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Molecular and Cellular Biology, January 2004, p. 217-227, Vol. 24, No. 1
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.1.217-227.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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

Tetsushi Iida^1, and Hiroyuki Araki^1*

Division of Microbial Genetics, National Institute of Genetics, and Department of Genetics, SOKENDAI, Mishima, Shizuoka 411-8540, Japan¹

Received 2 July 2003/ Returned for modification 12 August 2003/ Accepted 1 October 2003

	ABSTRACT

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Relocation of euchromatic genes near the heterochromatin region often results in mosaic gene silencing. In Saccharomyces cerevisiae, cells with the genes inserted at telomeric heterochromatin-like regions show a phenotypic variegation known as the telomere-position effect, and the epigenetic states are stably passed on to following generations. Here we show that the epigenetic states of the telomere gene are not stably inherited in cells either bearing a mutation in a catalytic subunit (Pol2) of replicative DNA polymerase (Pol ) or lacking one of the nonessential and histone fold motif-containing subunits of Pol , Dpb3 and Dpb4. We also report 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 demonstrate that without Pol and ISW2/yCHRAC, the epigenetic states of the telomere are frequently switched. Furthermore, we reveal that Pol and ISW2/yCHRAC function independently: Pol operates for the stable inheritance of a silent state, while ISW2/yCHRAC works for that of an expressed state. We therefore propose that inheritance of specific epigenetic states of a telomere requires at least two counteracting regulators.

	INTRODUCTION

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The eukaryotic genome is packaged into either euchromatin or heterochromatin. Heterochromatin is defined cytologically as the fraction of the genome that is constitutively condensed throughout the cell cycle (25) and corresponds to regions of highly repeated DNA such as centromeres or telomeres (13). In this region, DNA-mediated metabolisms, such as transcription, recombination, and replication, are generally inactive. In the case of chromosomal DNA replication, heterochromatin replicates in late S phase, while euchromatic regions replicate in early S phase (27).

In a simple eukaryote, Saccharomyces cerevisiae, although its genome 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 and telomeric regions are replicated in late S phase (17, 47), and these silenced regions are associated with histone hypoacetylation (5, 8, 23, 39). The silencing process has three phases, establishment or assembly of a silent chromatin, its maintenance through the cell cycle, and its inheritance to daughter cells, as described by Lau et al. (35). Factors that are involved in these phases of silencing are identified from studies of mutant strains that lose silencing. Mutations in SIR2, SIR3, and SIR4 eliminate the silencing at HM loci, whereas mutations in SIR1 partially reduce it, because Sir1 is required only for establishment, whereas Sir2, Sir3, and Sir4 are required for both establishment and maintenance (45). Sir2, Sir3, and Sir4 form a Sir complex at HM loci (40). The Sir complex does not directly bind DNA but rather interacts with DNA-binding factors, including Rap1, Orc, and Abf1, that bind to silencer DNA elements sandwiching HM loci (28). rDNA encoding 35S rRNA consists of 100 to 200 times the number of tandem repeats in a 9-kb unit on chromosome XII and is localized in the nucleolus. In rDNA silencing, the RENT complex, which is distinct from the Sir complex and which consists of Sir2, Net1, and Cdc14, represses homologous recombination as well as transcription of transgenes in the rDNA repeats (50, 55).

When a wild-type gene is located near a telomere in budding yeast, it is subjected to telomere position effect (TPE) variegation, which provides heritable silent and expressed states and reversible switching between the epigenetic states (22). The silent state of the telomeric gene is attributable to a heterochromatin-like structure that consists of several proteins, such as Sir proteins and hypoacetylated histones, that spread from the telomeric ends (1, 23). The Sir complex but not Sir1 interacts with tandemly reiterated Rap1 molecules bound to the telomere repeat sequence and spreads into subtelomeric DNA to form a silent heterochromatin-like structure. Spreading of Sir proteins into the HM loci and subtelomeric DNA is probably facilitated by the interaction of Sir3/Sir4 with hypoacetylated histones H3/H4 and Sir2, a NAD-dependent histone deacetylase (14, 23, 24, 54), and blocked by Dot1-catalyzed methylation of histone H3 (33, 36, 42).

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

One of the replicative polymerases, DNA polymerase (Pol ) (56), is composed of a catalytic-subunit Pol2, Dpb2, Dpb3, and Dpb4 in S. cerevisiae, and the subunit composition of Pol is evolutionarily conserved in eukaryotes (37, 38). Although Pol2 and Dpb2 are essential for DNA replication (56), the histone fold motif-containing subunits, Dpb3 (3) and Dpb4 (44), are dispensable and their function remains unclear. A counterpart of Dpb4, p17 in human cells, was recently reported to be a subunit of not only Pol but also CHRAC (chromatin accessibility complex) (38, 46). CHRAC consisting of ISWI, Acf1, and two small subunits was purified from Drosophila and human cells and shown to catalyze chromatin remodeling (12, 46). In budding yeast, a similar complex was reported to catalyze chromatin remodeling although small subunits had not been detected (19, 30, 31, 59).

