Depletion of H2A-H2B Dimers in Saccharomyces cerevisiae Triggers Meiotic Arrest by Reducing IME1 Expression and Activating the BUB2-Dependent Branch of the Spindle Checkpoint

Corresponding author: Andrew K. Vershon, 190 Frelinghuysen Rd., Piscataway, NJ 08854., vershon{at}waksman.rutgers.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the yeast Saccharomyces cerevisiae, diploid strains carrying homozygous hta1-htb1 mutations express histone H2A-H2B dimers at a lower level than do wild-type cells. Although this mutation has only minor effects on mitotic growth, it causes an arrest in sporulation prior to the first meiotic division. In this report, we show that the hta1-htb1 mutant exhibits reduced expression of early and middle-sporulation-specific genes and that the meiotic arrest of the hta1-htb1 mutant can be partially bypassed by overexpression of IME1. Additionally, deletions of BUB2 or BFA1, components of one branch of the spindle checkpoint pathway, bypass the meiotic arrest. Mutations in the other branch of the pathway or in the pachytene checkpoint are unable to suppress the meiotic block. These observations indicate that depletion of the H2A-H2B dimer blocks sporulation by at least two mechanisms: disruption of the expression of meiotic regulatory genes and activation of the spindle checkpoint. Our results show that the failure to progress through the meiotic pathway is not the result of global chromosomal alterations but that specific aspects of meiosis are sensitive to depletion of the H2A-H2B dimer.

MEIOSIS is a highly regulated developmental process that ensures the accurate transmission of genetic material. In the yeast Saccharomyces cerevisiae, meiosis is controlled by a tightly regulated transcriptional cascade (SMITH and MITCHELL 1989 ; CHU and HERSKOWITZ 1998 ; HEPWORTH et al. 1998 ). Initiation of the cascade requires activation of IME1 (SMITH and MITCHELL 1989 ), which encodes a global transcription factor for early meiosis-specific genes required for DNA replication and recombination (MANDEL et al. 1994 ). IME1 is regulated by an unusually large promoter (>2 kb) consisting of regulatory elements that sense both nutritional and genetic signals (SAGEE et al. 1998 ).

Meiosis in yeast is also regulated by checkpoints that monitor key events and prevent cells from progressing through the pathway until these events are completed. For instance, double-strand breaks generated by meiotic recombination are signals that activate the pachytene checkpoint pathway (reviewed in ROEDER and BAILIS 2000 ). The pachytene checkpoint prevents activation of NDT80, a transcription factor required for expression of middle-meiosis-specific genes, until all double-strand breaks have been repaired (CHU and HERSKOWITZ 1998 ; HEPWORTH et al. 1998 ). A second checkpoint, the spindle checkpoint, monitors attachment of spindles to kinetochores. Activation of the latter checkpoint prevents transition to anaphase until all chromosomes are properly attached, thereby preventing chromosome missegregation (SHONN et al. 2000 ).

Regulation of chromatin structure contributes to, and is influenced by, these pathways in unknown ways. Yeast chromosomes undergo many changes during the sporulation pathway, the most dramatic occurring in meiotic prophase when genetic recombination increases >1000-fold (PETES et al. 1991 ). Unlike at other times in the yeast life cycle, chromatin condenses dramatically during this period, such that the chromosomes can be visualized by standard fluorescence microscopy (ESPOSITO et al. 1991 ). It is also during this period that chromosomes align with their homologs in synaptonemal complexes (SCs), which contain, in addition to the highly condensed chromatin, central proteinaceous cores associated with meiotic recombination and/or chiasmata formation (DRESSER and GIROUX 1988 ; ROEDER 1995 , ROEDER 1997 ; KLECKNER 1996 ).

