessthey are exposed to starvation conditions. Nitrogen starva-tion induces a meiotic program in diploids that provokesthe formation of an ascus containing four spores, two ofeach mating type. When nutrient levels are restored, thesehaploid spores grow into a oracells that are again capable ofmating to form a diploid, starting the life cycle over again.

Although haploid yeast cells in the laboratory are usu-ally genetically constructed to be stable a or a cells, yeast inthe wild switch their mating type nearly every cell cycle(Fig. 3A). Mating type switching is provoked by an endog-enous cell-cycle-regulated endonuclease activity (HO) thatinduces a site-specific double-strand break at the MAT lo-cus. A gene conversion event (in which the donor DNAsequence remains unchanged, but the recipient DNA isaltered) transposes the opposite mating type informationfrom one of two constitutively silent donor loci, HMLa orHMRa, to the MAT locus, in which the mating type–de-termining genes, a1 and a2, or a1 and a2, are expressed.Strains capable of mating type switching are called homo-thallic. This name reflects the fact that a single vegetativeMATa cell can produce MATa progeny, and vice versa, al-lowing offspring to mate with each other.

In the laboratory it is useful to have strains with stablemating types, and thus laboratory yeast generally contain amutant HO endonuclease gene (ho –). These cells fail toinduce a double-strand cleavage at the MAT locus, andtherefore cannot switch mating type. These heterothallic

cells are stable haploid strains of either a or a mating type,unless placed in proximity of cells of the opposite matingtype, in which case the two haploid cell types will mate toform a diploid.

Importantly, the two stable haploid cell types do notrequire silencing for viability, yet they must repress the genesat the homothallic mating type loci, HML and HMR, toretain their ability to mate. If repression fails and cells ex-press both sets of mating type genes at once, then a haploidwill behave as if it were diploid, suppressing mating com-petence and generating a sterile haploid strain (Fig. 3B). Themechanisms that repress the two homothallic mating typeloci, HML and HMR, have become a classic system for thestudy of heterochromatin-mediated repression.

3 YEAST HETEROCHROMATIN IS PRESENTAT THE SILENT HM MATING LOCI ANDAT TELOMERES

The three mating type loci, HMLa, MAT, and HMRa, arelocated on one small chromosome, Chr III, and contain theinformation that determines a or a mating type in yeast.The silent loci, HMLa (�12 kb from the left telomere) andHMRa (�23 kb from the right telomere), are situated be-tween short DNA elements called E and I silencers (Fig.3B,C). In a wild-type cell, the silent cassettes are active onlyonce copied and integrated into the MAT locus, which lackssilencer elements. The transfer of HMLa information intoMATa results in an a mating type (MATa) cell, whereas thetransfer of HMRa information into MAT results in the amating type (MATa) (Fig. 3B). This shows that the promot-ers and genes at the HM loci are completely intact, andremain repressed because of their position between the Eand I silencers. Deletion of the flanking silencer sequencesindeed allows expression of the silent information, gener-ating a nonmating state.

By scoring for haploid sterility, mutations that impairsilencing at the HM loci were isolated (Rine et al. 1979).This allowed identification of the silent information regu-latory proteins, Sir1, Sir2, Sir3, and Sir4, as being essentialfor the full repression of HM loci (Rine and Herskowitz1987). Mutations in sir2, sir3, or sir4 caused a complete lossof mating, which is attributable to a loss of HM repression.In sir1 mutants, only a fraction of MATa cells were unable tomate. Taking advantage of the partial phenotype of sir1deficient cells, it could be shown that the two alternativestates (mating and nonmating) are heritable through suc-cessive cell divisions in genetically identical cells (Pillus andRine 1989). This provided a clear demonstration that mat-ing type repression displays the hallmark characteristic ofepigenetically controlled repression. Later genetic studiesshowed that the amino termini of histones H3 and H4,






ahaploid

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Starvation

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Germination Germination

αhaploid

a/αdiploid

aa

αα

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A

B GFP pore =nuclear envelope

Shmoo

Figure 2. The life cycle of budding yeast. (A) Yeast cells divide mitotically in both haploid and diploid forms.Sporulation is induced in a diploid by starvation, whereas mating occurs spontaneously when haploids of oppositemating type are in the vicinity of each other. This occurs by pheromone secretion, which arrests the cell cycle in G1 ofa cell of the opposite mating type, and after sufficient exposure to pheromone the mating pathway is induced. Thediploid state represses the mating pathway. (B) In response to pheromone, haploid cells distort toward cells of theopposite mating type. These are called shmoos. The nuclear envelope is shown in green fluorescence, showingdistortions of the nucleus.






Mating typeswitching

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O = ORC binding site = silenced chromatin region

a factor

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a haploid

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HMRa

A

B

C

D

Figure 3. Mating type switching in yeast. (A) Homothallic yeast strains are able to switch mating type after onedivision cycle. The switch occurs before DNA replication so that both mother and daughter cells assume the newmating type. (B) In a wild-type population of yeast, this allows rapid conjugation between daughter cells to form adiploid. (C) The position of the silent and expressed mating type loci on Chr III are shown here. The active MATlocus is able to switch through gene conversion roughly once per cell cycle because of cleavage by the HO endo-nuclease. The percentages indicated show the frequency with which the gene conversion event replaced the MATlocus with the opposite mating type information. The directionality of switching is guaranteed by the recombinationenhancer on the left arm of Chr III. (D) Repression at the silent mating type loci HMR and HML is mediated by twosilencer DNA elements that flank the silent genes. These silencers are termed E (for essential) or I (for important)(Brand et al. 1987) and provide binding sites for Rap1 (R), Abf1 (A), and origin recognition complex (O). Artificialsilencers can be created using various combinations of the redundant binding sites, although their efficiency is lessthan that of the native silencers. HMLa and HMRa are 12 kb and 23 kb, respectively, from the telomeres of Chr III.Telomeric heterochromatin domains at Chr III are silenced independently from the HM loci in a process that isinitiated at the telomeres through binding sites for Rap1.





repressor activator protein 1 (Rap1), and the origin recog-nition complex (ORC) are also components of silent mat-ing locus heterochromatin (reviewed by Rusche et al.2003). These latter two DNA binding factors have otheressential functions in the nucleus, namely, the regulationof ribosomal protein gene expression or the initiation ofDNA replication, and, thus, only “moonlight” as corepres-sors. Although less well studied, the same appears true forAbf1, a third silencer binding factor.

A similar position-dependent repression occurs imme-diately adjacent to the yeast telomeric repeat DNA (C1 – 3A/TG1-3) found at the ends of all yeast chromosomes. Asmentioned above, the variegated but heritable repressionof subtelomeric reporter genes such as URA3 and ADE2 iscalled TPE (Gottschling et al. 1990). TPE shares the HMrequirement for Rap1, Sir2, Sir3, Sir4, and the histone ami-no termini (Aparicio et al. 1991; Thompson et al. 1994a),and the repression mechanisms have proven to be closelyrelated. However, given that subtelomeric reporters canswitch at detectable rates between silent and expressedstates, unlike HM loci, telomere-proximal gene repressionappears to be more similar to fly PEV (see Elgin and Reuter2013).

4 Sir PROTEIN STRUCTURE AND EVOLUTIONARYCONSERVATION

The known chromatin binding factors that are essential forSir mediated silencing are Sir2, Sir3, and Sir4, whereas Sir1enhances the efficiency of repression at HM loci, but is notfound at telomeres. The Sir2-3-4 proteins work as a trimericcomplex with 1:1:1 stoichiometry (Cubizolles et al. 2006).Both Sir3 and Sir4 are able to bind nucleosomes and DNAindependently, yet the Sir holo-complex remains a trimerwhen bound to nucleosomes. Moreover, Sir3 and Sir4 eachhavehomo- andheterodimerizationmotifs in theircarboxyltermini. Mutation or deletion of their interaction domainsdisrupts silencing in vivo (Murphy et al. 2003; Rudner et al.2005; Ehrentraut et al. 2011; Oppikofer et al. 2013).

Sir protein expression levels are tightly regulated and asingle extra copy of the SIR4 gene impairs repression, asdoes strong induction of SIR2 (Cockell et al. 1998). On theother hand, increasing levels of Sir3 protein alone extendsthe spreading of Sir3 along nucleosomes, and with it, tran-scriptional repression (Renauld et al. 1993; Hecht et al.1996). Balanced overexpression of all three proteins greatlyimproves silencing efficiency at telomeres, and allows re-pression of reporters located in euchromatic regions thatare flanked by silencer elements, which at normal Sir pro-tein levels are not repressed (Maillet et al. 1996).

Although Sir2, Sir3, and Sir4 are equally essential forthe structural integrity of the Sir complex and therefore,

for both the establishment and maintenance of silentchromatin, each has a different function. Sir2 provides anicotinamide dinucleotide (NAD)-dependent histone de-acetylase activity that is essential for repression in a wild-type background (Imai et al. 2000), whereas Sir3 and Sir4fulfill structural roles without obvious enzymatic activities.Sir3 is a member of the AAA+ ATPase family, which lacksATPase activity. It is largely responsible for the specificity ofSir complex binding to nucleosomes because of its selectiveaffinity for nucleosomes with unacetylated histone H4 ly-sine 16 (H4K16) and unmethylated histone H3 lysine 79(H3K79) (Johnson et al. 1990; Altaf et al. 2007; Oppikoferet al. 2011). The sensitivity of Sir3 to these histone modi-fications helps restrict the binding of the Sir complex toappropriate sites.

