Noncompetitive Counteractions of DNA Polymerase {varepsilon} and ISW2/yCHRAC for Epigenetic Inheritance of Telomere Position Effect in Saccharomyces cerevisiae

Relocation of euchromatic genes near the heterochromatin regionoften results in mosaic gene silencing. In Saccharomyces cerevisiae,cells with the genes inserted at telomeric heterochromatin-likeregions show a phenotypic variegation known as the telomere-positioneffect, and the epigenetic states are stably passed on to followinggenerations. Here we show that the epigenetic states of thetelomere gene are not stably inherited in cells either bearinga mutation in a catalytic subunit (Pol2) of replicative DNApolymerase ${varepsilon}$ (Pol ${varepsilon}$ ) or lacking one of the nonessential and histonefold motif-containing subunits of Pol ${varepsilon}$ , Dpb3 and Dpb4. We alsoreport a novel and putative chromatin-remodeling complex, ISW2/yCHRAC,that contains Isw2, Itc1, Dpb3-like subunit (Dls1), and Dpb4.Using the single-cell method developed in this study, we demonstratethat without Pol ${varepsilon}$ and ISW2/yCHRAC, the epigenetic states ofthe telomere are frequently switched. Furthermore, we revealthat Pol ${varepsilon}$ and ISW2/yCHRAC function independently: Pol ${varepsilon}$ operatesfor the stable inheritance of a silent state, while ISW2/yCHRACworks for that of an expressed state. We therefore propose thatinheritance of specific epigenetic states of a telomere requiresat least two counteracting regulators.

INTRODUCTION

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Introduction

The eukaryotic genome is packaged into either euchromatin orheterochromatin. Heterochromatin is defined cytologically asthe fraction of the genome that is constitutively condensedthroughout the cell cycle (25) and corresponds to regions ofhighly repeated DNA such as centromeres or telomeres (13). Inthis region, DNA-mediated metabolisms, such as transcription,recombination, and replication, are generally inactive. In thecase of chromosomal DNA replication, heterochromatin replicatesin late S phase, while euchromatic regions replicate in earlyS phase (27).

In a simple eukaryote, Saccharomyces cerevisiae, although itsgenome is too small to define heterochromatin cytologically,the HMR and HML cryptic mating-type loci, ribosomal DNA (rDNA)repeats, and telomeres are loci affected by silencing (22, 26,52). Like heterochromatin in higher eukaryotes, HM loci andtelomeric regions are replicated in late S phase (17, 47), andthese silenced regions are associated with histone hypoacetylation(5, 8, 23, 39). The silencing process has three phases, establishmentor assembly of a silent chromatin, its maintenance through thecell cycle, and its inheritance to daughter cells, as describedby Lau et al. (35). Factors that are involved in these phasesof silencing are identified from studies of mutant strains thatlose silencing. Mutations in SIR2, SIR3, and SIR4 eliminatethe silencing at HM loci, whereas mutations in SIR1 partiallyreduce it, because Sir1 is required only for establishment,whereas Sir2, Sir3, and Sir4 are required for both establishmentand maintenance (45). Sir2, Sir3, and Sir4 form a Sir complexat HM loci (40). The Sir complex does not directly bind DNAbut rather interacts with DNA-binding factors, including Rap1,Orc, and Abf1, that bind to silencer DNA elements sandwichingHM loci (28). rDNA encoding 35S rRNA consists of 100 to 200times the number of tandem repeats in a 9-kb unit on chromosomeXII and is localized in the nucleolus. In rDNA silencing, theRENT complex, which is distinct from the Sir complex and whichconsists of Sir2, Net1, and Cdc14, represses homologous recombinationas well as transcription of transgenes in the rDNA repeats (50,55).

When a wild-type gene is located near a telomere in buddingyeast, it is subjected to telomere position effect (TPE) variegation,which provides heritable silent and expressed states and reversibleswitching between the epigenetic states (22). The silent stateof the telomeric gene is attributable to a heterochromatin-likestructure that consists of several proteins, such as Sir proteinsand hypoacetylated histones, that spread from the telomericends (1, 23). The Sir complex but not Sir1 interacts with tandemlyreiterated Rap1 molecules bound to the telomere repeat sequenceand spreads into subtelomeric DNA to form a silent heterochromatin-likestructure. Spreading of Sir proteins into the HM loci and subtelomericDNA is probably facilitated by the interaction of Sir3/Sir4with hypoacetylated histones H3/H4 and Sir2, a NAD-dependenthistone deacetylase (14, 23, 24, 54), and blocked by Dot1-catalyzedmethylation of histone H3 (33, 36, 42).

For the maintenance and inheritance of silencing, a silent statemust be propagated when chromosomal DNA is replicated. A previousstudy showed that the trans activator Ppr1 overcomes a silentstate of the URA3 gene integrated near telomere in G₂/M-phasearrested cells, but not in G₁- or early S-phase arrested cells(2), suggesting that progression through the S phase is requiredfor switching from a silent to an expressed state in TPE. Furthermore,Zhang et al. (63) recently showed that mutant forms of the replicationprotein PCNA are defective in silencing as well as in interactingwith CAF-1, a replication-coupled chromatin assembly factor,and they suggested that DNA replication machinery is linkedto chromatin assembly and silencing.

