INTRODUCTION

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Two molecules each of the core histones H2A, H2B, H3, and H4 tightly associate with 146 bp of DNA to form a nucleosome, the fundamental repeat structure of chromatin (38; for a review, see reference 73). Numerous genetic and biochemical studies have shown that core histones also contribute to gene activation (6, 16) and repression (18, 35) and have direct roles in position-dependent gene silencing (for reviews, see references 12 and 37). The latter point has been most clearly demonstrated in the budding yeast Saccharomyces cerevisiae, in which genes adjacent to telomeres and the mating type genes present at the silent HM loci are subject to position-dependent transcriptional repression. Mutations within the N-terminal residues of histones H3 and H4 disrupt this gene silencing (22, 41, 49, 71). Furthermore, these same N-terminal regions make direct contact with SIR gene products required for silencing in S. cerevisiae (14). Thus, chromatin structures responsible for gene silencing include nucleosomes as important components.

It is presently unknown how many proteins mediate assembly of histones on DNA in vivo or how such factors contribute to gene silencing. One factor involved in these processes is a three-subunit protein complex termed chromatin assembly factor I (CAF-I). CAF-I was initially purified from human somatic cell nuclear extracts on the basis of its ability to assemble histone octamers during DNA replication in vitro (62, 66; for reviews, see references 26 and 32). CAF-I-like activities have also been detected in extracts of Drosophila and Xenopus embryos (25). Purification of yeast CAF-I led to identification of the genes encoding the three yeast CAF-I subunits, each of which is homologous to its human counterpart (28). These genes have been termed CAC1, CAC2, and CAC3 (for chromosome assembly complex). Deletion of any of the three CAC genes results in decreased telomeric gene silencing (9, 28). cac mutants display decreased stability of the transcriptionally silent state at both telomeres and the silent HML locus (8, 42). However, cac mutants have no known growth defects. These data indicate that yeast CAF-I contributes to position-dependent gene silencing but is not the only cellular factor responsible for chromatin assembly.

Loss of SIR2, SIR3, or SIR4 gene function in S. cerevisiae completely abolishes gene silencing at both HM loci and telomeres (1, 55). These genes encode proteins that are structural components of silenced heterochromatin (15, 67) and are not known to be involved in the assembly of histones on DNA. Indeed, mutants specifically defective in histone deposition have not been described. There could be several reasons for this. Formation of nucleosomes from newly synthesized histones during DNA replication is an essential process because progression through S phase of the cell cycle in the absence of histone H2B or H4 synthesis causes lethality (13, 30). However, if the process of chromatin formation were performed by multiple, partially redundant factors, recovery of mutations in this process would be difficult in standard genetic screens because of the weak phenotypes of single mutants. In this case, disruption of multiple factors would be required to observe strong phenotypes resulting from chromatin malformation or malfunction.

We describe here a novel phenotype of cac mutants: the residual gene silencing in these cells is sensitive to mutations in the HIR1, HIR2, and HIR3 genes and to changes in histone gene dosage. The three HIR-encoded proteins act to restrict transcription of three of the four histone gene pairs to the G₁/S phase transition of the yeast cell cycle and to regulate the HTA1-HTB1 locus in response to altered levels of the gene products of this locus, histones H2A and H2B (43, 48, 60, 65). The HIR gene products regulate the histone HTA1-HTB1 promoter through a negative cis-acting site, although the Hir proteins themselves do not appear to directly contact DNA. hir mutants display a Hir phenotype: HTA1-HTB1 transcription is not repressed (i) outside of the G₁/S phase; (ii) when cells are treated with hydroxyurea (HU), which inhibits DNA synthesis and causes accumulation of unassembled histone proteins; or (iii) when the HTA and HTB genes are present on high-copy-number plasmids. Although hir mutants had no defects in gene silencing when CAF-I was intact, we observed a synergistic loss of telomeric and HM gene silencing in cac hir double mutants. These data suggest that pathways responsible for formation of heterochromatin in the absence of CAF-I are easily perturbed by changes in histone levels or histone stoichiometry. Furthermore, we provide evidence that the Hir proteins also have a role in cooperating with CAF-I to ensure proper cell growth and viability.

	MATERIALS AND METHODS

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Plasmids. To make the cac3::LEU2 deletion allele, a HindIII-BamHI fragment of pJJ283 (23) containing the LEU2 gene was inserted into ClaI- and BglII-cut pPK96 (28) to generate pPK112. pPK112 was digested with ApaI and SacI prior to transformation.