Sir4 is the largest (152 kDa) and the least conserved ofthe Sir proteins, yet it forms a stable heterodimer with Sir2(Moazed et al. 1997; Strahl-Bolsinger et al. 1997) and en-hances Sir2 deacetylase activity (Tanny et al. 1999; Cubi-zolles et al. 2006). Structural information on the Sir4interface indicates that most of the Sir2-interaction domainof Sir4 (aa 737–839) is buried in a pocket formed by thepoorly conserved amino terminus and the carboxy-termi-nal catalytic domain of Sir2 (Hsu et al. 2013). The Sir3-binding domain within Sir4 is contained in the parallelcoiled-coil structure at its extreme carboxyl terminus,which also serves as a binding site for other proteins(Chang et al. 2003; Rudner et al. 2005).

4.1 The “Scaffold” Role of Sir4

The affinity of Sir4 for both Sir2 and Sir3 already suggestedthat it might act as a scaffold for the assemblyof the silencingcomplex. Much of this scaffolding role is achieved by thecarboxy-terminal half of Sir4, which is sufficient for repres-sion at HM loci (Kueng et al. 2012). Best studied is theextreme carboxy-terminal coiled-coil domain of Sir4 (aa1257–1358), that forms a continuous parallel homodimerwith two Sir3 binding sites on its outer surface (Chang et al.2003). Mutations within the dimerization motif disruptboth Sir3 binding and silencing (Murphy et al. 2003). How-ever, this same carboxy-terminal coiled-coil domain alsobinds yKu70 and Rap1, which recruit Sir4 to telomeric re-peats or HM silencers (Moretti et al. 1994; Tsukamoto et al.1997; Mishra and Shore 1999; Luo et al. 2002). Finally, Sir4binds the second yKu subunit, called yKu80, through bothyKu80’s amino and carboxyl termini, and interacts withEsc1 (establishes silent chromatin 1) through its partition-ing and anchoring domain (PAD; Sir4 aa 950–1262; Ansariand Gartenberg 1997; Andrulis et al. 2002). The yKu80and Esc1 interactions serve to tether Sir4, and the silentchromatin it binds, to the nuclear periphery (Gartenberg






et al. 2004; Taddei et al. 2004). The Sir4 PAD domain alsobinds ubiquitin binding protein 10 (Ubp10), a histone H2Bdeubiquitylase that reduces levels of H2B K123ub at telo-meres (Gardner et al. 2005). Loss of H2B K123ub in turnreduces histone H3K79 methylation, which directly inter-feres in Sir3-nucleosome binding (Armache et al. 2011;Oppikofer et al. 2011).

The Sir4 amino terminus is necessary for silencing insubtelomeric domains, but not at HM loci. It appears toboth regulate the efficiency of recruitment by bindingyKu80 and provide linker DNA protection when bound toreconstituted nucleosomes (Kueng et al. 2012). The aminoterminus is also heavily phosphorylated in vivo in a cell-cycle-dependent manner, allowing modification of repres-sion by the cyclin-dependent kinase Cdc28 (Kueng et al.2012). These observations highlight the role of Sir4 as amultifaceted scaffold that recruits, binds, and regulates thebinding of various factors that impinge on Sir-mediatedrepression.

4.2 Evolutionary Conservation of Sir2 and Sir3

As mentioned above, the Sir2 deacetylase is well conserved,with homologs in all species extending from eubacteria andarchaea to man (Fig. 4). Many species have multiple Sir2family members, although some members are cytoplasmicand serve primarily to deacetylate nonhistone proteins (de-tailed in Seto and Yoshida 2014). S. cerevisiae has five Sir2-related deacetylases (SIR2 and HST1–4), but only Sir2functions together with Sir3 and Sir4 in silent chromatin,in which it targets the amino-terminal tails of histones H3and H4.

The Sir2 family is defined by a conserved catalytic do-main in which deacetylation is coupled to the breakdown ofNAD+. The coupling of NAD hydrolysis with deacetylationproduces O-acetyl-ADP-ribose, an intermediate that mayhave a function of its own (Tanner et al. 2000). The Sir2-likeNAD-dependent histone deacetylases (HDACs) are impli-cated in transcriptional repression in many distant speciessuch as fission yeast and flies, although they lack the otherSir proteins (reviewed in Chopra and Mishra 2005). There-fore, it is thought that an ancient Sir2 deacetylase evolved toacquire unique interaction interface with the species-spe-cific factor Sir4, in budding yeast.

S. cerevisiae Sir2 plays an important role beyond TPEand HM locus silencing in that it suppresses nonreciprocalrecombination in the highly repetitive rDNA locus (Gott-lieb and Esposito, 1989). In this context Sir2 does not func-tion as part of the Sir2-3-4 complex, but associates with analternative group of factors that regulate exit from mitosis(the regulator of nucleolar silencing and telophase exit orRENT complex containing the phosphatase Cdc14, Net1/

Cfi1, and the mitotic monopolin proteins, Lrs4 and Csm1;Mekhail et al. 2008; Chan et al. 2011). These proteins arealso involved in the maintenance of rDNA repeat stability.

Sir3 contains several conserved domains, as the geneitself arose from an ancient version of ORC1, a subunit ofthe ORC that is found in all eukaryotes. The carboxy-ter-minal half of Sir3 contains a large AAA+ ATPase domain,much like all ORC subunits and their loading protein,Cdc6 (Norris and Boeke 2010). AAA+ domain proteinsgenerally hydrolyze ATP to drive the assembly and disas-sembly of macromolecular complexes. Sir3, however, hasan altered nucleotide binding pocket that precludes nu-cleoside binding (Ehrentraut et al. 2011). Sir3 and Orc1further share a conserved amino-terminal BAH (bromo-adjacent homology) domain that binds nucleosomes(Armache et al. 2011), and a carboxy-terminal wingedhelix domain that mediates dimerization (Oppikoferet al. 2013). Interestingly, whereas the BAH domain of yeastSir3 recognizes histone H4 deacetylated at K16, the BAHdomain of evolutionarily related HsORC1 recognizes his-tone H4 dimethylated at K20 (H4K20me2), linking hetero-chromatin with origin function in vertebrates (Beck et al.2012).

Whereas Orc1 is found in all eukaryotic species, Sir3 isonly found in budding yeast species that underwent whole-genome duplication approximately 100 million years ago(Hickman et al. 2011). In very closely related budding yeastspecies that have both Sir3 and Orc1 orthologs, Sir3 medi-ates TPE and mating type repression. However, in Kluyver-omyces lactis, which lacks Sir3, Orc1 appears to assumeSir3’s role in repression. Indeed, the carboxy-terminalwinged helix-turn-helix domain of Sir3 and Orc1 in thesetwo yeasts has a similar dimerization function, which islacking in Orc1 proteins from other species.

Sir1 and Sir4, in contrast to Sir2 and the Sir3/Orc1 fam-ily, are present only in species closely related to S. cerevisiae(i.e., the Saccharomycetaceae family). Intriguingly, despitethe restricted evolutionary distribution of Sir1, it containsa functionally defined OIR (ORC-interacting region) do-main that associates both with the Orc1 BAH domain andwith Sir4 (Hickman et al. 2011).

5 SILENT CHROMATIN IS DISTINGUISHEDBY A REPRESSIVE STRUCTURE THATSPREADS THROUGH THE ENTIREDOMAIN

Repression of gene activity in euchromatin often requiresthe presence of a repressive protein or complex that recog-nizes a specific sequence in the promoter of a gene, thuspreventing the active engagement of the transcriptionmachinery. In contrast, heterochromatic repression occurs






SIRT7 H. sapiens

SIR7 D. melanogaster

SIRT6 H. sapiens


SIRT4 H. sapiens


SIRT5 H. sapiens

HST3 S. cerevisiae

HST4 S. cerevisiae

SIRT1 H. sapiens


SIRT2 H. sapiens

SIRT2 D. melanogaster

SIRT3 H. sapiens

HST2 S. cerevisiae

SIR2 S. cerevisiae

SIR2 S. bayanus

HST1 S. cerevisiae

SIR2 K. lactis

SIRT1(Ia)

SIRT4(II)

SIRT6/7(IV)

SIRT2/3(Ib)

SIRT5(III)

NPD E. coli

HST2 S. pombe

SIR2 S. pombe

HST4 S. pombe

SIR4 K. lactis

SIR4 S. bayanus

SIR4 S. cerevisiae

ORC1 S. pombe

ORC1 D. melanogaster

ORC1 H. sapiens

ORC1 K. lactis

ORC1 S. bayanus

ORC1 S. cerevisiae

SIR3 S. bayanus

SIR3 S. cerevisiae

DNAa E. coli

0.05

0.05

0.05

HST3/4(Ic)

SIR3/ORC1

SIR2

SIR4

Figure 4. Sir protein family trees. Sir2 is the founding member of a large family of NAD-dependent deacetylases. TheSir2 family of proteins is highly conserved, found in multiple isoforms in organisms that range from bacteria to man.In the latter, there are both nuclear and cytoplasmic isoforms. Homologs of Sir2, Sir3, and Sir4 from Saccharomycesbayanus, Kluyveromyces lactis, Schizosaccharomyces pombe, D. melanogaster, and Homo sapiens were collected fromUniProt and were aligned using ClustalW2 alignment. The phylogenetic tree was created using neighbor joining.Sir2 classification is according to Frye (2000). K. lactis has 4 SIR2 orthologs (homologs to S. cerevisiae Sir2, Hst2,Hst3, and Hst4), but the HST homologs were omitted in the tree for clarity. For S. bayanus, only the Sir2 homolog isannotated to date. S. cerevisiae homologs are in red. Sir3 arose through a gene duplication of a gene encoding anancient Orc1, and Sir4 is a rapidly evolving protein that is only found in related budding yeasts. The related proteinsshown are not exhaustive, particularly for Sir2.






through a different mechanism that is not promoter spe-cific: Repression initiates at specific nucleation sites, yetspreads continuously throughout a domain, silencing anyand all promoters in the region (Fig. 5) (Brand et al. 1985;Renauld et al. 1993). The correlation of transcriptional re-pression with Sir binding was confirmed by the use of ChIP,which showed that Sir2, Sir3, and Sir4 proteins interactphysically with chromatin throughout the subtelomericdomain of silent chromatin and spread continuously in-ward from the chromosomal end (Strahl-Bolsinger et al.1997). Evidence that this induces a repressive, less accessiblechromatin structure in vivo comes from other approaches.For instance, it was shown that the DNA of silenced chro-matin was not methylated efficiently in yeast cells that ex-press a bacterial dam methylase, although the enzyme

readily methylated sequences outside the silent region.This suggests that heterochromatin restricts access tomacromolecules like dam methyltransferase (Gottschling,1992). Similarly, the �3 kb HMR locus in isolated nucleiis preferentially resistant to certain restriction endonucle-ases (Loo and Rine 1994), and nucleosomes were shownto be tightly positioned between two silencer elements cre-ating nuclease resistant domains at silent, but not active,HM loci (Weiss and Simpson, 1998). The reduced accessi-bility of yeast silent chromatin to nucleolytic attack is alsoobserved in vitro when Sir-nucleosome complexes are re-constituted from recombinant proteins (Martino et al.2009).