One of the replicative polymerases, DNA polymerase ${varepsilon}$ (Pol ${varepsilon}$ ) (56),is composed of a catalytic-subunit Pol2, Dpb2, Dpb3, and Dpb4in S. cerevisiae, and the subunit composition of Pol ${varepsilon}$ is evolutionarilyconserved in eukaryotes (37, 38). Although Pol2 and Dpb2 areessential for DNA replication (56), the histone fold motif-containingsubunits, Dpb3 (3) and Dpb4 (44), are dispensable and theirfunction remains unclear. A counterpart of Dpb4, p17 in humancells, was recently reported to be a subunit of not only Pol ${varepsilon}$ but also CHRAC (chromatin accessibility complex) (38, 46).CHRAC consisting of ISWI, Acf1, and two small subunits was purifiedfrom Drosophila and human cells and shown to catalyze chromatinremodeling (12, 46). In budding yeast, a similar complex wasreported to catalyze chromatin remodeling although small subunitshad not been detected (19, 30, 31, 59).

Because several histone fold motif-containing proteins havebeen reported to interact with histones (10), we examined whetherthese nonessential subunits may play a role in chromatin configuration.Using the single-cell method, we found that deletions of Dpb3and Dpb4 confer defects of TPE in different manners. This isbecause Dpb4 is shared by Pol ${varepsilon}$ and the ISW2/CHRAC complex, aputative chromatin remodeling factor counteracting Pol ${varepsilon}$ forTPE. From these observations, we propose a model to maintainchromatin structure when chromosomal DNA is replicated.

MATERIALS AND METHODS

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Materials and Methods

Strains and media. The yeast strains used in this study are listed in Table 1.YPDA (YPD with 0.04 g of adenine/liter) and synthetic complete(SC) media were used as previously described (22), except that100 mg of adenine/liter was added but leucine was not.

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TABLE 1. Yeast strains used in this study

Plasmid construction. YEp112-DPB3 was constructed by subcloning the BamHI-KpnI DPB3fragment from YCp111-DPB3 into the BamHI-KpnI site of YEplac112(20). YEp112-DPB4 was constructed by subcloning the BamHI-HindIIIDPB4 fragment from pRS315DPB4 (gift of A. Sugino) into the BamHI-HindIIIsite of YEplac112. pUCDPB4 was constructed by subcloning the2.2-kb BamHI-SphI DPB4 fragment generated by PCR into the BamHI-SphIsite of pUC18. YEp112-DPB3DPB4 was constructed by subcloningthe 1.7-kb BglII-SphI DPB4 fragment from pUCDPB4 into the BamHI-SphIsite of YEp112-DPB3.

Assay for silencing of telomeric URA3 and ADE2. To assay telomeric silencing, the strains with telomeric URA3and ADE2 at the telomeres of the left arm of chromosome VIIand the right arm of chromosome V, respectively, were used (strainslisted in Table 1). All the strains harbor YCplac111 (LEU2),because cells used in this study (W303 background) show slightlyslow growth on synthetic medium. To assay silencing of telomericURA3, freshly grown yeast cells were taken from SC medium anddiluted to a concentration of 2 x 10⁷ cells/ml. Then, 10-foldserial dilutions (2 x 10⁷ to 2 x 10² cells/ml) were made, and5-µl cell aliquots from each of the dilutions were spottedonto SC, SC-Ura, and SC 5-fluoroorotic acid (5-FOA) plates.The inoculated plates were incubated at 25°C for 3 days(SC and SC-Ura plates) or 4 days (SC 5-FOA plates), and pictureswere taken. To assay silencing of telomeric ADE2, about 100yeast cells of each strain from SC medium were spread onto SCplates. The plates were incubated at 25°C for 10 days, andpictures were taken.

Single-cell telomeric silencing assay. Cells (YTI448 and its isogenic derivatives) carrying the ${alpha}$ 2 geneat the right arm of chromosome V were freshly grown in YPDAmedium, streaked onto YPDA agar containing ${alpha}$ -factor, and incubatedat 30°C for 4 h. Among the 200 cells from the culture, thepopulation of shmoo cells was defined as the silenced cells(off cells). The off cells were incubated in the absence of ${alpha}$ -factor at 30°C until they started to bud, when the cellswere reexposed to ${alpha}$ -factor and the morphology of their daughterswas observed. Daughters of the budding cells from the originalculture (on cells) were separated, and their morphology in thepresence of ${alpha}$ -factor was observed. Five batches of either 20or 100 cells from each strain were used for the assay. To followeach generation (pedigree assay), the procedures for on andoff cells were successively repeated for the original 20 cells.Although several a-specific genes are derepressed in itc1 ${Delta}$ andisw2 ${Delta}$ cells (49), the a1- ${alpha}$ 2 complex represses the haploid-specificgenes and consequently makes cells insensitive to ${alpha}$ -factor. Therefore,this assay is also useful for itc1 ${Delta}$ and isw2 ${Delta}$ cells.

Immunoprecipitation. Immunoprecipitation was performed according to the methods describedby Zachariae et al. (62) and Takayama et al. (58) by using ananti-Flag antibody (M2)-conjugated agarose and lysis buffer(50 mM HEPES-KOH [pH 7.5], 300 mM KCl, 0.05% Tween 20, 0.005%NP-40, 10% glycerol, 1x complete protease inhibitor cocktail[Boehringer-Mannheim], 1% Sigma protease inhibitor [Sigma],2 mM ß-glycerophosphate, 2 mM NaF, 0.4 mM Na₃VO₄,0.5 mM Na-pyrophosphate) containing 5 mg of bovine serum albumin/ml.From the immunocomplex, the proteins were eluted by incubationwith 100 µg of 3x Flag peptides (Sigma) per ml. Superose6 column (Amersham Biotech) chromatography of the eluates wasperformed in the lysis buffer.