Because several histone fold motif-containing proteins have been reported to interact with histones (10), we examined whether these nonessential subunits may play a role in chromatin configuration. Using the single-cell method, we found that deletions of Dpb3 and Dpb4 confer defects of TPE in different manners. This is because Dpb4 is shared by Pol and the ISW2/CHRAC complex, a putative chromatin remodeling factor counteracting Pol for TPE. From these observations, we propose a model to maintain chromatin structure when chromosomal DNA is replicated.

	MATERIALS AND METHODS

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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 that 100 mg of adenine/liter was added but leucine was not.

Assay for silencing of telomeric URA3 and ADE2. To assay telomeric silencing, the strains with telomeric URA3 and ADE2 at the telomeres of the left arm of chromosome VII and the right arm of chromosome V, respectively, were used (strains listed in Table 1). All the strains harbor YCplac111 (LEU2), because cells used in this study (W303 background) show slightly slow growth on synthetic medium. To assay silencing of telomeric URA3, freshly grown yeast cells were taken from SC medium and diluted to a concentration of 2 x 10⁷ cells/ml. Then, 10-fold serial dilutions (2 x 10⁷ to 2 x 10² cells/ml) were made, and 5-µl cell aliquots from each of the dilutions were spotted onto 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 pictures were taken. To assay silencing of telomeric ADE2, about 100 yeast cells of each strain from SC medium were spread onto SC plates. The plates were incubated at 25°C for 10 days, and pictures were taken.

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

Immunoprecipitation. Immunoprecipitation was performed according to the methods described by Zachariae et al. (62) and Takayama et al. (58) by using an anti-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 incubation with 100 µg of 3x Flag peptides (Sigma) per ml. Superose 6 column (Amersham Biotech) chromatography of the eluates was performed in the lysis buffer.

ChIP assay. Chromatin immunoprecipitation (ChIP) analysis was performed essentially as described in reference 29. DNA purified from whole-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. Quantification was carried out with a LumiVision Analyzer instrument (AISIN).

	RESULTS

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Pol is required for TPE. To determine whether Pol is involved in the maintenance of chromatin structure, we first investigated whether the deletions of DPB3 and DPB4 influence silencing of the URA3 gene integrated near the telomere of chromosome VII (22) (Fig. 1A). Expression of URA3 leads to cell growth on medium lacking uracil as well as to cell death on medium containing 5-FOA. In this assay, dpb3 and dpb4 mutant cells displayed partial expression of URA3, and this expression was more evident in dpb3 cells, while the expression of URA3 at the original locus was not affected (Fig. 1B, SC 5FOA). Moreover, the expression level in the dpb3 dpb4 double mutant was similar to that in dpb4 (Fig. 1B), indicating that the dpb4 mutation is epistatic to the dpb3 mutation. Furthermore, the pol2-11 mutation (9, 41) conferred partial expression of telomeric URA3 and this defect was suppressed by simultaneous introduction of high-copy-number DPB3 and DPB4 (Fig. 1C). Since Dpb3 and Dpb4 form a subassembly in vitro (15) and neither one of them suppressed the pol2-11 mutation (Fig. 1C), Dpb3 and Dpb4 seem to interact with Pol2 as a complex in vivo. This result further suggests that Dpb3, Dpb4, and Pol2 function for TPE as Pol .

View larger version (66K): [in this window] [in a new window]   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), YTI266 (dpb4), YTI268 (dpb3 dpb4) 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 ura3), YTI472 (dpb4 ura3), YTI297 (WT URA3), YTI468 (dpb3 URA3), and YTI469 (dpb4 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.

View larger version (43K): [in this window] [in a new window]   FIG. 2. Defects of TPE variegation in dpb3, dpb4, and dls1 mutations. (A) A colony color assay for cells carrying telomere-linked ADE2 (VR::ADE2-TEL). Freshly grown yeast cells of YTI249 (WT), YTI250 (dpb3), YTI266 (dpb4), YTI442 (dls1), and YTI443 (dpb3 dls1) 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 and dpb4 cells. Freshly grown yeast cells of YTI249 (WT), YTI250 (dpb3), and YTI266 (dpb4) 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.