The relatively simple organization of histone genes in yeast provides an ideal system for genetically probing the regulation of chromatin structure and dynamics in meiosis. S. cerevisiae contains two genes for histone H2A (HTA1 and HTA2) and two for histone H2B (HTB1 and HTB2; HEREFORD et al. 1979 ). These genes are located in two divergently transcribed H2A-H2B gene pairs: HTA1-HTB1 on chromosome IV and HTA2-HTB2 on chromosome II. Haploid mutants lacking the HTA1-HTB1 locus (hta1-htb1, HTA2-HTB2) express 60% of the wild-type level of H2A and H2B transcripts and hence produce suboptimal levels of the dimer (NORRIS and OSLEY 1987 ). This depletion leads to general changes in chromosome structure, as confirmed by genetic and biochemical studies (NORRIS and OSLEY 1987 ; CLARK-ADAMS et al. 1988 ; NORRIS et al. 1988 ; HIRSCHHORN et al. 1992 ; TSUI et al. 1997 ). Moreover, during the mitotic cycle the mutant exhibits a wide array of pleiotropic phenotypes, which include changes in transcription, chromosome segregation, heat-shock response, G₀ arrest, and cell-cycle progression (NORRIS and OSLEY 1987 ; CLARK-ADAMS et al. 1988 ; HIRSCHHORN et al. 1992 ). Histone depletion has also been shown to both increase and decrease expression of genes and to alter the structure of chromatin at the centromeres (KIM et al. 1988 ; SAUNDERS et al. 1990 ; WYRICK et al. 1999 ).

Even more profound effects were observed during sporulation in diploid yeast cells bearing homozygous hta1-htb1 mutations (hta1-htb1/hta1-htb1, HTA2-HTB2/HTA2-HTB2), possibly reflecting the more dramatic changes in chromosomal structure seen in this phase of the life cycle (NORRIS and OSLEY 1987 ). Although capable of vegetative growth, the homozygous mutant was almost completely deficient in progression through the sporulation pathway. The enhanced sensitivity of sporulation was not due to differences in HTA2 and HTB2 mRNA expression in the two developmental stages, since the expression patterns were very similar in mitosis and meiosis (TSUI et al. 1997 ). Additionally, the failure to sporulate was not due to a specific requirement for the histone subtypes encoded by the HTA1-HTB1 locus. Instead, the sporulation pathway per se appeared to be more sensitive to depletion of H2A-H2B dimers.

Further analysis demonstrated that the hta1-htb1 mutant was capable of completing premeiotic DNA replication, commitment to meiotic recombination, and completion of reciprocal exchanges, but arrested before the first meiotic division (TSUI et al. 1997 ). Electron microscopy on thin-sectioned cells at their terminal phenotypes failed to demonstrate any obvious disruptions in spindle pole bodies or microtubules. The meiotic block was not bypassed in backgrounds homozygous for spo13, rad50, or rad9 mutations, but was bypassed in the presence of subinhibitory concentrations of hydroxyurea, a drug known to inhibit DNA elongation. These data led us to hypothesize that the deposition of H2A-H2B dimers in the mutant was unable to keep pace with the replication fork during meiotic replication, thereby leading to a disruption in nucleosome structure or deposition that interfered with chromosome segregation during the meiotic divisions. Alternatively, the slowing down of DNA synthesis by hydroxyurea may simply allow more time for the accumulation of meiosis-specific genes.