The extent to which either yeast or metazoan hetero-chromatin is hypercondensed to hinder access to trans-

HMsilentchromatin

Silencer SilencerRepressed domain

Telomericsilent chromatin

Sir2

Ac

Ac

Ac Ac

cccccccccccccccccccccccccccccccAcAccAcAcAcAAAcAcAcAAAcAcAAAAcAcAcAcAcAccAAAcAcAcAcAcAcAAAcAcAcAcAcAcAAAAcAcAcAcAcAcAcAA

ccccccccccccccAcAcAcAcAAccccccccccccccccccccccccAcAccAcAcAcAcAcAAAcAccAcAcAcAAAAcAccAcAcAAcAAAcAccAcAcAcAcAAAcAcAcAcAcAcAcAA

Ac

yKu70yKu80

Rap1Deacetylatednucleosome

Acetylgroup

Sir4

Sir3

Rap1yKu Raa

ncer Reepressee d domaed Sileina

yKu

22Sir2

Sir4

Sir3

4AcetylgroupAAAA t lAA t lAA t lA

Ac

Figure 5. Model for yeast heterochromatin at telomeres and the HM loci. The telomere and HM silencer mechanismsfor nucleating Sir complex spreading both use Rap1, Sir2, Sir3, and Sir4. Yet they differ in that telomeres also rely onyKu, whereas the HM silencer elements use the factors ORC, Abf1 and Sir1. Telomeric heterochromatin is thought tofold back onto itself to form a cap that protects the telomere from degradation and whose condensation and foldingsilences genes. In the case of HM heterochromatin, the repressed domain between the silencer elements consists ofclosely spaced nucleosomes that form a condensed structure. Both the telomeric and HM silent regions are inac-cessible to a number of transcription factors and degradative enzymes.






cription factors sterically is less certain. Surprisingly, therepressive complex formed by the interaction of Sir pro-teins and histones appears to be dynamic because Sirproteins can be incorporated into HM silent chromatineven when cells are arrested at a stage in the cell cyclewhen heterochromatin assembly generally does not occur(Cheng and Gartenberg 2000). This may explain whySir-bound heterochromatin can serve as a binding sitefor certain transcription factors (e.g., the heat shock tran-scriptional activator, HSF1) even in its repressed state (Se-kinger and Gross 1999). Although such studies argue thatheterochromatin does not simply hinder access for all non-histone proteins, no obvious transcription occurs, andengaged RNA polymerases cannot be detected experimen-tally. Experiments by Chen and Widom (2005) argue thatthe step that is specifically prevented by yeast heterochro-matin is formation of a complex of RNA polymerase II(RNA Pol II) with the promoter-binding transcription fac-tors TFIIB and TFIIE. Consistently, a drop in RNA Pol IIbinding was seen in a system in which silencing was in-duced by the controlled expression of Sir3. Thus, silentyeast chromatin may allow turnover of Sir factors andsome transcription factors, yet it selectively impedes thebinding of specific elements of the basal transcription ma-chinery, thereby blocking mRNA production.

6 DISTINCT STEPS IN HETEROCHROMATINASSEMBLY

The assembly of heterochromatin in budding yeast involvesa series of molecular steps, starting with a site-specific nu-cleation step. This requires DNA recognition by a sequence-specific DNA binding factor. Next, heterochromatin spreadsfrom the initiation site, limited by specific boundary mech-anisms. A change in higher organization of the repressedchromatin then occurs, which is distinct from the simplebinding of Sir factors. Finally, yeast silent chromatin issequestered near the nuclear envelope, generating a subnu-clear compartment that favors heterochromatin-mediatedrepression by promoting its duplication. Although the as-sembly of heterochromatin at telomeres varies in some as-pects from its assembly at HM loci, both embody a verysimilar principle: the presence of specific DNA bindingfactors that nucleate the spread of general repressors. Inboth cases, the spreading requires active deacetylationby Sir2. These mechanisms are described in the followingparagraphs.

6.1 HM Heterochromatin

The silent mating loci HML and HMR are bracketed byshort DNA elements termed silencers (Fig. 3), which

provide binding sites for at least two, and in most cases,all three multifunctional nuclear factors, namely Rap1,Abf1, and the ORC complex (Brand et al. 1987). The dele-tion of HMR-E, which has three recognition sites, has amuch stronger effect on silencing than deletion of HMR-I, which has only two, whereas at HML, the two silencershave more equal roles. The factors bound to silencers areable to cooperate with a distant silencer through Sir pro-teins to promote repression, possibly by forming a loopeddomain (Hofmann et al. 1989). This would explain thecooperative effects of the E and I silencers on the initiationof repression (Valenzuela et al. 2008), and the effects ofthe silencers on nucleosome spacing throughout the locus(Weiss and Simpson 1998).

Redundancy of silencer element function is a hallmarkof heterochromatic repression, and redundancy is alsofound within silencer elements: DNA binding sites forany two of the three silencer binding factors allow repres-sion (Brand et al. 1987). This redundancy likely stemsfrom the factors that they recruit. For example, Rap1 isable to recruit either Sir4 or Sir3 (Moretti et al. 1994; Luoet al. 2002; Chen et al. 2011), Abf1 interacts with Sir3, andORC has high affinity for Sir1, which in turn binds Sir4(Triolo and Sternglanz 1996). Thus, each of the silencer-binding factors leads to the recruitment of Sir4 and/orSir3, and in turn, the Sir2-3-4 complex. Although target-ing Sir2 artificially can also nucleate repression, none ofthe HM silencer elements nucleates repression by firstrecruiting Sir2. The redundancy among Rap1, Abf1, andORC (at silencers) or the Ku heterodimer (at telomeres),thus reflects the ability of each nucleator to bind Sir3 orSir4 and, in turn, to recruit the entire Sir complex to thesilencer or telomeric repeat element. Thus, sequence-spe-cific recognition is at the heart of position-dependentrepression.

Sir1, which bridges from ORC to Sir4, is unique amongthe Sir factors. Unlike the others, Sir1 does not spreadwith the Sir complex beyond the silencers (Fig. 5) (Ruscheet al. 2002). Moreover, once it helps establish silencing,Sir1 is no longer needed for the stable maintenance ofthe repressed state (Pillus and Rine 1989). This arguesthat Sir1 primarily serves in the establishment step ofrepression, most likely through its ability to bind theDNA-bound ORC and Sir4 (Triolo and Sternglanz,1996). Its role in nucleation was shown by tethering theprotein artificially through a Gal4 DNA binding domain toGal4 binding sites, which replaced the HMR-E silencer. Inthis context, GBD-Sir1 could efficiently nucleate repres-sion, rendering the silencer and its binding factors unnec-essary (Chien et al. 1993), although the other Sir proteinsand intact histone tails were still required for transcription-al repression.






6.2 Telomeric Heterochromatin

At telomeres, an RNA-based enzyme called telomerasemaintains a simple but irregular TG-rich repeat of 300–350 bp in length, which provides 16 to 20 consensus sitesfor Rap1 binding. This array of Rap1 binding sites forms anonnucleosomal cap on the chromosomal end, and plays acritical role in telomere length maintenance (Kyrion et al.1992; Marcand et al. 1997). Along the telomeric repeat,Rap1 binds its consensus through a core DNA binding do-main and Sir4 through its carboxy-terminal domain, evenin the absence of the other Sir proteins or the H4 aminoterminus. Point mutations that disrupt the Rap1-Sir4 in-teraction disrupt TPE, although the effect on HM repres-sion is very slight (Buck and Shore 1995). Rap1 also bindsSir3 through its carboxy-terminal domain, and mutation ofthis interface has similar effects on silencing (Chen et al.2011). However, because the loss of Sir4 prevents other Sirproteins from binding to telomeric chromatin (Luo et al.2002), Sir4 is apparently the crucial link between nucleationfactors and the ensuing silent chromatin structure (Fig. 6).

Equally potent for the nucleation of repression at telo-meres is the DNA end-binding complex yKu70/yKu80. TheyKu heterodimer also recruits Sir4, and loss of yKu stronglyderepresses TPE. Conversely, a targeted GBD-yKu fusionefficiently nucleates repression at silencer-compromisedreporter genes. The requirement for yKu at telomeres canbe bypassed by eliminating the Rap1-interacting factor,Rif1, which competes for the interaction of Sir4 with theRap1 carboxy-terminal domain (Fig. 6) (Mishra and Shore1999). This illustrates the redundancy between the twotelomeric nucleation factors, yKu and Rap1, based on theiraffinity for Sir4.