ChIP assay. Chromatin immunoprecipitation (ChIP) analysis was performedessentially as described in reference 29. DNA purified fromwhole-cell extracts or coprecipitated with anti-c-myc antibodies(MBL) was analyzed by PCR. Primers specific for URA3 (ura3F[5'-CGAAAGCTACATATAAGGAACGTGC-3'] and ura3R [5'-CCCATGTCTCTTTGAGCAATAAAGC-3'])were used to amplify the fragments, which were resolved on 2%agarose gels and stained with ethidium bromide. Quantificationwas carried out with a LumiVision Analyzer instrument (AISIN).

RESULTS

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Results

Pol ${varepsilon}$ is required for TPE. To determine whether Pol ${varepsilon}$ is involved in the maintenance ofchromatin structure, we first investigated whether the deletionsof DPB3 and DPB4 influence silencing of the URA3 gene integratednear the telomere of chromosome VII (22) (Fig. 1A). Expressionof URA3 leads to cell growth on medium lacking uracil as wellas to cell death on medium containing 5-FOA. In this assay,dpb3 ${Delta}$ and dpb4 ${Delta}$ mutant cells displayed partial expression of URA3,and this expression was more evident in dpb3 ${Delta}$ cells, while theexpression of URA3 at the original locus was not affected (Fig.1B, SC 5FOA). Moreover, the expression level in the dpb3 ${Delta}$ dpb4 ${Delta}$ double mutant was similar to that in dpb4 ${Delta}$ (Fig. 1B), indicatingthat the dpb4 ${Delta}$ mutation is epistatic to the dpb3 ${Delta}$ mutation. Furthermore,the pol2-11 mutation (9, 41) conferred partial expression oftelomeric URA3 and this defect was suppressed by simultaneousintroduction of high-copy-number DPB3 and DPB4 (Fig. 1C). SinceDpb3 and Dpb4 form a subassembly in vitro (15) and neither oneof them suppressed the pol2-11 mutation (Fig. 1C), Dpb3 andDpb4 seem to interact with Pol2 as a complex in vivo. This resultfurther suggests that Dpb3, Dpb4, and Pol2 function for TPEas Pol ${varepsilon}$ .

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FIG. 1. DPB3 and DPB4 are involved in the regulation of TPE variegation. (A) Schematic diagram of the telomeric URA3 gene at chromosome VII. An open box and a wavy line represent the open reading frames of URA3 and the telomere, respectively. (B) Expression of URA3 at the telomere (VIIL::URA3-TEL) and internal chromosomal loci. To examine expression of the telomeric URA3 gene, the isogenic strains YTI249 (WT), YTI250 (dpb3 ${Delta}$ ), YTI266 (dpb4 ${Delta}$ ), YTI268 (dpb3 ${Delta}$ dpb4 ${Delta}$ ) and YTI258 (pol2-11) were used. To examine expression of the URA3 gene at the internal chromosomal locus, the isogenic strains YTI279 (WT ura3), YTI471 (dpb3 ${Delta}$ ura3), YTI472 (dpb4 ${Delta}$ ura3), YTI297 (WT URA3), YTI468 (dpb3 ${Delta}$ URA3), and YTI469 (dpb4 ${Delta}$ URA3) were used. Cells freshly grown in SC medium were placed onto the indicated plates, after 10-fold serial dilution, and further incubated at 25°C for 3 days. The numbers under the photographs indicate the estimated number of cells placed on a spot. (C) Suppression of the pol2-11 mutation by increased dosage of DPB3 and DPB4. Wild-type (YTI249) and pol2-11 (YTI258) strains carrying a high-copy-number plasmid (YEplac112) (20) with DPB3, DPB4, or DPB3 DPB4 were subjected to a telomeric silencing assay as described for panel B. YCpPOL2 (YCp22POL2) (4) and YCp (YCplac22) (20) are CEN-based plasmids with and without POL2, respectively. Serial dilutions of cultures were inoculated onto the SC plates containing leucine and lacking tryptophan in the absence or presence of 5-FOA and incubated at 25°C. Note that the growth of pol2-11 mutant cells being slower than is apparent in panel B is probably attributable to the difference in the medium used for the plates. The numbers under the photographs indicate the estimated number of cells placed on a spot.

We also observed, in parallel, the expression of telomeric ADE2on chromosome V (51), which shows phenotypic variegation ofa silent (red) and an expressed (white) state by TPE and providesresultant red- and white-sector colonies (22) in wild-type cells(Fig. 2A). As expected, dpb3 ${Delta}$ cells formed white colonies asmutants defective in structural components of telomeric or subtelomericchromatin (1) (Fig. 2A). Surprisingly, dpb4 ${Delta}$ cells formed nonsectoringcolonies with a solid light-pink color. Partial expression ofADE2 in each cell or a mixture of silent and expressed cellscaused by frequent switching between alternative epigeneticstates might confer a solid light-pink color. These possibilitieswere distinguished by the single-cell colony assay (see below).