However, these models fail to describe how these chromosomal changes might directly or indirectly inhibit meiotic chromosomal segregation. Specifically, it remained unclear how the alterations in histone dimers might interface with meiotic transcriptional regulatory pathways and checkpoints. In this report, we show that the depletion of H2A-H2B dimers results in a failure to fully induce several sporulation-specific genes. It also appears to activate a specific branch of the spindle checkpoint pathway. Depletion of the H2A-H2B dimers therefore appears to cause a meiotic block by affecting specific rather than global processes in meiosis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Media, growth conditions, and yeast strains:
The genotypes and sources of the strains used in this study are listed in Table 1. Vegetative cells were grown in YEPD (1% yeast extract, 2% peptone, 2% dextrose) or SD (0.67% Difco yeast nitrogen base without amino acids and 2% dextrose) supplemented with nutrients essential for auxotrophic strains as described previously (SHERMAN et al. 1986 ). Synchronous sporulation was carried out by inoculating logarithmic cells at an OD₆₀₀ of 0.1 in YEPA (1% yeast extract, 2% peptone, 2% potassium acetate), growing the culture at 30° to an OD₆₀₀ of 0.8–1.2 (16 hr), collecting cells by centrifugation, washing with 2% potassium acetate, resuspending cells at an OD₆₀₀ of 2.0 in SM (2% potassium acetate, 5 µg/ml histidine, 30 µg/ml leucine, 5 µg/ml uracil), and incubating cells with vigorous shaking at 30°.

Plasmids and ß-galactosidase assays:
pAM500 consists of a Sau3AI fragment containing the IME1 gene cloned into the Sau3AI site of the YEp24 high-copy vector (SMITH and MITCHELL 1989 ). pAV79 contains a HOP1-lacZ fusion reporter, pAV124 contains a HOP1-lacZ fusion in which the URS1 site in the HOP1 promoter is disrupted by a 5-bp substitution, and pAV130 contains a HOP1-lacZ fusion reporter in which the UAS_H site in the HOP1 promoter is disrupted by an 8-bp substitution (VERSHON et al. 1992 ). HOP1-lacZ expression from each of the three reporter constructs was assayed during meiosis by selecting three independent transformants of strains DN1000 and DN1197 and synchronously sporulating the cells. After transfer to SM, aliquots were taken every 2 hr and assayed for ß-galactosidase activity as described previously (KELEHER et al. 1988 ).

Northern blots:
A total of 10 ml of synchronously sporulating cells were collected at each time point. RNA preparation and Northern analysis were carried out as described previously (BROWN and MACKEY 1997 ; COLLART and OLIVIERO 1997 ). The PC4 probe was generated directly by random primed labeling of a plasmid containing the gene. All other probes were generated by random primed labeling of PCR fragments. Each experiment represents a single blot that was stripped and probed for all indicated genes.

Microscopy:
Nomarski and fluorescence microscopy were performed at x400 magnification using a Zeiss Axioplan microscope. Staining by 4,6'-diamidino-2-phenylindole (DAPI) was performed as described previously (WILLIAMSON et al. 1983 ). Progression through meiosis I and II was determined by fluorescence microscopy of DAPI-stained cells and progression through sporulation was determined by Nomarski microscopy of live cells. Single time-point experiments represent the average of at least three different experiments, with at least 500 cells counted for each strain in each experiment. For meiotic time-point experiments, at least 300 cells were counted for each strain at each time point.

Nuclei preparation:
To purify nuclei from yeast, we used a slight modification of the preparation described by NELSON and FANGMAN 1979 . Logarithmically growing or sporulating [6 hr for the wild-type strain (DN1000) and 12 hr for the hta1-htb1 mutant (DN1018)] cells were collected by centrifugation and washed one time in sterile water. Each pellet was then washed in 30 ml of dithiothreitol solution [10 mM dithiothreitol, 20 mM potassium phosphate (pH 7.0), 1 M sorbitol] and resuspended at a concentration of 4 ml/g of initial pellet in S buffer [1 mg/ml zymolyase T-100 (50,000 units/mg), 0.5 mM CaCl₂, 20 mM potassium phosphate (pH 7.0), 0.5 mM phenylmethylsulfonyl fluoride, and 1.1 M sorbitol]. The cells were incubated at 30° for 70 min or until fully spheroplasted, collected by centrifugation, washed one time in digestion buffer [0.1 mM CaCl₂, 20 mM PIPES (pH 6.3), 1 M sorbitol], and resuspended in 0.25 ml of digestion buffer per gram of initial pellet. The spheroplasts were then added slowly to 40 volumes of solution A [9% (wt/wt) Ficoll 400, 20 mM PIPES (pH 6.3), 0.5 mM CaCl₂, 1 mM phenylmethylsulfonyl fluoride], gently mixed, and centrifuged at 20,000 x g for 20 min. The pellet was resuspended in solution B [1 M sorbitol, 20 mM PIPES (pH 6.3), 0.1 mM CaCl₂, 1 mM phenylmethylsulfonyl fluoride] and centrifuged at 12,000 x g for 10 min. The resulting pellet was resuspended in 2 ml solution B per gram of initial pellet. Aliquots (500 µl) were stored at -80°.