There is a clear correlation between the amount of Rap1bound at telomeres that is Rif1-free and the efficiency ofsilencing. Deletion of RIF1 gene or lesions in Rap1 thatblock Rif1 binding leads to relatively stable increases intelomere length. Wild-type cells inheriting these longertelomeres show increased frequency of repression of report-er genes such as URA3 or ADE2 integrated at the telomereson Chr VIIL or Chr VR (Kyrion et al. 1993). Moreover, in adiploid strain containing both elongated and wild-typetelomeres on Chr VIIL, the elongated telomere with in-creased repression did not affect repression at a wild-typelength telomere (Park and Lustig 2000). Thus, the effect oftelomere length on the frequency of silencing occurs in cis.Moreover, the frequency of switching from a derepressed toa repressed state (white colonies to red in the case of sub-telomeric ADE2 gene) also depends on the length of itsadjacent telomere; a longer telomeric repeat imposed alower frequency of switching from derepressed to repressedstates (Park and Lustig 2000). Thus, both Rap1 and yKu,

which provide recruitment sites for Sir3 and Sir4, can belimiting for the nucleation of TPE.

7 THE CRUCIAL ROLE OF HISTONE H4K16ACETYLATION AND ITS DEACETYLATIONBY Sir2

The molecular interactions between the Sir proteins havebeen well characterized. Sir4 interacts strongly with Sir2 andseparately with Sir3 in vivo and in vitro (Moazed et al. 1997;Strahl-Bolsinger et al. 1997; Hoppe et al. 2002). When co-ordinately expressed in insect cells, Sir2, Sir3, and Sir4 pro-teins can be isolated as a stable complex with a 1:1:1stoichiometry (Cubizolles et al. 2006). Nonetheless, Sir3has a special role in this process because Sir3 can form astable extended multimer in vitro (Liou et al. 2005) and itsoverexpression extends the subtelomeric silent domainfrom its normal �3 kb to �15 kb from the telomericend, coincident with the binding of Sir3 (Renauld et al.1993; Hecht et al. 1996).

The platform on which the Sir complex spreads consistsof nucleosomes with deacetylated histone H3 and H4amino termini (Braunstein et al. 1996; Suka et al. 2001),and the manner in which Sir3 interacts with histones helpsexplain how spreading occurs (Fig. 7). Sir3 binds the de-acetylated histone H4 amino terminus in a highly selectivemanner both in vitro and in vivo (Johnson et al. 1990;Johnson et al. 1992; Carmen et al. 2002; Yang et al.2008a). In this regard, the most important histone regionis contained in residues 16–29 of histone H4, and lysine 16must be positively charged (unmodified or substituted byarginine) for Sir3 to bind (Johnson et al. 1990,1992).

A cocrystal structure of the Sir3 amino-terminal BAHdomain explains this specificity quite well (Armache et al.2011). Sixteen residues in the Sir3 BAH domain interactwith H4 tail residues 13 to 23 which are held in a rigidconformation, primarily through electrostatic interactionswith amino acid side chains. A negatively charged bindingpocket of BAH Sir3 accommodates the side chains of un-modified H4K16 and H4H18. Indeed, acetylation ofH4K16 could potentially disrupt most of the electrostaticcontacts in this pocket. In contrast, the Sir2-Sir4 subcom-plex binds with slight preference to nucleosomes bearingan acetylated H4K16 residue, at least in the absence ofNAD+ (Oppikofer et al. 2011). This is consistent with acet-ylated histone H4K16 being a preferred and crucial targetfor the Sir2 enzyme (Imai et al. 2000; Suka et al. 2002;Cubizolles et al. 2006). The AAA+ domain of Sir3 alsobinds unmodified nucleosomes in vitro (Ehrentraut et al.2011) and requires that all four H4 acetylation sites(K5, K8, K12, and K16) are deacetylated for optimal bind-ing (Carmen et al. 2002) and for telomeric silencing






AcAcAc Ac Ac AcAcAc

Ac Ac Ac

Ac

Ac Ac Ac AcRecruitment ofSir4 with bound Sir2, and ofSir3 to TG repeatbound Rap1and yKu

Sir2-mediateddeacetylationof histoneH4K16 in nearbynucleosomes

Spreading ofthe Sir complexalong nearby nucleosomes

Folding of asilent telomereinto a higher-order structure

TG1-3 repeats

Rif1 Rif1yKu

Sir4

Sir3Sir2

Rap1 binding sites Subtelomeric regions

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Ac

Rif1

Sir2

Sir2

Rif1

Step 1

Step 2

Step 3

Step 4

Figure 6. Model for stepwise assembly of heterochromatin in yeast. (Step 1) At telomeres, Rap1 and yKu recruit Sir4even in the absence of Sir2 or Sir3. Only Sir4 can be recruited in the absence of the other Sir proteins, and its bindingis antagonized by Rif1 and Rif2 (Mishra and Shore 1999). (Step 2) Sir4-Sir2 and Sir4-Sir3 interact strongly creatingSir complexes along the TG repeats. Sir2 NAD-dependent histone deacetylase activity is stimulated by complexformation and Sir2 deacetylates the acetylated histone H4K16 residue in nearby nucleosomes. (Step 3) Sir complexesspread along the nucleosomes, perhaps making use of the O-acetyl-ADP-ribose intermediate produced by NADhydrolysis (Liou et al. 2005). Sir3 and Sir4 bind the deacetylated histone H4 tails. Although the deacetylated histoneH3 amino-terminal tail also binds Sir3 and Sir4 proteins, it is not shown here. (Step 4) The silent chromatin“matures” at the end of M phase to create an inaccessible structure. This may entail higher-order folding andsequestering at the nuclear envelope.






(Thompson et al. 1994a). Because histones are naturallydeacetylated throughout telomeric heterochromatin, theSir3 carboxyl terminus may contribute to the stability ofthe Sir3 complex through its interaction with the fully de-acetylated histone tail.

On addition of NAD+, the Sir2-catalyzed deacetylationof H4K16ac generates a by-product called O-acetyl-ADP-ribose, as well as a deacetylated histone H4 tail (illustrated inFig. 5 of Seto and Yoshida 2014). Intriguingly, not only thegeneration of the high-affinity binding site for Sir3, butapparently also production of the intermediate metaboliteO-acetyl-ADP-ribose, enhances the affinity of Sir2-3-4complexes for chromatin (Johnson et al. 2009; Martinoet al. 2009). The generation of O-acetyl-ADP-ribose en-hances the interaction of Sir3 with Sir4-Sir2 in vitro (Liouet al. 2005), favors the oligomerization of Sir proteins onnucleosomal arrays (Onishi et al. 2007), and enhances pro-tection of the linker DNA from micrococcal nuclease diges-tion (Oppikofer et al. 2011). This is consistent with geneticstudies showing that any mutation of histone H4K16 dis-rupts telomere repression, even substitution of lysine by thesimilarly charged amino acid arginine, or glutamine, whichmimics the uncharged nature of acetylated lysine.

Interestingly, the basic region of aa 16–24 within theH4 amino terminus also promotes nucleosomal arraycompaction in vitro, suggesting that the acetylation stateof H4K16 may regulate the higher-order folding of thenucleosomal fiber (Shogren-Knaak et al. 2006). Thus, his-tone H4K16 deacetylation by Sir2 may actively promote

silent chromatin formation in several ways: First, a confor-mational change may be triggered in the Sir complex by theby-product O-acetyl-ADP-ribose; second, the affinity ofthe Sir complex for chromatin increases thanks to genera-tion of a high-affinity Sir3 site; and third, even when Sircomplexes are not bound, nucleosomal arrays may com-pact because of contact between the H4 tail and the adja-cent nucleosomal face. From this it was clear that controlover Sir-mediated silencing would lie in the acetylation/deacetylation cycle of histone H4.

We summarize here the different steps for the initiationand spreading of heterochromatin in an environment en-riched for acetylated histone H4, which is likely to be de-posited immediately after replication (Fig. 6). At telomeres,Rap1 and yKu recruit Sir4, and Sir4 forms a dimer with Sir2to deacetylate histone H4 and H3 amino-terminal tails ofnearby nucleosomes. Sir3 is recruited by its affinity for Sir4,but also for Abf1 and Rap1. The deacetylation of the histoneH4 tail produces a high affinity Sir3 binding site on thenucleosome, which favors assembly of a Sir2-3-4 complex.The interactions of Sir3 with Sir4, Sir3 with the H4 tail andthe nucleosomal core, and Sir4 nonspecifically with linkerDNA, all seem to contribute to the stable binding of the Sircomplex to the nucleosomal fiber. The action of Sir2 onadjacent acetylated nucleosomes appears to trigger thespread of the complex along adjacent histone tails. Finally,the long-range folding of the chromatin fiber may stabilizethe repressed state. Most of these events are likely to be verysimilar at HM loci, although there the initial recruitment of

Boundary and insulatorfunction inyeast

Sas2 acetyltransferase

Rap1 binding sites Subtelomeric regions

· Reb1· Tbf1· HsCtf1

· VP16· Bdf1· Htz1

· tRNA genes· H3K79me· pore tethering

STT OP

Ac Ac

acetylSas2

Ac

AcAc

Ac

ccccAcAcAcAcAcAc

cccccccAccccccccAcAcAcAcAc

Ac

y

Ac

yrase

cccccccAcccccccAcAcAcAcAc

cccccccAcAcAcAcAcAcAc

cccccccAcAcAcAcAcAc

ccccccccccccccccccccccccccAAAAAAAAcAcAc

ertransf

cccccccAcAcAcAcAcAc

cccccccAccccccccAcAcAcAcAcAc

ng sites Subt nsS

c regionelomericte

cccccccAcAcAcAcAcAc

Figure 7. Heterochromatin boundary function in budding yeast. Spreading of heterochromatin through deacety-lation of histone H4K16 by Sir2 is limited by the competing activity of Sas2 histone acetyltransferase, whichacetylates H4K16 in adjacent euchromatin, thus preventing Sir3 binding. Methylation of K79 in histone H3 inadjacent euchromatin also affects the spreading of heterochromatin. In addition, factors such as Reb1, Tbf1, andmammalian or viral factors Ctf1 or VP16; nuclear pore tethering; and the presence of tRNA genes may also mediateboundary function. It is conceivable that several of these factors function through the recruitment of histoneacetyltransferases, like Sas2.