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FIG. 2. Defects of TPE variegation in dpb3 ${Delta}$ , dpb4 ${Delta}$ , and dls1 ${Delta}$ mutations. (A) A colony color assay for cells carrying telomere-linked ADE2 (VR::ADE2-TEL). Freshly grown yeast cells of YTI249 (WT), YTI250 (dpb3 ${Delta}$ ), YTI266 (dpb4 ${Delta}$ ), YTI442 (dls1 ${Delta}$ ), and YTI443 (dpb3 ${Delta}$ dls1 ${Delta}$ ) strains were spread onto SC plates and further incubated at 25°C for 10 days. (B) High copy number of SIR3 does not enhance the telomere silencing in dpb3 ${Delta}$ and dpb4 ${Delta}$ cells. Freshly grown yeast cells of YTI249 (WT), YTI250 (dpb3 ${Delta}$ ), and YTI266 (dpb4 ${Delta}$ ) strains harboring a high-copy-number plasmid with the SIR3 gene (YEp-SIR3) were spread as described for panel A. Wild-type transformants with YEp-SIR3 form two types of sectoring colonies (red sector or white sector), and cells from each type colony were spread onto plates.

One of the replication proteins, PCNA, is suggested to be involvedin the assembly of a silent chromatin (establishment) throughCAF-1 (chromatin assembly factor 1) (63). To determine whetherDpb3 and Dpb4 function in the CAF-I-PCNA chromatin assemblypathway, we constructed double mutants bearing cac1 ${Delta}$ and eitherdpb3 ${Delta}$ or dpb4 ${Delta}$ . The CAC1 gene encodes one of three subunits ofCAF-1. As shown in Table 2, dpb3 ${Delta}$ and dpb4 ${Delta}$ mutations enhancedthe telomere silencing defect of cac1 ${Delta}$ cells, indicating thatthe roles of Dpb3 and Dpb4 in TPE are distinct from the CAF-I-PCNAchromatin assembly pathway.

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TABLE 2. Effect of dpb3 ${Delta}$ and dpb4 ${Delta}$ mutations with cac1 ${Delta}$ on telomere silencing^a

It is also known that the increased dosage of Sir3 enhancesthe telomere silencing if the assembly of a silent chromatinis defective because Sir3 is a limiting factor for assemblyof the Sir complex (48). We therefore introduced high-copy-numberSIR3 into dpb3 ${Delta}$ and dpb4 ${Delta}$ cells and examined TPE using the colonycolor assay. While the wild-type cells harboring high-copy-numberSIR3 frequently formed red-sectored colonies (enhancement ofsilencing), high-copy-number SIR3 did not affect the colonycolor of dpb3 ${Delta}$ and dpb4 ${Delta}$ cells (Fig. 2B), indicating that Sir3is not a limiting factor in these cells.

Deletions of Dpb3 and Dpb4 show different switching patterns. To monitor the switching between a silent and an expressed statein each cell division, we developed the single-cell telomericsilencing assay. In this assay, the ${alpha}$ -factor sensitivity of acells bearing the ${alpha}$ 2 gene (32) near the telomere of chromosomeV ( ${alpha}$ 2-TEL) (Fig. 3A) was used. When the telomere ${alpha}$ 2 gene is expressed(on), a cells ( ${alpha}$ 2-TEL) behave like a/ ${alpha}$ diploid cells, which areinsensitive to ${alpha}$ -factor and consequently continue to divide (16,45). Conversely, the ${alpha}$ 2-repressed cells (off) are sensitive to ${alpha}$ -factor and arrest in the G₁ phase with a shmoo shape (Fig.3B). We first followed three successive divisions of each cellto examine whether the switching depends upon a previous eventas a mating type switch (26) in budding yeast cells and foundthat cells switch their telomere states in an independent manner(Fig. 3C). Thus, we observed a single division of independentcells to measure their switching rate. We found that the switchingrate from off to on (29%) is higher than that from on to off(4.3%) in wild-type cells (Table 3). In dpb4 ${Delta}$ cells, both switchingrates increased, whereas in dpb3 ${Delta}$ cells the switching rate fromoff to on specifically increased. These results suggest thatDpb4 plays roles in switching in both directions, while Dpb3is only involved in switching from off to on.

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FIG. 3. Single-cell assay of TPE. (A) Schematic diagram of telomeric ADE2 and ${alpha}$ 2 genes at chromosome V. Open boxes and a wavyline represent the open reading frames of the reporter genes and the telomere, respectively. (B) Expression of the telomeric ${alpha}$ 2 gene (VR:: ${alpha}$ 2-TEL) in the YTI448 ( ${alpha}$ 2-TEL WT) strain and the isogenic control YTI280 (WT) strain without ${alpha}$ 2-TEL. Cells were exposed to ${alpha}$ -factor on YPDA plates at 30°C for 5 h. Pear-shaped cells, i.e., shmoos (cells with ${alpha}$ 2 unexpressed), and dividing cells (cells with ${alpha}$ 2 expressed) in each strain were magnified. The arrows indicate shmoo cells. (C) Pedigree assays of cells from strain YTI448 (WT) with ${alpha}$ 2 expressed (on) or silent (off). The procedure is described in detail in Materials and Methods.

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TABLE 3. Effect of dpb3 ${Delta}$ , dpb4 ${Delta}$ , and dls1 ${Delta}$ mutations on frequency of switching between silent and expressed states of telomere-linked ${alpha}$ 2^a

In this assay, the population of silenced cells among the dpb4 ${Delta}$ mutant cells was higher than that among the wild-type cells(Table 3), whereas the number of FOA-resistant cells (silencedcells) was reduced by the dpb4 ${Delta}$ mutation (Fig. 1B). This apparentdisparity is probably caused by counting cells grown on FOAplates. To form a colony on an FOA plate, the cells should keepa silent state for several divisions. However, dpb4 ${Delta}$ cells wereswitching their epigenetic states at a high frequency (Table3) and consequently hardly keeping a silent state.