Micrococcal nuclease digestions and indirect end labeling:
Micrococcal nuclease (MNase) digestions were performed as described (NORRIS et al. 1988 ). For indirect end labeling, MNase digestions were performed as previously described (HIRSCHHORN et al. 1992 ), except that concentrations of 0, 0.04, 0.2, or 1 unit of MNase were used to digest 250 µl of nuclei at 30° for 15 min. DNA was purified as previously described (BLOOM and CARBON 1982 ), except that two phenol and chloroform and isoamyl alcohol (25:24:1) extractions were performed after the MNase and RNAase A digestions and DNA were resuspended in 40 µl of water. Subsequent restriction digestions with BamHI, BstBI, PvuII, or PflMI were performed as previously described (BLOOM and CARBON 1982 ), except that 10 µl of each sample was digested with 16–20 units of enzyme overnight at the appropriate temperature. Samples were electrophoresed through a 1.1% agarose gel and Southern blotted as previously described (BROWN 1997 ). Analysis of CEN3 chromatin was performed as previously described (PINTO and WINSTON 2000 ). IME1 DNA was detected using a 410-bp probe beginning at -2015 (for PflMI-digested DNA) and a 230-bp probe beginning at +198 (for PvuII-digested DNA).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Depletion of the H2A-H2B dimers does not cause dramatic changes in chromatin structure during sporulation:
A reduction in histone dimers might be expected to alter the number, positioning, or accessibility of nucleosomes on DNA. These variables can be monitored by treating purified nuclei with micrococcal nuclease and subjecting the digested fragments to agarose gel electrophoresis. When carried out on vegetative hta1-htb1 haploids, this protocol has been shown to generate a canonical nucleosome ladder superimposed on a smeared background (NORRIS et al. 1988 ). In the SK-1 strain background used in this study, there again was no dramatic alteration in the positioning or accessibility of nucleosomal DNA after depletion of the dimer during vegetative growth (Fig 1A). During sporulation, however, there appeared to be a slight increase in general nuclease accessibility in the mutant strain compared with the wild-type strain, as manifested by a 1.5-fold increase in the proportion of DNA digested to mononucleosomes (Fig 1A).

View larger version (98K): In this window In a new window Download PPT slide   Figure 1. The chromatin of the hta1-htb1 mutant in meiosis is more susceptible than that of the wild-type strain to digestion by micrococcal nuclease. Wild-type (DN1000 lanes 1–4 and 9–12) and hta1-htb1 mutant (DN1018 lanes 5–8 and 13–16) nuclei were prepared from logarithmic mitotic cells (lanes 1–8) and from cells grown in sporulation media (6 hr DN1000 or 12 hr DN1018; lanes 9–16). The nuclei were then digested with micrococcal nuclease for the indicated times. DNA was purified and separated on a 1.5% agarose gel (lane 17 in A contains a DNA ladder). (A) Total chromatin was visualized with ethidium bromide. A Southern blot of the same samples was hybridized with probes for (B) IME1, (C) NDT80, and (D) CEN3.