Yeast strains. The genotypes of yeast strains used in this study are shown in Table 1. All strains used were derived from strain W303 (70) by transformation or by crosses with other strains in this background, with the exception of the spt21::HIS3 allele, which was backcrossed from strain GNX193-1B (44) to W303 strains six times prior to the construction of the strains used here. Previously described deletion alleles include the following: cac1::LEU2, cac2::TRP1, cac3::URA3, cac3::hisG-URA3-kan-hisG, and URA3-VIIL (28); hir1::HIS3 and hir3::HIS3 (53); sir1::LEU2 (21); (hht1-hhf1)::LEU2 (plasmid pUK192 [39]); (hht2-hhf2)::HIS3 (pUK431 [39]); hhf2::LEU2 (pPK21 [29]); (hta2-htb2)::TRP1 (pJH21 [16]); and hml::LEU2 (pJH455 [76]).

Genetic procedures and media. Standard procedures were used for genetic crosses and tetrad analysis. Standard yeast media used for crosses and for scoring genetic marker segregations were as described elsewhere (24). YPAD is yeast extract-peptone-dextrose (YPD) medium supplemented with adenine at 50 mg/liter. 5-Fluoro-orotic acid (FOA) was added in all cases to synthetic media at a concentration of 1 mg/ml.

Viability and growth assays. Viability tests were conducted by plating serial dilutions of cells growing exponentially at 30°C in YPD broth onto prewarmed YPD plates and incubating at 30 and 37°C. Samples (80 µl fixed in phosphate-buffered saline-3.7% formaldehyde) were also counted with a hemocytometer immediately before duplicate platings were performed to determine total cell numbers. Generation times were determined in YPD medium at 30 or 37°C by following the absorbance at 660 nm; for several cac hir double mutants, generation times were determined by counting cell numbers with a hemocytometer. Because the trp1-1 allele present in the W303 strain background causes slow growth at 16°C, we performed control experiments to show that cac hir double mutants had growth defects at this temperature that were independent of the status of the TRP1 gene (data not shown).

Telomeric silencing assays. To measure telomeric silencing, the URA3 gene inserted next to the chromosome VIIL telomere was used as previously described (28); originally described in reference 11). We found that equivalent results were obtained by using log-phase cells (A₆₀₀, ~0.6) from liquid medium or cells taken from plates and adjusted to an A₆₀₀ of ~0.6 (see, e.g., reference 57). To assess quantitatively the proportion of silenced cells in a population, cells were plated on synthetic medium with or without FOA (either complete or selective for plasmids for the data in Table 4), and the number of FOA-resistant colonies per viable cell plated was determined after 8 days of incubation at 30°C. Small FOA-resistant microcolonies formed by the cac strains were counted under a dissecting microscope. Colonies on synthetic complete (or selective)-medium plates were counted after 3 days to assess the number of viable plated cells. To correct for variations in the potency of the FOA in different batches of plates, the fraction of FOA-resistant cells was normalized to that of the wild-type strain for each repetition of the experiment. In experiments using strains carrying plasmids, duplicate transformants of each strain tested gave the same results. In some cases, visual, semiquantitative estimates of telomeric silencing were obtained by spotting 5-µl quantities of 10-fold serial dilutions of cells onto medium with or without FOA; however, data from these spot tests were not used to obtain the numerical values presented.

Quantitative mating assays. Quantitative assays were performed as described elsewhere (7), using strains 216 (MATa his1) and 217 (MAT his1) as the testers. Patch mating tests (see Fig. 3C) were performed by growing cell patches overnight on YPD medium at 30°C and then replica plating the patches either onto synthetic dextrose agar plates onto which had been spread a fresh culture of the appropriate mating tester or onto YPD plates as a control for cell growth. Patches were photographed after 2 days of growth at 30°C.

Dam methylase accessibility assays. These assays were performed essentially as described elsewhere (33), except that the enzyme DpnII (New England Biolabs) was used instead of MboI. In this case, DNA was first digested with HindIII and then reaction mixtures were adjusted with the buffer recommended by the manufacturer for digestion with DpnII.