The frequent switching between alternative epigenetic statesalso suggest that the solid-pink colony of dpb4 ${Delta}$ cells in thecolony color assay (Fig. 2) is a mixture of silent and expressedcells. Furthermore, two daughter cells that originated fromthe same mother cell and individually showed a silent and anexpressed state appeared (alternative switching [Table 3]),suggesting that the telomere states switch either in the daughtercells before budding or in the mother cell at or after DNA replication.

Pol ${varepsilon}$ and ISW2/yCHRAC share Dpb4. The epistasis of dpb4 ${Delta}$ to dpb3 ${Delta}$ , together with different switchingpatterns in these mutants, suggested that Dpb4 is shared byPol ${varepsilon}$ and an unknown complex that play counteracting roles forTPE. We therefore attempted to purify protein complexes containingDpb4. For this purpose, we immunoprecipitated Dpb4 using cellextracts bearing 5Flag-tagged Dpb4 and an anti-Flag antibody.Dpb4-5Flag was eluted with 3x Flag peptide, and the eluateswere separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Following silver staining, we observedseven distinct polypeptides (Fig. 4A, lanes 1 and 2). Westernblot analysis with anti-Pol ${varepsilon}$ , anti-Dpb3, and anti-Flag antibodiesidentified Pol2, Dpb2, Dpb4-5Flag, and Dpb3 as p250, p80, p35,and p34, respectively (Fig. 4A, lanes 3 to 8). The remainingthree polypeptides were identified by peptide mass fingerprintinganalysis as Itc1 for p160, Isw2 for p130, and the product encodedby novel gene YJL065c for p20. These results were further confirmedby the introduction of a 13myc tag to Itc1 and Isw2 and a 5Flagtag to YJL065c, which resulted in a shifting up of the p160,p130, and p20 bands (Fig. 4B, lanes 2 to 4 and 8). Isw2 (59)is an ATPase subunit of the ISW2 ATP-dependent chromatin remodelingcomplex that is highly related to Drosophila ISWI (34). Itc1(19) is also a subunit of the ISW2 chromatin-remodeling complexthat contains an N-terminal WAC (WSTF/Acf1/cbp146) motif (6)that may be responsible for the functions of the ISWI complexes.YJL065c encodes a histone fold motif-containing polypeptidethat has 31% identity (49% similarity) to Dpb3 over 151 aminoacids (data not shown), which we named DLS1 (Dpb3-like subunitof ISW2/yCHRAC complex). To determine whether these polypeptidesform a complex, we performed gel filtration column chromatographyanalysis of the immunoprecipitates. The Pol ${varepsilon}$ complex elutedin fractions 15 to 17 at a relative molecular mass of 670 kDa(Fig. 4C). On the other hand, Itc1, Isw2, Dpb4-5Flag, and Dls1coeluted in fractions 11 to 13, encompassing the relative molecularmasses of 1,000 to 670 kDa, suggesting that these four proteinsform a complex distinct from Pol ${varepsilon}$ . These complexes were alsoisolated separately when we employed Dpb3-5Flag or Dls1-5Flaginstead of Dpb4-5Flag (Fig. 4B, lanes 6 and 8). Similar resultswere obtained by the immunoprecipitation of Dpb4-5Flag fromDPB3- or DLS1-deleted cell extracts (Fig. 4D, lanes 3 and 4).These results thus suggest that Dpb4 binds to either Pol2/Dpb2or Isw2/Itc1 through its partnered histone motif protein, Dpb3or Dls1. Since the composition of this novel complex (ISWI homolog,a WAC motif protein, and two histone fold motif proteins) issimilar to that of chromatin accessibility complex (CHRAC) (60),a conserved chromatin-remodeling complex in higher eukaryotes,we named this complex yeast CHRAC-like complex (yCHRAC). Meanwhile,recent characterization of the ISW2 complex revealed that ISW2exists mainly as a four-subunit complex (A. D. McConnel andT. Tsukiyama, personal communication). Thus, we designate thiscomplex ISW2/yCHRAC. Interestingly, human Pol ${varepsilon}$ and CHRAC alsoshare a common subunit, p17 (38, 46), suggesting that the relationshipbetween Pol ${varepsilon}$ and the CHRAC-like complex (four-subunit ISW2 complex)is evolutionarily conserved.