The histone mutant fails to properly express genes required at different stages of the meiotic pathway:
The hta1-htb1 mutant might fail to sporulate because of a defect in the expression of an important sporulation gene. We therefore synchronously sporulated wild-type and hta1-htb1 diploids, prepared RNA at various times, and performed Northern blot analyses using probes directed against a number of meiosis-specific genes. Because the histone mutant arrests at the G2/MI border, we first examined the expression of genes required at the middle stages of the meiotic pathway. NDT80, along with some of its targets, CLB1, SMK1, and SPS1, exhibited between a 2.5- and 3.2-fold reduction in expression in the hta1-htb1 mutant background (Fig 2, lanes 1–5 vs. 6–11; XU et al. 1995 ; CHU and HERSKOWITZ 1998 ; HEPWORTH et al. 1998 ). Additionally, these genes showed a 6-hr delay in reaching their maximum level of expression.

View larger version (55K): In this window In a new window Download PPT slide   Figure 2. The hta1-htb1 mutant exhibits reduced expression of meiosis-specific genes that is partially bypassed by expression of IME1 from a GAL promoter. Wild-type (DN1568 lanes 1–5 and DN1546 lanes 12–16) and hta1-htb1 (DN1570 lanes 6–11 and DN1569 lanes 17–21) cells were synchronously sporulated and RNA was prepared from cells taken at various time points. Northern blot analyses were then performed on the RNA using probes for the indicated early and middle meiotic genes. PC4, an uncharacterized transcript expressed constitutively during meiosis, was used as a loading control. Lanes 12–21 contain RNA from cells that carry an integrated pGAL-IME1 construct.

To determine whether expression of genes that are induced before NDT80 in the meiotic pathway might be similarly affected, we examined expression of HOP1 using a HOP1-LacZ fusion reporter. ß-Galactosidase activity was expressed 50-fold less in hta1-htb1 cells than in wild-type cells (Fig 3A). The HOP1 promoter consists of two main elements, UAS_H and URS1, which regulate its transcription (VERSHON et al. 1992 ). The UAS_H site functions as a constitutive activator site and is bound by the general transcription factor Abf1 (GAILUS-DURNER et al. 1996 ). The URS1 site is bound by the Ume6/Sin3/Rpd3 complex that represses expression during mitosis and by the Ume6/Ime1/Rim11 complex that induces expression during the early stages of meiosis (RUBIN-BEJERANO et al. 1996 ; KADOSH and STRUHL 1997 , KADOSH and STRUHL 1998 ). When the URS1 site was deleted from the HOP1-LacZ fusion reporter, both the wild-type and the mutant strains exhibited similar transcriptional activation, suggesting that Abf1 binds to the UAS_H site and activates transcription normally in the histone mutant (Fig 3B). By contrast, when the UAS_H site was deleted, the wild-type strain showed a normal temporal pattern of HOP1 expression (but at lower levels than with the full promoter), while the mutant failed to express HOP1 at all (Fig 3C). These results suggest that in the histone mutant, HOP1 expression is not efficiently induced through the URS1 site.

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. HOP1 expression is reduced in the hta1-htb1 mutant due to improper function of the URS1 regulatory site. Wild-type and hta1-htb1 cultures carrying a pHOP1-LacZ fusion reporter containing (A) the full promoter (pAV79), (B) a mutant URS1 site (pAV124), or (C) a mutant UASH site (pAV130) were switched to sporulation conditions and assayed for ß-galactosidase activity. The values at each time point are an average of three independent transformants and the standard deviation was <10% of the value. •, wild type (DN1000); , hta1-htb1 (DN1197).

Finally, we examined the expression of IME1, a meiosis-specific transcription factor whose expression is required for the induction of early meiotic genes (SMITH and MITCHELL 1989 ; MANDEL et al. 1994 ). Because IME1 acts through the URS1 site to induce HOP1, it is possible that the decrease in HOP1 expression in the histone mutant was due to a reduction in IME1 expression. Although IME1 was induced in the early stages of meiosis, we observed a 3-hr delay in its induction and a 1.6-fold reduction in its expression in the hta1-htb1 background (Fig 2). Furthermore, the Northern blot illustrates that expression of IME2, a second target of IME1, is reduced 1.7-fold in the histone mutant. Interestingly, we also found that the expression of IME2 is not repressed at later stages of meiosis, as it is in wild-type cells (Fig 2).