View this table: [in this window] [in a new window]   TABLE 2. Effect of dpb3 and dpb4 mutations with cac1 on telomere silencinga

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

View larger version (13K): [in this window] [in a new window]   FIG. 3. Single-cell assay of TPE. (A) Schematic diagram of telomeric ADE2 and 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 2 gene (VR::2-TEL) in the YTI448 (2-TEL WT) strain and the isogenic control YTI280 (WT) strain without 2-TEL. Cells were exposed to -factor on YPDA plates at 30°C for 5 h. Pear-shaped cells, i.e., shmoos (cells with 2 unexpressed), and dividing cells (cells with 2 expressed) in each strain were magnified. The arrows indicate shmoo cells. (C) Pedigree assays of cells from strain YTI448 (WT) with 2 expressed (on) or silent (off). The procedure is described in detail in Materials and Methods.

View this table: [in this window] [in a new window]   TABLE 3. Effect of dpb3, dpb4, and dls1 mutations on frequency of switching between silent and expressed states of telomere-linked 2a

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

Pol and ISW2/yCHRAC share Dpb4. The epistasis of dpb4 to dpb3, together with different switching patterns in these mutants, suggested that Dpb4 is shared by Pol and an unknown complex that play counteracting roles for TPE. We therefore attempted to purify protein complexes containing Dpb4. For this purpose, we immunoprecipitated Dpb4 using cell extracts bearing 5Flag-tagged Dpb4 and an anti-Flag antibody. Dpb4-5Flag was eluted with 3x Flag peptide, and the eluates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following silver staining, we observed seven distinct polypeptides (Fig. 4A, lanes 1 and 2). Western blot analysis with anti-Pol , anti-Dpb3, and anti-Flag antibodies identified Pol2, Dpb2, Dpb4-5Flag, and Dpb3 as p250, p80, p35, and p34, respectively (Fig. 4A, lanes 3 to 8). The remaining three polypeptides were identified by peptide mass fingerprinting analysis as Itc1 for p160, Isw2 for p130, and the product encoded by novel gene YJL065c for p20. These results were further confirmed by the introduction of a 13myc tag to Itc1 and Isw2 and a 5Flag tag 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 remodeling complex that is highly related to Drosophila ISWI (34). Itc1 (19) is also a subunit of the ISW2 chromatin-remodeling complex that 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 polypeptide that has 31% identity (49% similarity) to Dpb3 over 151 amino acids (data not shown), which we named DLS1 (Dpb3-like subunit of ISW2/yCHRAC complex). To determine whether these polypeptides form a complex, we performed gel filtration column chromatography analysis of the immunoprecipitates. The Pol complex eluted in fractions 15 to 17 at a relative molecular mass of 670 kDa (Fig. 4C). On the other hand, Itc1, Isw2, Dpb4-5Flag, and Dls1 coeluted in fractions 11 to 13, encompassing the relative molecular masses of 1,000 to 670 kDa, suggesting that these four proteins form a complex distinct from Pol . These complexes were also isolated separately when we employed Dpb3-5Flag or Dls1-5Flag instead of Dpb4-5Flag (Fig. 4B, lanes 6 and 8). Similar results were obtained by the immunoprecipitation of Dpb4-5Flag from DPB3- or DLS1-deleted cell extracts (Fig. 4D, lanes 3 and 4). These results thus suggest that Dpb4 binds to either Pol2/Dpb2 or Isw2/Itc1 through its partnered histone motif protein, Dpb3 or Dls1. Since the composition of this novel complex (ISWI homolog, a WAC motif protein, and two histone fold motif proteins) is similar 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 ISW2 exists mainly as a four-subunit complex (A. D. McConnel and T. Tsukiyama, personal communication). Thus, we designate this complex ISW2/yCHRAC. Interestingly, human Pol and CHRAC also share a common subunit, p17 (38, 46), suggesting that the relationship between Pol and the CHRAC-like complex (four-subunit ISW2 complex) is evolutionarily conserved.

View larger version (75K): [in this window] [in a new window]   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 (-Pol ), anti-Dpb3 (-Dpb3), or anti-Flag (-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), and YTI458 (DPB4-5FLAG dls1). The protein band corresponding to Itc1 often smeared, probably because of modifications, as mentioned previously (21).

ISW2/yCHRAC counteracts Pol for TPE.