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FIG. 4. Dpb4 is involved in two distinct complexes. (A) Anti-Flag immunoprecipitations were performed on protein extracts prepared from cells of YTI189 (wild-type allele, -) and YTI367 (5Flag-tagged allele, +). The immunoprecipitated proteins were separated by SDS-PAGE (4 to 20% acrylamide) and visualized with silver stain (left panel). The immunoprecipitates were probed with anti-Pol ${varepsilon}$ ( ${alpha}$ -Pol ${varepsilon}$ ), anti-Dpb3 ( ${alpha}$ -Dpb3), or anti-Flag ( ${alpha}$ -Flag) antibodies (right panels). (B) Anti-Flag immunoprecipitates from the cell extracts were separated by SDS-PAGE (4 to 20% acrylamide) and visualized with silver stain. The extracts were prepared from the indicated cells. In the left panel, a plus indicates a 5Flag-tagged gene and a minus indicates a wild-type allele. Strains used were YTI189 (no tag), YTI367 (DPB4-5FLAG), YTI460 (DPB4-5FLAG ITC1-13MYC), YTI461 (DPB4-5FLAG ISW2-13MYC), YTI365 (DPB3-5FLAG), and YTI459 (DLS1-5FLAG). (C) Silver staining of Superose 6 fractions (30 µl) separated by SDS-PAGE (5 to 20% acrylamide). The arrows indicate the positions of molecular weight markers, thyroglobulin (670 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), and RNase A (13 kDa). (D) Anti-Flag immunoprecipitations were performed on extracts prepared from cells of the indicated genotypes. +, epitope-tagged allele; -, wild-type deletion allele; wt, wild-type allele. The strains used were YTI189 (no tag), YTI367 (DPB4-5FLAG), YTI369 (DPB4-5LAG dpb3 ${Delta}$ ), and YTI458 (DPB4-5FLAG dls1 ${Delta}$ ). The protein band corresponding to Itc1 often smeared, probably because of modifications, as mentioned previously (21).

ISW2/yCHRAC counteracts Pol ${varepsilon}$ for TPE. Since Dpb4 is shared by Pol ${varepsilon}$ and ISW2/yCHRAC, dpb4 ${Delta}$ cells lackboth complexes, while dpb3 ${Delta}$ cells lack Pol ${varepsilon}$ but not ISW2/yCHRAC.We thus expected that ISW2/yCHRAC would counteract Pol ${varepsilon}$ forTPE by the reasons already described. As expected, the itc1 ${Delta}$ and dls1 ${Delta}$ mutations enhanced telomere silencing and restoredit in dpb3 ${Delta}$ cells to the level in dpb4 ${Delta}$ cells, whereas they didnot affect it in dpb4 ${Delta}$ cells (Fig. 5A). On the other hand, theisw2 ${Delta}$ mutation slightly enhanced the telomere-silencing defectin dpb4 ${Delta}$ mutant cells, suggesting that the Isw2 protein solely,not as a complex, or a putative complex of Isw2 with the otherWAC domain protein (6), such as a product from YPL216w, playsa putative positive role in TPE. Moreover, in contrast to thedpb3 ${Delta}$ mutation, the dls1 ${Delta}$ mutation increased the switching ratefrom on to off but did not affect that from off to on (Table3). As observed in the dpb3 ${Delta}$ and dpb4 ${Delta}$ mutations (Fig. 1B), thedls1 ${Delta}$ mutation did not affect the expression level of internalURA3 (Fig. 5B). Consistently, in the colony color assay, dls1 ${Delta}$ cells formed colonies with solid-red color while dpb3 ${Delta}$ dls1 ${Delta}$ formedcolonies with solid-pink color, which suggests that dpb3 ${Delta}$ dls1 ${Delta}$ cells switch between alternative states at as a high frequencyas dpb4 ${Delta}$ cells do (Fig. 2A). These results strongly suggest thatPol ${varepsilon}$ and ISW2/yCHRAC independently regulate epigenetic inheritanceof silent and expressed states of TPE, respectively.

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FIG. 5. ISW2/yCHRAC counteracts Pol ${varepsilon}$ for TPE. The numbers under the photographs indicate the estimated number of cells placed on a spot. (A) Expression of the telomere-linked URA3 gene (VIIL::URA3-TEL) in the isogenic strains YTI249 (WT), YTI456 (itc1 ${Delta}$ ), YTI452 (isw2 ${Delta}$ ), YTI442 (dls1 ${Delta}$ ), YTI266 (dpb4 ${Delta}$ ), YTI458 (dpb4 ${Delta}$ itc1 ${Delta}$ ), YTI454 (dpb4 ${Delta}$ isw2 ${Delta}$ ), YTI444 (dpb4 ${Delta}$ dls1 ${Delta}$ ), YTI250 (dpb3 ${Delta}$ ), YTI457 (dpb3 ${Delta}$ itc1 ${Delta}$ ), YTI453 (dpb3 ${Delta}$ isw2 ${Delta}$ ), YTI443 (dpb3 ${Delta}$ dls1 ${Delta}$ ), and YTI268 (dpb3 ${Delta}$ dpb4 ${Delta}$ ) was assayed as described in the legend to Fig. 1. ITC1, ISW2, and DLS1 were deleted using TRP1, which makes cells grow slightly faster. Apparent weak suppression of dpb4 ${Delta}$ by itc1 ${Delta}$ and dls1 ${Delta}$ is caused by TRP1-marker effect. (B) Expression of URA3 at the internal chromosomal locus in dls1 ${Delta}$ mutant cells. The isogenic strains YTI279 (WT ura3), YTI473 (dls1 ${Delta}$ ura3), YTI297 (WT URA3), YTI470 (dls1 ${Delta}$ URA3), and YTI249 (WT URA3-TEL) were examined as described for panel A.