Ectopic expression of IME1 from a GAL promoter, but not from its own promoter, bypasses meiotic arrest:
On the basis of the observed reduction in expression of both IME2 and HOP1, we reasoned that it might be possible to bypass the meiotic arrest of the histone mutant by overexpressing IME1 from a high-copy plasmid. To test this hypothesis, we transformed the pAM500 2µ plasmid that carries the entire coding and promoter regions of IME1 into both the wild-type and the hta1-htb1 strains and subjected the cells to sporulation conditions. The hta1-htb1 mutants carrying the IME1 plasmid continued to arrest primarily with a single nucleus (Fig 4A). Because this might simply reflect a lack of transcription from the plasmid IME1 gene, we constructed an hta1-htb1 mutant in an isogenic gal80 background, carrying the IME1 gene under the control of the GAL1 promoter and subjected this strain to sporulation conditions. In this strain background, the histone mutant showed a 2.5-fold increase in the number of cells that complete the first or second meiotic divisions (Fig 4B).

View larger version (17K): In this window In a new window Download PPT slide   Figure 4. Expression of IME1 from a GAL promoter partially bypasses the meiotic arrest of the histone mutant. Cells were switched to sporulation conditions, samples were collected at the indicated times, and the percentage of cells that had progressed through either the first or the second meiotic division was determined by fluorescence microscopy. (A) Expression of IME1 from a high-copy plasmid. •, wild type (DN1000); , hta1-htb1::ura3 (DN1197); , hta1-htb1::ura3 + pIME1. (B) Expression of IME1 from a GAL1 promoter. •, gal80::LEU2 (DN1568); , pGAL-IME1 gal80::LEU2 (DN1546); , hta1-htb1::URA3 gal80::LEU2 (DN1570); , hta1-htb1::URA3 pGAL-IME1 gal80::LEU2 (DN1569). At least 300 cells were counted for each strain at each time point.

Northern blot analysis of both the wild-type and the hta1-htb1 strains carrying pGAL-IME1 showed strong expression of IME1 early in meiosis (Fig 2, lanes 12–21). Ectopic expression of IME1 allowed for strong expression of the downstream target genes, such as IME2, as well as genes expressed later in the pathway, such as NDT80 and its targets (Fig 2, lanes 6–11 vs. 17–21). Comparing the histone mutant alone to the histone mutant containing pGAL-IME1, we observed an 2-fold increase in early gene expression and a 2.7- to 5.5-fold increase in middle-gene expression. These observations suggest that the bypass caused by ectopically expressing IME1 is likely due to increased expression of meiosis-specific genes. However, it does not account for why there was only a partial bypass of the sporulation defect.

The chromatin structure of IME1 is not grossly disrupted in the histone mutant:
One explanation for the reduced expression of IME1 is that depletion of the H2A-H2B dimers causes changes in the chromatin structure of the IME1 promoter. To examine this possibility, we probed a Southern blot of our MNase-digested samples with an IME1 promoter-specific probe. As with total chromatin, the wild-type strain and the hta1-htb1 mutant exhibited similar patterns of digestion, with the histone mutant containing 2.1-fold more DNA digested to the monosome level than the wild type (Fig 1B). We examined a region of 3 kb up and downstream of IME1 in more detail by an indirect end-labeling protocol (Fig 5A and Fig B). The histone mutant again exhibited essentially the same pattern of digestion as wild-type cells. Taken together, our MNase experiments show that there were no dramatic alterations in the position of the nucleosomes or accessibility of the DNA in the IME1 promoter. We obtained similar results at the NDT80 promoter (Fig 1C). However, in comparison to most other yeast promoters, the IME1 promoter is very large and contains multiple regulatory sites. It is possible that this promoter is particularly sensitive to alterations in the histone levels and that the minor changes in chromatin structure may indeed impair gene expression.