	RESULTS

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The CAC2/HIR1 subfamily of WD repeat proteins. The CAC2 and CAC3 genes encode the smaller two subunits of yeast CAF-I (28). Cac2p and Cac3p contain WD repeat motifs found in many eukaryotic proteins (45). Cac3p (previously described in the literature as Msi1p [19, 28, 56]) is a member of the histone-binding p48 subfamily of WD proteins which includes the p48 subunit of human CAF-I, the yeast histone acetyltransferase-associated protein Hat2p, and the human histone acetyltransferase-associated protein p46 (28, 50, 74, 75). All members of the p48 subfamily share conserved residues outside the canonical WD repeat signature and are members of at least one multisubunit protein complex that either binds or modifies histone proteins (28, 50, 69, 72, 74, 75, 79). Cac2p is not a member of the p48 subfamily. However, examination of database entries showed that the yeast protein most similar to Cac2p is Hir1p, which contains WD motifs in its N-terminal half (34, 60). The yeast Cac2p and Hir1p proteins both have conserved human homologs, the CAF-I p60 subunit and the HIRA protein, respectively (27, 34). Comparison of CAC2, HIR1, and their human homologs showed that they encode a distinct subfamily of WD proteins (Fig. 1). Each of the seven WD repeats in these proteins includes conserved residues other than those that define a generic WD motif consensus, and each repeat contains distinctly conserved residues not shared by the other six repeats (Fig. 1). Furthermore, the N-to-C-terminal order of the WD repeats in these four proteins is conserved.

View larger version (66K): [in this window] [in a new window]   FIG. 1.   The CAC2/HIR1 subfamily of WD repeat proteins. The yeast CAC2 gene (28), the human CAF-I p60 gene (27), the human HIRA gene (34), and the yeast HIR1 gene (34, 60) were aligned by using the PILEUP program (Genetics Computer Group, Madison, Wis.). Each protein sequence is shown starting from the initiator methionine through the seven WD motifs depicted on each line. Amino acids identical in at least three proteins are shadowed in black; conservative changes are underlined. A WD motif consensus from a large number of proteins is illustrated between repeats 3 and 4 (45), with shadowed H's representing hydrophobic residues and DPGN representing a region that often includes those amino acids.

Growth defects in cac hir mutants. Loss of single CAC or HIR genes does not result in growth defects, nor does loss of multiple members within the same group (9, 28, 60) (Fig. 2). However, most cac hir double mutants grew somewhat more slowly than wild-type or single-mutant cells at 30°C, as reflected by decreased colony sizes on plates; this phenotype was exacerbated at low (16°C) (data not shown) and high (37°C) (Fig. 2A) temperatures, although there was no specific morphological defect (e.g., cell cycle arrest phenotype) associated with growth at 37°C. These colony phenotypes arose only when deletions of CAC and HIR genes were combined, and they were most pronounced in cac2 hir2 double mutants and in triple mutants with deletions of both HIR1 and HIR2, for which growth defects were noted at both 30 and 37°C (Fig. 2B). cac hir double-mutant cells also had longer generation times than cac or hir single-mutant cells in liquid YPD medium. For example, cac2 or hir2 single-mutant strains doubled every 90 to 100 min at both 30 and 37°C, whereas cac2 hir2 double mutants had a 125-min generation time at 30°C and a 195-min generation time at 37°C (Table 2). A cac2 hir1 hir2 triple mutant had an even longer generation time, doubling every 225 min at 30°C. Also, cac hir colonies were smaller after tetrad dissection, including those double mutants that do not have significantly decreased colony sizes or increased doubling times at 30°C in liquid culture (e.g., cac1 hir1 mutants [Fig. 2C]). In contrast, small tetrad colony phenotypes were never observed on deletion of multiple members of either the CAC or HIR gene family (data not shown).

View larger version (63K): [in this window] [in a new window]   View larger version (52K): [in this window] [in a new window]   View larger version (75K): [in this window] [in a new window]   FIG. 2.   Growth defects of cac hir mutants. Cultures were streaked onto YPD plates for distribution into single colonies, and the plates were incubated for 3 to 5 days. (A) cac hir strains grown at 37°C; (B) triple and quadruple cac hir deletion strains grown at 30 and 37°C. Strains used were W303 (HIR+ CAC+), W3031 (hir1), W3032 (hir2), W30312(FOA) (hir1 hir2), PKY035 (cac1), PKY031 (cac2), PKY034 (cac3), PKY102 (cac1 hir1), PKY103 (cac2 hir1), PKY132 (cac3 hir1), PKY136 (cac1 hir2 ), PKY137 (cac2 hir2), PKY103 (cac3 hir2), PKY110 (cac1 hir1 hir2), PKY111 (cac2 hir1 hir2), and PKY112 (cac1 cac2 hir1 hir2). (C) Slow growth of cac1 hir1 spores after germination. Strain PKY102 (MAT cac1::LEUZ hir1::HIS3) was mated to PKY090 (MATa URA3-VIIL). The resulting diploid was sporulated, and tetrads were dissected. The viable progeny, shown here after 3 days of growth on YPAD medium at 30°C, are of two sizes. Scoring of markers revealed that all of the smaller progeny were His+ Leu+, indicating that they were cac1 hir1 double mutants (data not shown). Note that the number of complete tetrads with a 3:1 ratio of large to small colonies is below the expected 2:3 frequency for tetratypes; this is presumably because of the proximity of CAC1 to the chromosome XVI centromere.