View larger version (91K): [in this window] [in a new window]   FIG. 5. ISW2/yCHRAC counteracts Pol 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), YTI452 (isw2), YTI442 (dls1), YTI266 (dpb4), YTI458 (dpb4 itc1), YTI454 (dpb4 isw2), YTI444 (dpb4 dls1), YTI250 (dpb3), YTI457 (dpb3 itc1), YTI453 (dpb3 isw2), YTI443 (dpb3 dls1), and YTI268 (dpb3 dpb4) 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 by itc1 and dls1 is caused by TRP1-marker effect. (B) Expression of URA3 at the internal chromosomal locus in dls1 mutant cells. The isogenic strains YTI279 (WT ura3), YTI473 (dls1 ura3), YTI297 (WT URA3), YTI470 (dls1 URA3), and YTI249 (WT URA3-TEL) were examined as described for panel A.

View larger version (53K): [in this window] [in a new window]   FIG. 6. Association of Sir3 with telomeric URA3 in dpb3, dpb4, or dls1 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), YTI465 (SIR3-13MYC, dpb4), YTI466 (SIR3-13MYC, dls1), and YTI467 (SIR3-13MYC, sir2) 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 (ura3rvs) loci is shown. (D) Sir3 associations with the telomeric URA3 and endogenous ura3rvs 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, dpb4, dls1, and sir2 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), YTI266 (dpb4), and YTI442 (dls1). 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

Top Abstract Introduction Materials and Methods Results Discussion References

So far, we do not know whether Pol and ISW2/yCHRAC function for TPE directly or indirectly. However, we prefer the model of their direct involvement in maintenance or inheritance of chromatin structures because they do not influence the events that have been thought to affect TPE so far, telomere length and expression of Sir proteins (Fig. 6B and F). This is further strengthened by the recent observation that Dls1 as well as Dpb3 associates with the telomeric region in a chromosome-wide ChIP assay of yeast chromosome VI (K. Shirahige, personal communication). In recent studies, both human Pol (18) and ACF1-SNF2H (11), a human counterpart of Itc1-Isw2, were shown to be localized to late-replicating chromatin such as heterochromatin, and ACF1-SNF2H is required for efficient DNA replication in the heterochromatin region. We therefore suggest that Pol and the CHRAC-like complex (four-subunit ISW2 complex) in eukaryotic cells regulate duplication of the heterochromatin configuration that includes epigenetic information during or after DNA replication by their counteractions.

Since the small subunits of Pol are dispensable for the cell growth, their functions had not been well elucidated. The finding of their involvement in TPE suggests that Pol participates in not only chromosomal DNA replication but also duplication of chromatin structure. PCNA is also known to be involved in the maintenance of silencing as well as DNA replication, in cooperation with CAF-1 (63). Therefore, several replication proteins at replication forks seem to be involved in duplication of chromatin structure. Interestingly, Pol and ISW2/yCHRAC share a subunit, Dpb4. Since Pol and ISW2/yCHRAC are counteracting for TPE, it is likely that the ratio between Pol and ISW2/yCHRAC is kept constant, and consequently the silencing level of telomere is maintained constant.

Isw2 and Itc1 were shown to participate in repression of early meiotic genes and INO1 in mitotic cell cycle, which is mediated by the interaction with Ume6 (19, 21, 57) and PHO3, probably mediated by the interaction with a transcriptional regulator(s) other than Ume6 (31). Recently, it was also reported that Isw2 and Itc1 are required for full repression of a-type specific genes in haploid and a/ diploid cells and this repression may be mediated by an a-type specific transcriptional regulator(s) (49) (it does not affect the single-cell telomere silencing assay). In the case of TPE, ISW2/yCHRAC works for derepression of URA3, ADE2, and 2, suggesting that it works irrespective of transcription regulators. Thus, it is likely that in TPE, ISW2/yCHRAC is directly involved in remodeling the chromatin structure, as shown in the association of Sir3 (Fig. 6D and E).

dpb3 cells displayed a partial defect in HMR silencing (our unpublished result, which does not affect the single-cell telomere assay [Table 3]) that also requires the Sir complex, and a previous study (53) reported that the mutation in DPB3 also caused a defect in rDNA silencing, which requires a distinct silencing complex, the RENT complex (55). Thus, Pol seems to maintain the configuration of common components of the heterochromatin-like structure, such as histones. Actually, YBL1 and YBL1-YCL1 (7), which are mouse counterparts of Dpb4 and Dpb4-Dls1, directly interact with histones in vitro. Therefore, it is conceivable that Pol and ISW2/yCHRAC directly regulate chromatin configuration through the interaction between histones and their histone fold subunits, Dpb4, Dpb3, and Dls1. From this viewpoint, although we could not coimmunoprecipitate them (our unpublished results), Pol and ISW2/yCHRAC may interact and respectively maintain hypoacetylated and hyperacetylated histones on chromatin and consequently 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 providing information 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 the Ministry 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, Kobe 650-0047, Japan.

	REFERENCES

Top Abstract Introduction Materials and Methods Results Discussion References

 
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