View larger version (80K): In this window In a new window Download PPT slide   Figure 5. Indirect end-labeling analysis of IME1 (A and B) and CEN3 (C and D) chromatin structure. Nuclei from wild-type (DN1000) and hta1-htb1 (DN1018) cells were isolated from logarithmically growing cells, digested with increasing amounts of MNase, and analyzed by indirect end labeling as described in MATERIALS AND METHODS. (A) PflMI-digested DNA probed with a 410-bp probe adjacent to the restriction site. (B) PvuII-digested DNA probed with a 230-bp probe adjacent to the restriction site. (C) BamHI-digested DNA probed with a 616-bp probe adjacent to the restriction site. (D) BstBI-digested DNA probed with a 173-bp probe adjacent to the restriction site. Probes are indicated as open bars.

Depletion of H2A-H2B dimers triggers one branch of the meiotic spindle checkpoint:
Another attractive model to explain the sporulation defect is that depletion of dimers leads to DNA damage, which then induces the pachytene checkpoint and the observed arrest (reviewed in ROEDER and BAILIS 2000 ). We therefore examined whether deletion of RAD17, RAD24, RED1, MEK1, or DOT1, all of which play roles in the pachytene checkpoint, would bypass the meiotic block in the hta1-htb1 background (GRIFFITHS et al. 1995 ; LYDALL and WEINERT 1995 ; SIEDE et al. 1996 ; XU et al. 1997 ; BAILIS and ROEDER 2000 ; SAN-SEGUNDO and ROEDER 2000 ). The double mutants arrested primarily with single nuclei and with few observable spores, i.e., before the first meiotic division (Fig 6A). This argues against the preceding DNA-damage checkpoint model.

View larger version (17K): In this window In a new window Download PPT slide   Figure 6. Assay for the ability of mutants in the pachytene (A) and spindle (B) checkpoint pathways to bypass the meiotic arrest caused by the hta1-htb1 mutant. The indicated strains were subjected to sporulation conditions. Progression through the meiotic divisions was monitored as in Fig 4 and completion of sporulation was monitored by Nomarski microscopy of live cells. The data represent the average of at least three separate experiments. At least 500 cells were counted for each strain in each experiment. Solid bars indicate cells containing meiosis I or meiosis II nuclei; shaded bars indicate cells containing two or more spores. (C) Spindle checkpoint mutants do not restore the timing of sporulation. The meiotic divisions were monitored as in Fig 4. At least 300 cells were counted for each strain at each time point. •, wild type (DN1000); , hta1-htb1::URA3 (DN1018); , bub2::KanMX hta1-htb1::URA3 (DN1561); , bfa1::KanMX hta1-htb1::URA3 (DN1582).

A second checkpoint pathway, the spindle checkpoint, operates at a similar point in meiosis, preventing progression into anaphase if the spindle apparatus fails to connect appropriately with kinetochores (HOYT et al. 1991 ; LI and MURRAY 1991 ). The spindle checkpoint pathway is composed of two branches, the MAD branch and the BUB2/BFA1 branch (reviewed in GARDNER and BURKE 2000 ). When components of the MAD branch of the checkpoint are deleted in the histone mutant, the cells continue to arrest primarily prior to the first meiotic division (Fig 6B). This result suggests that the arrest does not operate through a Mad1-dependent spindle checkpoint. However, deletion of BFA1, and to an even greater extent deletion of BUB2, partially bypasses the meiotic defects of the histone mutant (Fig 6B). These results suggest that depletion of the H2A-H2B dimers triggers a specific branch of the spindle checkpoint pathway. Moreover, when BUB2 or BFA1 are deleted in the hta1-htb1 background, the double mutants exhibited essentially the same timing of the meiotic divisions (Fig 6C).