Synergistic effects of cac and hir mutations on telomeric gene silencing. To test whether the growth defects of cac hir double mutants were due to defective chromatin formation, we first analyzed telomeric chromatin structure and function. In S. cerevisiae, telomere-proximal genes are subject to position-dependent but gene-independent silencing, termed the telomeric position effect (TPE) (11). TPE can be quantitatively assayed by placing the URA3 gene adjacent to a telomere; the fraction of cells in a population that are resistant to the drug FOA, a metabolic poison for Ura⁺ cells, represents the level of silencing of the URA3 gene. In cac mutants, TPE is reduced but not abolished (9, 28). We quantitated the levels of TPE in cac mutants, hir mutants, and cac hir double mutants. hir1 mutants had no defects in TPE; in fact, there was a slight increase in the TPE in these strains (Fig. 3A). hir1 mutants were indistinguishable from hir2 or hir3 mutants in this assay (data not shown). In contrast, cac hir double mutants had TPE levels approximately 3 orders of magnitude below the levels observed for the partially derepressed cac mutants. These synergies were observed in multiple cac hir double-mutant combinations: deletion of cac1 or cac2 in combination with either hir1, hir2, or hir3 gave similar results, with the fraction of FOA-resistant colonies being in the range of 10⁴ to 10⁵ relative to wild-type cells (Fig. 3A and data not shown). cac3 mutants had a weaker TPE defect than cac1 or cac2 mutants (28), and cac3 hir double mutants were also slightly less derepressed than other cac hir combinations (data not shown).

View larger version (75K): [in this window] [in a new window]   FIG. 3.   Deletion of both CAC and HIR genes synergistically reduces silencing of a telomere-proximal URA3 gene, the ADE2 gene at HMR, and the a genes at the natural HML locus. (A) Quantitation of silencing in representative strains. PKY090 (CAC+), PKY106 (cac1), PKY117 (hir1), PKY361 (spt21), PKY329 (spt21 hir1), PKY360 (cac1 spt21), PKY302 (cac1 hir1), and JRY4470 (sir2) were used. Tenfold serial dilutions of each strain were spotted onto nonselective (YPAD) medium to observe the total number of cells and onto FOA medium to observe cells capable of telomeric silencing. For each independent experiment, the fraction of viable cells resistant to FOA (FOAR) was normalized to the value obtained for the wild-type (wt) strain; the averages of data from three experiments ± standard deviations are shown. <, no FOA-resistant colonies were observed (of 2 × 106 to 4 × 106 plated). (B) Strains containing the HMR+::ADE2 allele (68) were grown on YPD medium without additional adenine. A dark colony color indicates silencing of the ADE2 gene. Strains PKY268 (wild type [wt]), PKY269 (cac1), and PKY375 (cac1 hir1) were used. (C) Strains PKY730 (MATa hm1 sir1 cac2 hir3) (left) and PKY364 (MATa HML sir1 cac2 hir3) (right) were replica plated onto YPD medium to test for growth (upper panel) or onto synthetic dextrose medium spread with a lawn of mating tester strain 217 (MAT his1) to select for diploid formation (lower panel).

Synergistic effects of cac and hir mutations on HM gene silencing. Position-dependent transcriptional repression at the HML and HMR loci of S. cerevisiae ensures that only the a or genes at the MAT locus are expressed; defects in silencing at HML or HMR cause coexpression of a and  genes in the same cell, resulting in a reduced mating efficiency (for a review, see reference 37). Although cac mutants mate as efficiently as wild-type cells (9, 28) (Table 3), we asked whether combinations of cac and hir mutations cause synergistic silencing defects at HM loci. We quantitated mating of MATa strains as an assay for silencing of the  genes at HML, and we measured mating of isogenic MAT strains as an assay for silencing of the a genes at HMR. As previously observed, cac or hir gene deletions by themselves did not confer a mating defect (Table 3). However, in MATa cac hir double mutants (e.g., cac2 hir3 [Table 3]), approximately a 10-fold reduction in mating was observed, indicative of HML derepression. This demonstrates that the synergistic loss of position-dependent gene silencing in cac hir mutants is not limited to telomere-proximal genes.