Sir4 needs Rap1, Abf1, or ORC and Sir1. The question thenarises, what causes Sir complex spreading to stop?

8 BARRIER FUNCTIONS: HISTONEMODIFICATIONS RESTRICT Sir COMPLEXSPREADING

Because the acetylated histone H4K16 binds Sir2-4 tightly,whereas its deacetylation by Sir2 is crucial for the spreadingof heterochromatin, it is not surprising that interferingwith the cycle of H4K16 acetylation/deacetylation impedesheterochromatin propagation (Kimura et al. 2002; Suka etal. 2002). The yeast histone acetyltransferase (HAT) Sas2, amember of the highly conserved MYST class of HATs, mod-ifies K16 in bulk euchromatin. Therefore at the boundariesof silent chromatin, one expects to find acetylated histoneH4K16. Accordingly, if the SAS2 gene is deleted, or ifH4K16 is changed to arginine to simulate the deacetylatedstate, Sir3, Sir4, and Sir2 spread at low levels inward fromthe telomeric repeats, approximately fivefold further thanin a wild-type cell. This suggests that the spreading of sub-telomeric heterochromatin is controlled, at least in part,by the opposing activities of Sir2 and Sas2 on lysine 16of H4 (Fig. 7). Limiting the global amount of Sir2 (or ofthe Sir2-Sir4 complex) naturally limits spread by limitingdeacetylation.

At the HM loci, restricting the spread of silent chroma-tin is perhaps even more critical than at telomeres becausegenes important for growth are found on Chr III, and it isknown that silencing can spread bidirectionally from si-lencers into flanking DNA sequence. One boundary thatprevents further spreading of silencing from HMR, is atRNA gene (Donze and Kamakaka 2001). This boundaryfunction is likely to require the HAT activity that is associ-ated either with transcription or the transcriptional poten-tial of this locus. It is significant that one of these HATs isSas2, although the H3 HAT, Gcn5, can also affect boundaryfunction of the tRNA gene. This suggests that transcrip-tional activators, in general, can restrict Sir complex prop-agation by recruiting HATs. Consistently, in subtelomericeuchromatin regions, boundary activity has been attribut-ed to the general transcription factors, Reb1, Tbf1, and tothe acidic trans-activating domain of VP16 (Fourel et al.1999, 2001). These factors are likely to promote the hyper-acetylation of histones (Fig. 7).

Another modification that antagonizes the spread oftelomeric heterochromatin is histone H3K79 methylation,which is deposited by the lysine methyltransferase Dot1(Van Leeuwen et al. 2002; Ng et al. 2002). This histonelysine methyltransferase (KMT) was discovered in a screenfor factors whose overexpression caused loss of telomeric

silencing (Singer et al. 1998). However, Dot1 does notmethylate H3K79 in heterochromatin itself, but insteadin adjacent euchromatin and at active genes. Indeed, theartificial targeting of Dot1 to telomeric heterochromatinderepresses silencing by Sir proteins (Stulemeijer et al.2011), most likely by reducing the affinity of Sir3 fornucleosomes.

Surprisingly, the H4 tail is required for the bulk meth-ylation of H3K79 by Dot1. An elegant set of experiments(Altaf et al. 2007; Fingerman et al. 2007) has generated amodel that helps explain the interplay between the H4 tailand the demethylated state of K79 in heterochromatin.Namely, when H4K16 is deacetylated by Sir2, a high affinitybinding site for Sir3 is generated by a charged patch in theH4 tail (K16, R17, H18, R19, K20). Interestingly, Dot1 andSir3 compete for this charged patch, although Sir3 is sen-sitive to H4K16 acetylation, and Dot1 is not. Thus, in de-acetylated heterochromatin, Sir3 is a potent inhibitor ofDot1 binding. However, in adjacent euchromatin, in whichK16 is acetylated, Dot1 preferentially binds the chargepatch and methylates histone H3K79. The Sir3N-nucleo-some crystal structure confirmed genetic evidence, suggest-ing that Sir3 interacts with H3K79 and that it is in closeproximity to H4K16 (Armache et al. 2011). Both the bind-ing of Sir3 and that of the holo-SIR complex are, in turn,weakened by histone H3K79 methylation. Again, this couldbe reconstituted in vitro, in binding assays between Sir3 andreconstituted nucleosomes that bear either H4K16ac orH3K79me (Oppikofer et al. 2011). Thus, the weak bindingof Sir3 to H4K16ac in euchromatin favors Dot1 binding tothe H4 tail and subsequent H3K79 methylation, which, inturn, weakens interaction with Sir3. This provides a goodexample of interhistone interactions as discussed and illus-trated in Figure 12 of Allis et al. 2014.

In addition to the mechanism regulating H3K79 meth-ylation and H4K16 acetylation near boundaries, it was re-ported that in euchromatin the presence of the varianthistone H2A.Z and the RNA polymerase associated factorBdf1 (Meneghini et al. 2003), as well as the tethering ofDNA to nuclear pores (Ishii et al. 2002), generate bound-aries that limit the spread of silent chromatin. Although themechanisms by which these factors affect heterochromatinspreading are unknown, it is interesting to note thatsome inducible genes with H2A.Z-containing promoters,associate with nuclear pores on activation, and remain as-sociated there in a manner that facilitates their re-induction(Ishii et al. 2002; Brickner and Walter 2004). Thus, boun-dary function may reflect a chromatin state that allowsrapid recruitment of transcriptional activators, includingHATs, KMTs like Dot1, or nucleosome remodelers thatdirectly or indirectly disrupt histone interactions with het-erochromatin proteins (Fig. 7).






9 A ROLE FOR THE H3 AMINO-TERMINAL TAIL INHIGHER-ORDER CHROMATIN STRUCTURES

There is increasing evidence that the formation of hetero-chromatin involves a series of steps that include, but gobeyond, the binding of Sir proteins. When Sir protein ex-pression is induced artificially in G1, Sir proteins spreadfrom their initiation site by interacting with deacetylatedH4 amino termini, but silencing is still defective (Kirchma-ier and Rine 2006). Also, when the histone H3K56 acetyla-tion site is mutated, Sir protein spreading occurs, butsilencing is disrupted. Similarly, the presence of H3K56acetylation leads to increased accessibility of Sir-boundnucleosomes to micrococcal nuclease in vitro without im-pairing Sir protein association (Oppikofer et al. 2011).Thus, the establishment of silencing requires not only Sirprotein spreading, but also the deacetylation of H3K56, anevent that enables nucleosomes to bind DNA more tightlyto form a structure resistant to ectopically expressed bacte-rial dam methylase (Maas et al. 2006; Xu et al. 2007; Celicet al. 2008; Yang et al. 2008b).

Repression also involves the replacement of histone H3methylated at K4 and K79 with unmethylated histone H3(Katan-Khayakovich and Struhl 2005; Osborne et al. 2009).Whereas H3K56 deacetylation by Hst3 and Hst4, two ho-mologs of Sir2, takes place every S phase, demethylation ofH3K79 requires dilution by replication, and can take up tofour cell divisions. These changes may parallel the forma-tion of a compact, higher-order chromatin structure.

At first, the role of the H3 amino terminus in silencingappeared to be similar to that of the H4 amino terminusbecause the domain in each tail involved in silencing in vivowas shown to bind Sir3 and Sir4 in vitro (Hecht et al. 1995).However, although the H4 amino-terminal residues arerequired for the recruitment and spreading of Sir3 andthe remaining Sir complex, the H3 tail was required neitherfor recruitment nor for spreading of the Sir proteins. None-theless, deletion of the H3 amino terminus or mutation ofcritical residues 11–15 (T–G–G–K–A) led to altered to-pology, increased accessibility to bacterial dam methylaseexpressed in yeast, and less tightly folded chromatin (Sperl-ing and Grunstein 2009; Yu et al. 2011). Thus, although Sirproteins are clearly recruited by the H4 tail, they may sub-sequently interact with the H3 amino terminus to formcompacted chromatin.

10 TRANS-INTERACTION OF TELOMERES,AND PERINUCLEAR ATTACHMENTOF HETEROCHROMATIN

In budding yeast, as in many lower eukaryotes, telomerescluster together during interphase, in close association with

the nuclear envelope. This clustering was initially observedas prominent foci of Rap1 and Sir proteins, which weredetected above a diffuse nuclear background by immuno-staining (Fig. 8). Disruption of silencing by histone H4K16mutation, or interference in Rap1 or yKu function, led tothe dispersion of the Sir proteins from these clusters (Hechtet al. 1995; Laroche et al. 1998). Later it was shown that notonly telomeres, but also the silent HML and HMR loci areclosely associated with telomeres at the nuclear envelope.Binding to the nuclear envelope is mediated by redundantpathways that depend either on the telomere-bound yKufactor, or on components of silent chromatin itself. Inter-estingly, the interactions that lead to telomere clusteringcan be genetically separated from telomere anchoring tothe nuclear envelope, although both pathways involve Sirproteins (Ruault et al. 2011).