Chromatin structure in mutant cells. To form heterochromatin-like structure at telomeres, the Sircomplex associates with telomeres (1). We therefore examinedassociation of the Sir complex with the telomere in mutant cellsusing the ChIP assay. For this purpose, we replaced one of thegenes encoding a subunit of the Sir complex, the SIR3 gene,by the SIR3-13MYC allele. Although the resultant cells bearingSIR3-13MYC showed slightly reduced telomere silencing, a similareffect of dpb3 ${Delta}$ , dpb4 ${Delta}$ , and dls1 ${Delta}$ on TPE was observed (Fig. 6A).We also observed similar levels of the Sir3-13myc protein indpb3 ${Delta}$ , dpb4 ${Delta}$ , dls1 ${Delta}$ , and wild-type cells (Fig. 6B). Then, we performedthe ChIP assay. As shown in Fig. 6D and E, the Sir3 proteinassociated with telomeric DNA, while its association is slightlyreduced in dpb3 ${Delta}$ cells and is enhanced in dls1 ${Delta}$ cells. This isconsistent with populations of on and off cells in these mutant-cellcultures (Table 3). Thus, this suggests that alteration of heterochromatin-likestructure at the telomere in these mutant cells confers thedefect of TPE. Moreover, the telomere length of dpb3 ${Delta}$ and dpb4 ${Delta}$ cells is almost the same as that of the wild-type cells (43),and we further found that wild-type, dpb3 ${Delta}$ , dpb4 ${Delta}$ , and dls1 ${Delta}$ cellshave almost the same telomere length (Fig. 6F). Thus, thesemutations reduce or enhance the telomere silencing, irrespectiveof the telomere length. Therefore, it is suggested that Pol ${varepsilon}$ functions for TPE through heterochromatin structure.

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FIG. 6. Association of Sir3 with telomeric URA3 in dpb3 ${Delta}$ , dpb4 ${Delta}$ , or dls1 ${Delta}$ mutant cells. (A) Silencing of the telomeric URA3 gene in strains with or without 13MYC epitope-tag allele of SIR3. The isogenic strains YTI312 (no tag, WT), YTI446 (SIR3-13MYC, WT), YTI464 (SIR3-13MYC, dpb3 ${Delta}$ ), YTI465 (SIR3-13MYC, dpb4 ${Delta}$ ), YTI466 (SIR3-13MYC, dls1 ${Delta}$ ), and YTI467 (SIR3-13MYC, sir2 ${Delta}$ ) were examined as described for Fig. 1 and 2. The numbers under the photographs indicate the estimated number of cells placed on a spot. (B) Steady-state protein levels of Sir3-13myc and Sir2 in the strains were determined by Western blot analysis using anti-myc antibodies (MBL) and anti-Sir2 antibodies (Santa Cruz). (C) The PCR primer set to amplify either telomeric URA3 or endogenous ura3 (ura3 ${Delta}$ rvs) loci is shown. (D) Sir3 associations with the telomeric URA3 and endogenous ura3 ${Delta}$ rvs loci (IP, lanes 7 to 12) were examined by ChIP assay using anti-c-myc antibodies. DNA from whole-cell extract was amplified as a control (WCE, lanes 1 to 6). The ChIP assay was performed on WT, dpb3 ${Delta}$ , dpb4 ${Delta}$ , dls1 ${Delta}$ , and sir2 ${Delta}$ cells (see panel C). (E) Relative amounts of URA3-TEL DNA in IP samples were quantified and normalized to input DNA, which were amplified from the same ChIPs in each strain (see panel D). (F) Telomere length in the isogenic strains YTI249 (WT), YTI250 (dpb3 ${Delta}$ ), YTI266 (dpb4 ${Delta}$ ), and YTI442 (dls1 ${Delta}$ ). Yeast genomic DNA from each of the strains was digested with XhoI, separated on a 1.0% agarose gel, transferred to a nitrocellulose membrane, and hybridized with poly-TG and poly-CA probes that hybridize to telomere repeat sequences.

DISCUSSION

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Discussion

We suggest in this paper that Pol ${varepsilon}$ and ISW2/yCHRAC are involvedin TPE because mutants defective in subunits of Pol ${varepsilon}$ and ISW2/yCHRACshowed defect of TPE. Using the single-cell assay, we furtherfound that Pol ${varepsilon}$ and ISW2/yCHRAC counteract for TPE noncompetitively.This finding gives us a novel view of TPE as described below.To explain the stochastic nature of switching epigenetic states,competition of the assembly of a silent chromatin and the transactivatorhas been proposed (2). This proposal predicts that when theswitching rate for one direction is increased by a mutation,the other decreases and vice versa. This is not the case forPol ${varepsilon}$ and ISW2/yCHRAC, because Pol ${varepsilon}$ and ISW2/yCHRAC functionindependently: Pol ${varepsilon}$ operates for stable inheritance of a silentstate, while ISW2/yCHRAC works for that of an expressed state(Table 3). We therefore propose that Pol ${varepsilon}$ and ISW2/yCHRAC donot regulate the assembly of a silent chromatin and the transactivatorin a competitive way, as previously proposed (2), but rathermaintain or inherit proper configuration of the chromatin duringcell division, probably during DNA replication. Two lines ofevidence further support this idea. First, one of the replicationproteins, PCNA, is suggested to be involved in the assemblyof a silent chromatin (establishment) through CAF-1 (63). However,the roles of Pol ${varepsilon}$ and ISW2/yCHRAC seem to be distinct from thatof CAF-I-PCNA (Table 2). Second, the dpb3 ${Delta}$ , dpb4 ${Delta}$ , and dls1 ${Delta}$ cellsretain their silencing ability because mutations of Pol ${varepsilon}$ andISW2/yCHRAC do not decrease but increase the switching rates(Table 3) and Sir3 is associated near the telomere in the ChIPassay in these mutants (Fig. 6). Thus, Pol ${varepsilon}$ and ISW2/yCHRACseem not to affect the establishment of silencing. Note thatthe altered levels of association of Sir3 near telomere mightreflect the maintenance or inheritance but not the establishment(assembly) of a silent chromatin in these mutant cells (Fig.6D and E). This is further supported by the fact that high-copy-numberSIR3 did not enhance the telomere silencing in dpb3 ${Delta}$ and dpb4 ${Delta}$ cells (Fig. 2B), because the increased dosage of Sir3 enhancesthe telomere silencing if the assembly of a silent chromatinis defective (48).