View this table: [in this window] [in a new window]   TABLE 3.   Mating efficiencies of strains with different combinations of sir1, cac2, and hir3 deletions

Defects in telomeric chromatin structure. Position-dependent gene silencing at yeast telomeres and HM loci results in changes in chromatin structure as revealed by increased protection from nucleases and methylases (10, 36, 61). To test whether the reductions in telomeric gene silencing in cac hir mutants are related to chromatin structure, we expressed the bacterial dam methylase gene in various yeast strains and tested the DNA of these strains for digestion by methylation-sensitive restriction enzymes (Fig. 4). The DNA was hybridized with a URA3 DNA probe, which recognizes both the mutant ura3-1 gene at the normal chromosomal position and the URA3 gene inserted next to the chromosome VIIL telomere. Figure 4A illustrates the expected sizes of DNA fragments from both loci; previous work has established that the GATC sequence internal to the telomeric copy of the URA3 gene is largely protected from methylation in wild-type but not sir2 mutant cells, which lack telomeric silencing (10, 33). Methylase access at this sequence was assayed by digestion with the restriction enzyme DpnI, which requires methylation of the GATC recognition sequence for activity. Conversely, DpnII digestion of GATC sequences is inhibited by methylation. Sau3AI digestion of GATC sequences is insensitive to dam methylation and thus serves as a positive control for digestion.

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Increased accessibility of dam methylase to telomeric chromatin in a cac2 hir1 double mutant. (A) Diagram of the URA3 gene adjacent to the chromosome VIIL telomere [(TG)n] and the ura3-1 gene at the natural locus on chromosome V (33, 78). Restriction sites for HindIII and 5'-GATC-3' sequences are indicated by H3 and Sau, respectively. The GATC sequence that is more protected from methylation in wild-type cells than in sir cells is indicated by Sau*. Expected restriction fragments (A, B, C, and the telomeric fragment T) are shown schematically. (B) Strains expressing bacterial dam methylase were PKY300 (CAC+), PKY299 (cac1), PKY305 (cac2), PKY310 (cac2 hir1), PKY311 (hir1), and AJL387-5asir2 (sir2). DNA from each strain was digested with four different combinations of restriction enzymes prior to agarose gel electrophoresis and DNA hybridization with a URA3 probe. Shown from left to right, for each strain: digestion with HindIII alone, digestion with HindIII plus DpnI, digestion with HindIII plus DpnII, digestion with HindIII plus Sau3AI. "DpnI protection" indicates the fraction of total counts in the HindIII plus DpnI lanes present in the B restriction fragment, indicating protection of the GATC site internal to the telomeric URA3 gene from dam methylase. The values presented are averages of data from two independent experiments.

cac2 hir1

Effects of changes in histone gene copy number on telomeric gene silencing. We have considered two classes of models to explain the synergistic loss of chromatin-mediated gene silencing in cac hir and cac spt21 double mutants. First, the HIR genes and SPT21 could encode chromosome assembly factors that act in a pathway partially redundant to CAF-I, so that severe silencing defects are not observed until both classes of genes are mutated. A second possibility is that cac mutants are sensitive to the loss of these genes because nucleosome assembly pathways that operate in the absence of CAF-I are intrinsically sensitive to perturbations in histone levels. Both hir and spt21 mutations cause misregulation of histone synthesis: three of the four histone gene loci are derepressed in hir mutants (48, 65), while the histone HTA2-HTB2 and HHT2-HHF2 gene pairs are not fully activated in spt21 mutants (5). Thus, although hir and spt21 mutations cause misregulation of histone gene transcription in opposite directions, both sets of mutations share the property of altering histone levels and, in some instances, histone stoichiometry (59). Note that the two models proposed are not mutually exclusive.