Within silent chromatin, the anchoring function hasbeen assigned to the PAD of Sir4 (aa 950–1262) and itsinteraction with the nuclear envelope associated protein,Esc1 (Andrulis et al. 2002; Taddei et al. 2004). Sir4-Esc1interactions tether the Sir-repressed chromatin domain atperinuclear sites distinct from pores. Even in the absence ofa yKu anchoring pathway, the association of telomeres withthe nuclear periphery can be achieved by Sir4-Esc1 inter-actions, as long as silent chromatin is formed (Hediger et al.2002). Thus, rings of silent chromatin excised from theirchromosomal context by recombination and lacking TGrepeats, remain associated with perinuclear foci in a Sir-dependent manner (Gartenberg et al. 2004).

Initially, telomeres are recruited to the nuclear envelopeby yKu, given that yKu-dependent tethering occurs even inthe absence of silencing. This interaction with the nuclearenvelope, together with interactions in trans between telo-meres, generates a nuclear subcompartment that appearsto sequester Sir proteins from the rest of the nucleoplasm(Fig. 9). yKu mediated anchoring is achieved eitherthrough yKu-Sir4 interaction, or through the interactionof yKu with telomerase (Schober et al. 2009). The Est1subunit of telomerase binds specifically to an inner nuclearmembrane-spanning protein, Mps3, which is a member ofthe conserved SUN domain family of inner nuclear enve-lope proteins. Intriguingly, this interaction is cell-cycle spe-cific and mediates the link between yKu and the nuclearenvelope only in S phase, possibly to maintain anchoragewhile subtelomeric chromatin is disrupted by the replica-tion fork. In G1 phase, there appears to be a secondary yKuanchoring pathway, and Sir4 may bind Mps3 through anintermediary to help tether telomeres (Bupp et al. 2007).

Both pathways of telomere anchoring, through Sir4-Esc1 and S-phase yKu-Est1-Mps3 pathway, are controlledby post-translational modifications. Sir4 and both subunitsof yKu are modified by SUMO, a ubiquitin-like moiety,






deposited specifically by the E3 SUMO ligase Siz2. Remark-ably, loss of Siz2 led to the displacement of telomeres fromthe nuclear envelope (Ferreira et al. 2011). Silencing wasreduced only slightly in siz2 mutants, possibly becausetelomeres became abnormally long due to telomerasederegulation (see Section 12). Thus, the perinuclear com-partment created by telomere anchoring and silent chroma-tin appears to regulate telomere functions beyond silencing.

The perinuclear clustering generates a subnuclear com-partment that favors silencing (Fig. 9). Evidence support-ing this conclusion includes the fact that silencer-flankedHM constructs repress less efficiently when they are inte-grated far from telomeres (Thompson et al. 1994b; Mailletet al. 1996), and this can be reversed by artificially tetheringthe domain at the nuclear envelope through a targetedtransmembrane factor (Andrulis et al. 1998). Importantly,the ability to improve repression by peripheral tethering (orby being placed in telomere proximity) is lost when Sir3 andSir4 are no longer sequestered in foci (Taddei et al. 2009).Similarly, coordinate overexpression of Sir3 and Sir4 pro-teins ablates the positive effects of tethering. Thus, an un-even distribution of Sir proteins is the relevant feature oftelomere sequestration at the nuclear envelope for chroma-tin-mediated repression. Given that displaced Sir proteins

can repress promiscuously (Taddei et al. 2009), the seques-tration of Sir foci also positively reinforces active gene ex-pression. It is proposed that the assembly of newlyreplicated DNA into heterochromatin is likely to be favoredwhen DNA is replicated in a zone rich in silencing factors.

The crucial interaction that mediates telomere–telo-mere clustering, even in the absence of silencing, appearsto be Sir3. Whether this is mediated by Sir3 itself or ligandsof Sir3 remains to be determined, yet some form of clus-tering can occur even in the absence of Sir2 and Sir4(Ruault et al. 2011). Other factors also affect telomere–telomere interaction, namely, the other Sir proteins, theKu heterodimer, Asf1, Rtt109, Esc2, the Cohibin complex,and two factors involved in ribosome biogenesis, Ebp2 andRrs1. However, because these factors also affect heterochro-matin formation, one cannot rule out that they promoteclustering by promoting Sir3 recruitment to telomeres.

11 TELOMERE LOOPING

A further long-range interaction may stem from the foldingback of a single telomere on itself, which may allow silentchromatin to bypass subtelomeric boundary elements andstabilize repressed chromatin at subtelomeric genes (Figs. 5

A

α-poreα-Sir4

C

B

α-poreα-Sir4

D

WT

WT

2 µm

yku70Δ

Nop1

Rap1

2 µm

HML FISH +Tel Y' FISHα-pore IF

Figure 8. Sir proteins and Rap1 are found in foci at the nuclear periphery. (A) Rap1 (anti-Rap1, green) identifiesseven clusters representing all 64 telomeres in this diploid yeast cell nucleus, in which DNA is stained red. Telomeresare either perinuclear or adjacent to the nucleolus (blue, anti-Nop1). (B) Telomeric repeat DNA (red) and HML(green) is identified by fluorescent in situ hybridization. The two colocalize in �70% of the cases and both areadjacent to the nuclear envelope (anti-pore staining, blue). (C) The focal distribution of Sir4 (green) adjacent to thenuclear envelope (Mab414, red). (D) This pattern is lost in a yKu70 deletion strain, coincident with the loss oftelomeric silencing (Laroche et al. 1998).






and 6). Although Rap1 binding sites are found only withinthe first �300 bp of TG repeat DNA on the end of a telo-mere, ChIP showed that Rap1 is associated with nucleo-somes as far as �3 kb away from the TG repeat (Strahl-Bolsinger et al. 1997). Similarly, yKu is recovered for �3 kbfrom the chromosomal end to which it binds (Martin et al.1999). When silencing is disrupted by mutation of SIRgenes, both Rap1 and yKu are lost exclusively from themore internal subtelomeric sequences and not from theterminal TG repeats (Hecht et al. 1996; Martin et al.1999). This was interpreted as showing that the truncatedtelomere folds back, enabling TG-bound Rap1 and yKu tobind Sir proteins in trans (Figs. 5 and 6).

Evidence for telomere looping comes from the work ofde Bruin et al. (2001), who have exploited the inability ofyeast transcriptional activators, such as Gal4, to functionfrom a site downstream of the targeted gene. Strains wereconstructed in which the Gal4 upstream activating se-quence (UAS) element was placed beyond the 3′ termina-tion site of a reporter gene, and the construct was insertedeither at an internal chromosomal location or near a telo-mere. At an internal site, this construct could not be in-duced by activating Gal4, but in a subtelomeric context, theGal4 UAS could activate the promoter from a site 1.9 kb

downstream of the promoter. This was Sir3-dependent,arguing that the telomeric end can fold back in the presenceof Sir3, but not in its absence, to allow the Gal4 UAS toposition itself proximal to the transcription start site. Inthis way, silent chromatin appears to promote at least atransient folding of the chromosome end.

12 VARIABLE REPRESSION AT NATURALSUBTELOMERIC DOMAINS

We have set forth here a simplistic view of continuous silentchromatin emanating from the telomeric Rap1 bindingsites, yet the situation at native telomeres is significantlymore complex, largely because of the presence of naturalboundary elements found in subtelomeric repeat sequenc-es. Generally, when reporter constructs for telomeric re-pression are integrated, the subtelomeric repeat elementscalled X and Y′at telomeres are deleted, placing the reportergene and unique sequence immediately adjacent to TGrepeats. All native telomeres, on the other hand, contain acore subtelomeric repeat element, X, which is positionedbetween the TG repeat and the most telomere-proximalgene, and 50%–70% of native telomeres also contain at leastone copyof a larger subtelomeric element called Y′ (Fig. 10).

Pores

4. Sir complex spreading

3. Increased local Sir factor concentration

2. Sir4-Esc1and Yku-Mps3 anchoring

Nuclearenvelope

1. Nucleation = Sir4/Sir3 recruitment

Esc1 Esc1

yKu

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Sir Sir Sir4 Sir4 SirSir

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Sir

Sir Sir

Sir

Esc1

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Silencer sites

Silencers or TG

Peripheral attachmentgenerates self-propagatingcompartments of silent chromatin

Figure 9. Spontaneous formation of silencing subcompartments. A simple model for the formation of subnuclearcompartments is shown. (1) Sir4 is first recruited at the nucleation center by DNA binding proteins that can bindSir4. These include Rap1, ORC, Abf1, and yKu. (2) The presence of Sir4 at the locus will then bring it to the nuclearperiphery through one of the two Sir4 anchoring pathways (yKu or Esc1). (3) At the nuclear envelope, the high localconcentrations of Sir proteins will help silencing complexes assemble and spread. (4) The ability of silent loci toremain attached at the periphery increases the local concentration of Sir proteins and reinforces the silencing of otherloci within this region. Importantly, telomere-bound yKu can independently recruit telomeres to the nuclearenvelope just as Sir4 recruits silencer sequences.






Both X and Y′ elements contain binding sites for the tran-scriptional regulators Tbf1 and Reb1, which have beenshown to reduce the spread of silent chromatin (Fourelet al. 1999). However, Xelements also contain autonomous-ly replicating sequence (ARS) consensus sequences, andbinding sites for Abf1 and other transcription factors,which have the opposite effect: These re-initiate or boostthe repression of reporters placed on the centromere prox-imal side of these elements. The result is one of disconti-nuity in silencing at many native telomeres. This adds a levelof complexity to the model of continuous spreading out-lined in Figure 6. Pryde and Louis (1999) have proposed thatthe unrepressed Y′ element loops out when it is found be-tween two repressed domains, leading to discontinuity insilent domains without eliminating the need for nucleationand spreading from the TG repeats.