So far, we do not know whether Pol ${varepsilon}$ and ISW2/yCHRAC functionfor TPE directly or indirectly. However, we prefer the modelof their direct involvement in maintenance or inheritance ofchromatin structures because they do not influence the eventsthat have been thought to affect TPE so far, telomere lengthand expression of Sir proteins (Fig. 6B and F). This is furtherstrengthened by the recent observation that Dls1 as well asDpb3 associates with the telomeric region in a chromosome-wideChIP assay of yeast chromosome VI (K. Shirahige, personal communication).In recent studies, both human Pol ${varepsilon}$ (18) and ACF1-SNF2H (11),a human counterpart of Itc1-Isw2, were shown to be localizedto late-replicating chromatin such as heterochromatin, and ACF1-SNF2His required for efficient DNA replication in the heterochromatinregion. We therefore suggest that Pol ${varepsilon}$ and the CHRAC-like complex(four-subunit ISW2 complex) in eukaryotic cells regulate duplicationof the heterochromatin configuration that includes epigeneticinformation during or after DNA replication by their counteractions.

Since the small subunits of Pol ${varepsilon}$ are dispensable for the cellgrowth, their functions had not been well elucidated. The findingof their involvement in TPE suggests that Pol ${varepsilon}$ participatesin not only chromosomal DNA replication but also duplicationof chromatin structure. PCNA is also known to be involved inthe maintenance of silencing as well as DNA replication, incooperation with CAF-1 (63). Therefore, several replicationproteins at replication forks seem to be involved in duplicationof chromatin structure. Interestingly, Pol ${varepsilon}$ and ISW2/yCHRACshare a subunit, Dpb4. Since Pol ${varepsilon}$ and ISW2/yCHRAC are counteractingfor TPE, it is likely that the ratio between Pol ${varepsilon}$ and ISW2/yCHRACis kept constant, and consequently the silencing level of telomereis maintained constant.

Isw2 and Itc1 were shown to participate in repression of earlymeiotic genes and INO1 in mitotic cell cycle, which is mediatedby the interaction with Ume6 (19, 21, 57) and PHO3, probablymediated by the interaction with a transcriptional regulator(s)other than Ume6 (31). Recently, it was also reported that Isw2and Itc1 are required for full repression of a-type specificgenes in ${alpha}$ haploid and a/ ${alpha}$ diploid cells and this repression maybe mediated by an a-type specific transcriptional regulator(s)(49) (it does not affect the single-cell telomere silencingassay). In the case of TPE, ISW2/yCHRAC works for derepressionof URA3, ADE2, and ${alpha}$ 2, suggesting that it works irrespectiveof transcription regulators. Thus, it is likely that in TPE,ISW2/yCHRAC is directly involved in remodeling the chromatinstructure, as shown in the association of Sir3 (Fig. 6D and E).

dpb3 ${Delta}$ cells displayed a partial defect in HMR silencing (ourunpublished result, which does not affect the single-cell telomereassay [Table 3]) that also requires the Sir complex, and a previousstudy (53) reported that the mutation in DPB3 also caused adefect in rDNA silencing, which requires a distinct silencingcomplex, the RENT complex (55). Thus, Pol ${varepsilon}$ seems to maintainthe configuration of common components of the heterochromatin-likestructure, such as histones. Actually, YBL1 and YBL1-YCL1 (7),which are mouse counterparts of Dpb4 and Dpb4-Dls1, directlyinteract with histones in vitro. Therefore, it is conceivablethat Pol ${varepsilon}$ and ISW2/yCHRAC directly regulate chromatin configurationthrough the interaction between histones and their histone foldsubunits, Dpb4, Dpb3, and Dls1. From this viewpoint, althoughwe could not coimmunoprecipitate them (our unpublished results),Pol ${varepsilon}$ and ISW2/yCHRAC may interact and respectively maintainhypoacetylated and hyperacetylated histones on chromatin andconsequently counteract for the TPE.

ACKNOWLEDGMENTS

We thank J. Berman, S. Enomoto, D. E. Gottschling, D. Moazed,B. Stillman, and A. Sugino for providing various strains, plasmids,and antibodies; K. Shirahige and T. Tsukiyama for providinginformation before publication; and Y. Kamimura, S. Tanaka,and T. Tsukiyama for critical reading of the manuscript.

This study was partially supported by a grant-in-aid from theMinistry of Education, Culture, Sports, Science, and Technology,Japan.

FOOTNOTES

* Corresponding author. Mailing address: Division of Microbial Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan. Phone: (81) 55 981 6754. Fax: (81) 55 981 6762. E-mail: hiaraki{at}lab.nig.ac.jp.

Present address: Center for Developmental Biology, RIKEN, Kobe650-0047, Japan.

REFERENCES

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