View this table: [in this window] [in a new window]   TABLE 4.   Effects of histone overexpression on telomeric silencing in wild-type and cac2 cells

View larger version (68K): [in this window] [in a new window]   FIG. 5.   Changes in histone gene dosage and expression affect telomeric gene silencing in a cac mutant but not in wild-type cells. Telomeric silencing of the URA3-VIIL reporter was assayed as described in the legend to Fig. 3. For each strain, the fraction of viable cells resistant to FOA (FOAR) was normalized to the value obtained for the wild-type strain; the averages of values from n experiments ± the standard deviations are shown, except for the hhf2 cac2 strain, for which the averages of data from two experiments are presented. (A) Deletion of histone H3 and H4 genes. Strains PKY090 (wild type [wt]), PKY107 (cac2), PKY408 [(hht1-hhf1)], PKY409 [(hht1-hhf1) cac2], PKY410 [(hht2-hhf2)], PKY411 [(hht2-hhf2) cac2), PKY412 (hhf2), and PKY413 (hhf2 cac2) were used. (B) Deletion of the HTA2-HTB2 gene pair encoding histones H2A and H2B. Strains PKY090 (wt), PKY106 (cac1), PKY499 [(hta2-htb2)], and PKY500 (hta2-htb2) cac1] were used. (C) Deletion of the negative regulatory site in the HTA1-HTB1 promoter. Strains PKY090 (wt), PKY106 (cac1), PKY581 (HTA1neg), and PKY582 (HTA1neg cac1) were used.

Telomeric silencing is largely unaffected by misregulation of HTA1-HTB1 transcription. The previous data do not rule out the possibility that the Hir proteins also directly function with CAF-I in formation of heterochromatin. To test this possibility, we constructed strains in which the cis-acting negative site within the HTA1-HTB1 promoter was deleted (HTA1-neg mutants). The Hir proteins negatively regulate expression of this gene pair through this site (48); the resulting cells thus constitutively express the HTA1 and HTB1 genes, yet they contain functional Hir proteins. We measured the TPE in these strains in the presence and absence of the CAC1 gene to determine whether misregulation of the HTA1 and HTB1 genes in the presence of functional Hir proteins was sufficient to cause a synergistic loss of TPE in a cac1 mutant. We observed that the HTA1-neg mutation had no effect on TPE itself (Fig. 5C), consistent with the observation that hir mutants, which cannot negatively regulate HTA1 synthesis, also do not have TPE defects (Fig. 3). The HTA1-neg cac1 double mutant displayed a TPE level approximately twofold lower than that of a cac1 strain, a decrease similar to that observed in a cac mutant on overproduction of histones H2A and H2B (Table 4). However, the TPE in the HTA1-neg cac1 double mutant was much stronger than that observed in cac hir strains (Fig. 3). We conclude that misregulation of the HTA1-HTB1 gene pair does not contribute in a major way to the synergistic loss of telomeric silencing observed in cac hir strains.

	DISCUSSION

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Multiple contributions to heterochromatin formation. Yeast cells lacking CAF-I have reduced levels of telomeric gene silencing (9, 28). In this study, we found that cac mutants also have subtle defects in silencing at the silent mating type loci HMR and HML that can be detected with sensitive assays (Fig. 3C; Table 3), consistent with other recent studies (8). The extensive residual silencing observed in cac mutants suggests that CAF-I is partially redundant to other factors which participate in heterochromatin formation. The defect in silencing in cac mutants appears to be related to the stability of the silenced chromatin once it is formed, because cac mutants activate previously repressed telomere-proximal genes at a higher frequency (42) and more rapidly escape cell cycle arrest caused by exposure to alpha mating pheromone, a sensitive assay for the stability of HML silencing (8). These findings are consistent with the small colony size of FOA-resistant cac mutants in TPE assays (Fig. 3) (9, 28) and the variegated appearance of cac HMR::ADE2 strains on adenine-limited media (Fig. 3B). The synergistic reduction of HML silencing in sir1 cac double mutants (Table 3) (8) thus may result from a loss of factors that contribute to two aspects of silencing: establishment of the silenced state (Sir1p [51]) and maintenance of heterochromatin (CAF-I). However, because the defect in HML silencing in sir1 cac double mutants is partial, there must be other factors that contribute to silencing in the absence of Sir1p and CAF-I.

Role of HIR genes in silencing through regulation of histone synthesis. Why is silencing in cac mutants sensitive to the loss of the HIR genes? One possible explanation is that Hir proteins and CAF-I play partially redundant roles in the formation of heterochromatin and that the loss of both factors is required to observe dramatic decreases in silencing. Because hir mutants misregulate expression of a subset of the yeast histone genes (48, 60), a second hypothesis proposes that changing the absolute levels or the relative stoichiometry of the core histones would be detrimental to gene silencing in the absence of CAF-I.

Global defects in cac hir mutants. In addition to causing reductions in heterochromatic gene silencing, simultaneous deletion of the CAC and HIR genes revealed other phenotypes that sugge