There is large variation in the efficiency of TPE at dif-ferent native telomeres in budding yeast. If one inserts areporter gene near a telomere without deleting the subtelo-meric repeats, only about half of the telomeres appear to besubject to TPE (Pryde and Louis 1999). Empirically, it was

shown that telomeres containing only X subtelomeric ele-ments (rather than XY′ telomeres) are more likely to si-lence, possibly because the Y′ long terminal repeats bindfactors that prevent Sir spreading, whereas various tran-scription factors in the X element (e.g., Reb1, Tbf1, andAbf1) contribute to Sir factor nucleation (Mak et al. 2009).These subtelomeric elements are particularly enriched forfactors that regulate stress genes, whose effects on TPE maydiffer from their effects on transcription at nontelomericloci. For example, Reb1 has boundary activity at telomeres(Fourel et al. 1999), but is a gene activator at internal sites.The binding of such factors can explain the discontinuity ofSir-mediated repression at native telomeres, yet they do notappear to affect the repression of classical reporters forTPE, which are integrated without X or Y′ elements (Re-nauld et al. 1993).

Interestingly, many of the genes found in subtelomericdomains are repressed by the HDAC Hda1 and the repres-sor Tup1 (Robyr et al. 2002), and are induced only on stressconditions (Ai et al. 2002). In general, the 267 genes that arewithin 20 kb of budding yeast telomeres produce roughly

Telomere:X + Y'-containingends

TG repeats STAR STR Core XY' ORF1Y' ORF1A B C D

Y' X

X-containingends

STR Core XB C D

Artificialtelomeres

URA3 reporter

A

TG repeats

TG repeats

Sir-mediatedsilencing

Figure 10. The organization of native telomeres and their silencing patterns. Subtelomeric elements are shown withtheir major protein binding sites. Telomeres fall into the two general classes: X-containing or X+Y′-containing ends.The STAR and STR elements block the propagation of repression and leave a region of reduced repression within theY′or X element. This is not the case at artificially truncated telomeres in which there is a gradient of repression thatextends 3–4 kb from the TG repeat. Looping similar to that in Figures 5 and 6 is proposed for native telomeres, sothat repressed regions contact each other leaving unrepressed chromatin in between areas of contact. (Adapted fromPryde and Louis 1999.)






five times fewer mRNA molecules (average of 0.5/cell) thannontelomeric genes. Importantly, only 20 of these genesshow derepression upon loss of Sir3. This leads to a logicof genome organization in which genes that are rarely orconditionally induced are found near telomeres, in whichthey are repressed by a non-Sir mechanism, but are adjacentto domains repressed by Sir proteins and, therefore, alsotethered near the nuclear envelope.

13 INHERITANCE OF EPIGENETIC STATES

A universal characteristic of heterochromatin is that its si-lent state is passed from one generation to the next. Thisrequires the reassembly of a repressive chromatin structureon daughter strands soon after replication of the DNA tem-plate. Pioneering work on the role of the cell cycle in theestablishment or inheritance of the silent state was per-formed by Miller and Nasmyth, who studied the onsetand loss of silencing with a temperature-sensitive sir3ts mu-tant (Miller and Nasmyth 1984). A shift from permissivetemperature to nonpermissive temperature caused silenc-ing to be lost immediately, indicating that SIR3 was requiredfor maintenance of the repressed state. However, in the re-ciprocal experiment, shifting from nonpermissive temper-ature to permissive temperature (SIR3+) did not lead toimmediate restoration of repression; passage through thecell cycle was required. They concluded that an event in Sphase was required for establishment of heritably repressedchromatin. This requirement was later shown to involveevents in both S and G2/M phases (Lau et al. 2002).

Initially it was thought that origin firing from the si-lencer linked ARS elements might be a critical event in theestablishment or inheritance of silent chromatin, but be-cause there was no detectable initiation from the originsflanking the HML locus, this seemed an unlikely explana-tion. Indeed, an experiment showing that ORC can beefficiently replaced by a targeted GDB-Sir1 fusion proteinput to rest the notion that origin firing is essential for theinheritance of silent chromatin. In addition, recent exper-iments have shown that establishment of repression canoccur on DNA that does not replicate (Kirchmaier andRine 2001; Li et al. 2001). Nonetheless, on rings of silentchromatin that are excised from the genome either with orwithout silencers, Sir complex association is in continualflux, arguing that nucleation and/or stabilization providedby silencer elements at HM loci is needed to actively sup-press rapid decay (Cheng and Gartenberg 2000).

What occurs in S phase to enable the propagation ofsilent chromatin? One candidate event could be the deace-tylation of H4K16ac or H3K56ac, or else the suppression ofthe enzymes that deposit these modifications (Xu et al.2007; Neumann et al. 2009). Alternatively, chaperones

necessary for histone deposition (CAF1) may be requiredfor generating repressed chromatin after replication. Thedeacetylation of histone H3K56 can be achieved in vitroby Sir2 family members (Xu et al. 2007; Oppikofer et al.2011) and in vivo during late S phase by Hst3 and Hst4(Maas et al. 2006; Celic et al. 2008; Yang et al. 2008b). Thesetwo Sir2 paralogs are nuclear enzymes whose activities arerequired in S phase when newly synthesized DNA must beassembled into repressed chromatin, yet they are not struc-tural components of heterochromatin. Importantly, dis-ruption of the two genes weakens, but does not eliminate,Sir mediated silencing (Yang et al. 2008b).

Other studies have shown that robust silencing is notachieved until telophase, well beyond the S-phase windowof nucleosome assembly. It appears that prevention of themetaphase degradation of the cohesin subunit, Scc1, inhib-its stable repression (Lau et al. 2002). Propagation of repres-sion thus depends both on a critical S-phase component,and a further event that entails continual recruitment andloading of Sir proteins.

Intriguingly, when the proteins of telomeric hetero-chromatin were examined by ChIP in the transcriptionallyOFF and ON states, the major difference found betweenthem was the presence of H3K79 methylation at telomericchromatin in the ON state (Kitada et al. 2012). BecauseDot1 is recruited during transcription (Shahbazian et al.2005), this suggests a positive feedback loop in which theON state is triggered, possibly by decreased telomere length,to initiate transcription and K79 methylation. Dot1 wouldthen be responsible for maintaining the ON state throughits promotion of K79 methylation.

14 OTHER FUNCTIONS OF Sir PROTEINSAND SILENT CHROMATIN

Although the standard function of heterochromatin is thesilencing of adjacent genes, a closer examination of silenc-ing factors has uncovered a plethora of new functions thatcorrelate with silencing or require silencing factors. Partic-ularly in organisms in which repetitive centromeric DNAplays a crucial role in centromere function, it is clear thatheterochromatin contributes to centromere and kineto-chores function. Budding yeast, on the other hand, doesnot depend on silent chromatin for centromere function.Nonetheless, a number of other roles have been identifiedfor silent chromatin or silent chromatin factors, and theseare described in this section.

14.1 Suppression of Recombination

In Drosophila highly active rDNA repeats are adjacent tocentromeric heterochromatin, and in many higher eu-






karyotic species, nucleoli and condensed heterochromatinare spatially juxtaposed. It is significant, therefore, that yeastSir2, independent of the other Sir proteins, is geneticallyand physically associated with rDNA repeats (Gotta et al.1997). Importantly, the loss of Sir2 in budding yeast leads toa dramatic increase in rDNA recombination and reductionof the contiguous integrated array (Gottlieb and Esposito,1989). Instability of the rDNA has been also correlated withan accumulation of extrachromosomal rDNA circles, whicharise from unequal crossing over between sister chromatids(Kobayashi et al. 2004). These events that are normally sup-pressed by a complex called Cohibin, a V-shaped complexof two Lrs4 proteins and two Csm1 homodimers (Mekhailet al. 2008; Chan et al. 2011), which also mediates the

association of rDNA repeats with the two nuclear envelopeproteins, Heh1 (a human Man1 paralog) and Nur1. Loss ofeither Sir2 or the cohibin-Heh1 anchoring pathway leads toinstability of the rDNA repeat, followed by cell-cycle arrestor premature senescence (Fig. 11A) (Sinclair and Guarente1997; Kaeberlein et al. 1999). Although Sir2 can also silenceRNA Pol II genes integrated in the rDNA (Smith and Boeke1997), recent evidence separates this effect on transcriptionfrom the role of Sir2 in preventing rDNA recombination.

Exactly how the tethering of rDNA repeats to the nu-clear envelope reduces recombination is still unknown. Thesimplest explanation may be that it imposes a steric hin-drance on the binding of Rad52, a protein essential forhomologous exchange in yeast. Indeed, Rad52 is excluded

rDNA repeatinstability andcellularsenescence

Sir- and Sumo-mediatedanchoringregulates telomere length

Sir2

rDNA repeats

Excision and/orinheritance of

an ERC

Nucleolar fragmentationRelocalization of SIR proteins

Cell death

15–20divisions

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asymmetricsegregation

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Elongation

Protected telomere: no elongation,low recombination

Telomere released: TG extension,hyperrecombination

Figure 11. Secondary functions of Sir proteins and silent chromatin. (A) rDNA recombination leads to cellularsenescence in yeast. The rDNA is organized in an array of 140–200 direct repeats of a 9.1 kb unit (red block). Theseencode the 18S, 5.8S, 25S, and 5S rRNAs, and contain two Sir2 responsive elements downstream of the 5S gene andwithin the 18S gene. The rDNA repeats tend to be excised in aging yeast cells, and the circles accumulate in themother cell (Kaeberlein et al. 1999). This correlates with premature senescence and can be antagonized by Sir2,which helps suppress unequal recombination and ring excision. (B) Telomere anchoring and silent chromatincontribute to telomere homeostasis. Redundant pathways that tether yeast telomeres to the nuclear envelope includesumoylation targets, Sir4, yKu70, and yKu80 (Ferreira et al. 2011). The relevant Sumo E3 ligase is Siz2. Loss of Siz2,ablation of the Mps3 amino terminus, or deletion of Sir4 all lead to release of telomeres from the nuclear envelopeand longer steady state telomere length. Loss of Mps3 amino terminus or yKu also increases telomere recombination.This suggests that sequestration at the nuclear envelope may limit access for both recombination and telomeraseactivation mechanisms, and that loss of anchoring increases both pathways. Regulated desumoylation by Ulp1 mayplay a role in releasing telomeres from the periphery allowing efficient elongation in late S phase. Siz2-mediatedsumoylation is indicated by red circles.






from the nucleolus and when rDNA requires repair by re-combination, the damage is extruded from the nucleolus.Intriguingly, other mutations that reduce the efficiency ofrDNA excision, such as the elimination of the replicationfork barrier protein Fob1, extend replicative lifespan ofyeast cells, confirming that rDNA instability is indeed akey culprit for limiting cellular lifespan.

Because Sir2 is a NAD-dependent deacetylase, and be-cause NAD levels act as a metabolic thermostat, it was pro-posed that the effect of yeast Sir2 on lifespan might berelated to the extension of lifespan by calorie restriction, aconserved pathway that functions in many species. Howev-er, Sir2 and caloric restriction increase lifespan throughindependent pathways (Kaeberlein et al. 2004). It is alsorelevant to note that an accumulation of extrachromosomalrDNA rings has not been detected during aging in any otherspecies. This is probably because of the unique buddingmechanism through which yeast divides; the budding ge-ometry and rapid kinetics of mitosis in yeast leads to aninevitable retention of noncentromere-bearing DNA ele-ments in the mother cell (Gehlen et al. 2011).

14.2 Preventing Homologous Recombinationon Chr III

Switching mating type involves the generation of a doublestrand break induced by the HO endonuclease at the MATlocus. That break is then repaired with homologous se-quences that recombine from the heterochromatic silentHM loci. In vitro studies have shown that the holo-Sir com-plex and histone sequences involved in silencing preventcleavage by the HO endonuclease at HM loci, and impairthe early steps in strand invasion by the break induced atMAT. The nucleosome remodeling SWI/SNF complex,which displaces Sir3 from nucleosomes (Sinha et al. 2009),is needed to counteract the repression of strand invasionconferred by silent chromatin. This enables recombination-al repair of MAT through an appropriate HM donor.

The deletion of SIR3 enhanced general recombinationrates throughout the genome (Palladino et al. 1993). Morespecifically the loss of telomeric anchoring correlates withincreased rates of recombination between a telomeric se-quence and internal sequences (Marvin et al. 2009). Thelatter involves the integrity of yKu, but probably does notsimply reflect the loss of anchoring alone.

14.3 Chromosomal Cohesion

Cohesion of sister chromatids is made possible by a com-plex of proteins known as cohesin. Yeast heterochromatin,like that of more complex eukaryotes, is enriched in cohesinwhich holds these silent regions together in sister chroma-

tids. When Sir proteins were tethered to chromosomalsites, in which pairing of sister chromatids could be seenby fluorescence microscopy, it was evident that Sir2 alonecould mediate cohesion. Although this required the cohe-sin complex, it did not require the deacetylase activity ofSir2 indicating yet another function for Sir2 in chromo-somal mechanics (Wu et al. 2011). It remains to be seen ifthis involves components of the Cohibin complex (Chanet al. 2011).

14.4 Telomere Length Regulation

In yeast, modification of histone H2A serine 129 by phos-phorylation generates a modified form of H2A, calledgH2A. In other species, this serine acceptor site is onlypresent in an H2Avariant called H2AX. Histone H2A phos-phorylation is mediated by the checkpoint kinases, Tel1 orMec1, and occurs when these central checkpoint kinasesare recruited to DNA damage by a complex of replicationprotein A with ssDNA or by the Mre11 complex. Intrigu-ingly, even without exogenously induced damage, gH2Awas found by ChIP to be coextensive with and dependenton silent subtelomeric chromatin (Szilard et al. 2010;Kitada et al. 2011). The phosphorylation of H2A in telo-meres is mediated by Tel1 kinase with weaker contribu-tions from Mec1 kinase. This suggests that subtelomericdomains trigger a low-level checkpoint response, possiblyduring telomere replication. The persistence of gH2A insilent domains may stem from reduced histone turnover,given that the enzyme that dephosphorylates gH2A actsonly on nonnucleosomal H2A (Keogh et al. 2006). Pointmutations that remove the H2A phosphoacceptor residuerender telomeres slightly shorter in certain genetic back-grounds, again linking telomeric chromatin structure toaspects of telomerase control (Kitada et al. 2011).

As discussed above, telomerase activity is increasedwhen telomeres are released from the nuclear envelopebecause of the absence of Sir4 or the SUMO ligase Siz2(Palladino et al. 1993; Ferreira et al. 2011). This further linkstelomere length homeostasis to subtelomeric chromatinstatus, implicating both the Sir proteins and the bindingof the yKu complex to Mps3 (Fig. 11). Interestingly, activa-tion of Mec1 kinase by double-strand breaks leads to a par-tial release of Sir proteins and displacement of telomeres(Martin et al. 1999), although it is unclear what Sir proteinrelease achieves in this situation.

14.5 A Link with Replication Factors

Whereas passage through an event between early S andG2/M is required for silencing, this critical event is notreplication itself. Nevertheless, a number of replication






factors are involved in silencing, most likely through struc-tural roles that are independent of their roles in replication.ORC binding sites are found at each of the silencer E and Ielements, and ORC directly recruits Sir1 in the absence ofreplication. Moreover, ORC binds not only at the silencerelements, but also in the region between E and I at HMR ina manner that is dependent on Sir proteins.

Like ORC, the helicase complex Mcm2-7 is part of theprereplicative complex (pre-RC) that assembles on repli-cation origins before the initiation of replication in S phase.However, MCM proteins are found in abundance in yeastcells and may have functions in processes distinct fromDNA replication, such as silencing. Indeed, Sir2 interactsindirectly with proteins of the Mcm2-7 complex outside ofS phase through a carboxy-terminal protein bridge (53aa)found in another pre-RC component, Mcm10. Mutationsin this bridge disrupt the binding of Mcm2-7 with Sir2 andweaken silencing efficiency, but they do not disrupt repli-cation nor the association of Sir2 with chromatin (Liachkoand Tye, 2009). In a speculative model, it is suggested thatMcm10, which itself forms a ring-shaped hexamer, bindsSir2 away from chromatin, ensuring that Sir2 undergoes amodification that renders it more competent for silencing.In the absence of Mcm10 and the MCM complex, a lesscompetent Sir2 would be incorporated into chromatin,reducing repression. What this modification might be re-mains unknown, but it is noteworthy that in fruit flies,Mcm10 also interacts with Hp1, and that in yeast,Mcm10’s silencing function can be genetically separatedfrom its replication function.

Independent of Mcm10, there is a strong evolutionaryrelationship between silencing and certain replication fac-tors, revealed by a phylogenetic analysis of Orc1, the largestsubunit of the ORC complex and Sir3. K. lactis containsOrc1 that is found at replication origins and HMLa. Asmentioned above, K. lactis contains the Orc1 paralog ofSir3, but not Sir3. The BAH domain of Orc1, however, in-teracts with the deacetylated H4K16 just like S. cerevisiaeSir3, allowing Orc1 to spread along telomeric and HMLaheterochromatin in a manner that is dependent on Sir2 andSir4 (Hickman and Rusche 2010). Surprisingly, however,HMR in K. lactis is silenced by a different mechanism thatinvolves neither Orc1 nor Sir4.

14.6 Regulating Replication Origin Choice

Replication origins are primed in G1 phase of the cell cycleby the formation of a pre-RC that recognizes DNA replica-tion origins. Interestingly, Sir2 inhibits pre-RC assemblyat certain origins, including one that is found at HMR-E.This origin is sensitive to the presence of Sir2 and Sir3, argu-ing that the silencing mechanism itself influences origin

choice. In contrast, certain origins that are not in hetero-chromatin are sensitive to the presence of Sir2 only, indi-cating that Sir2 has a unique function in origin functionthat is independent of transcriptional silencing. This func-tion may be in nucleosome placement near the origins thatprevents pre-RC assembly and is controlled by the deace-tylation of histone H4K16 by Sir2 (Fox and Weinreich2008). This is but one among many aspects of chromatinstructure that affect origin function, particularly in highereukaryotic species.

15 SUMMARY

Combined genetic, biochemical, and cytological tech-niques have been exploited in budding yeast to show fun-damental principles at work during heterochromatin-mediated gene silencing. These principles include (1) themechanism of initiation, spreading, and barriers to spread-ing of heterochromatin; (2) the balance of heterochromatinfactors and their distribution within a subnuclear environ-ment; (3) higher-order folding of heterochromatin; and (4)the effects of cell-cycle involvement in its formation. Thein vivo and in vitro studies to date provide a strong mech-anistic basis for our understanding of the assembly ofheterochromatin from chromatin fibers in all eukaryotes.The in vitro systems currently being developed for the re-constitution of yeast heterochromatin promise to yield astructural reconstruction of Sir complexes bound to chro-matin, enabling a first glimpse at higher-order chromatinfolding.

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