Bypass of the hta1-htb1-induced checkpoint results in increased expression of middle-meiosis-specific genes:
We were interested in determining whether the partial bypass of the histone mutant arrest caused by deleting the checkpoint genes BUB2 and BFA1 was accompanied by a corresponding increase in the expression of meiosis-specific genes. When probed for IME1, HOP1, and IME2 on meiotic time course Northern blots, the histone and checkpoint double mutants showed minimal increases in gene expression compared to the histone mutant alone (Fig 7, lanes 12–18 and 19–24 vs. 5–11). Thus, as expected, abolishing that checkpoint does not affect early gene expression, which likely occurs prior to the induction of the checkpoint. However, when the same blot was probed for NDT80, we observed a 1.7-fold increase in expression in the bub2 histone double mutant and a 2.1-fold increase in the bfa1 histone double mutant (Fig 7, lanes 12–18 and 19–24 vs. 5–11). Furthermore, when the blot was probed for the Ndt80 targets CLB1, SMK1, and SPS1, the histone and checkpoint double mutants exhibited increases in expression of 1.6- to 1.9-fold, suggesting that a greater proportion of Ndt80 present in the double mutants is in the active form.

View larger version (62K): In this window In a new window Download PPT slide   Figure 7. Histone and spindle checkpoint double mutants exhibit increased middle-gene expression. Gene expression during sporulation in wild-type (DN1000 lanes 1–4), hta1-htb1 (DN1018 lanes 5–11), bub2 hta1-htb1 (DN1561 lanes 12–18), and bfa1 hta1-htb1 (DN1582 lanes 19–24) cells was assayed by Northern blot as described in Fig 2.

Depletion of H2A-H2B dimers does not appear to alter centromere structure:
One way in which the histone mutant may be triggering the spindle checkpoint is by disrupting the chromatin surrounding the centromeres. To address this question, we performed a Southern blot analysis on micrococcal nuclease-digested DNA using a 300-bp fragment immediately adjacent to CEN3 as a probe (Fig 1D). As was observed for total DNA, the histone mutant exhibited a slightly less distinct banding pattern and 1.9-fold more DNA digested to monosome levels. To examine the centromere structure more closely, we analyzed a region of 3 kb surrounding CEN3 using indirect end-labeling analysis (Fig 5C and Fig D). Again, we observed a similar banding pattern between the wild-type strain and the hta1-htb1 mutant. However, the bands appear less distinct in the histone mutant, especially to the right of CDEIII (Fig 5D). Thus, there may be a slight disruption in chromatin structure surrounding CEN3 in the hta1-htb1 mutant. However, this effect was again rather subtle.

Ectopic expression of IME1 and deletion of BUB2 in the same hta1-htb1 strain result in a greater level of bypass of the meiotic arrest:
We hypothesized that the decreased expression of IME1 and the activation of a meiotic checkpoint were two separate events that both contribute to the meiotic arrest of the histone mutant. A histone mutant strain containing either bub2 or pGAL-IME1 completed meiosis with an efficiency of 50% compared to 20% for the histone mutant alone (Fig 8). However, when the histone mutant carried both bub2 and pGAL-IME1, the strain sporulated with an efficiency of 65%. This result shows that both altered expression of meiosis-specific genes and the activation of a meiotic checkpoint pathway contribute to the meiotic defect of the hta1-htb1 mutant.

View larger version (26K): In this window In a new window Download PPT slide   Figure 8. Ectopic expression of IME1 and deletion of BUB2 in the same hta1-htb1 strain result in an even greater level of bypass than does either treatment alone. Cells were switched to sporulation conditions, and samples were collected at the indicated times, stained with DAPI, and examined by fluorescence microscopy. At least 300 cells were counted for each strain at each time point. •, wild type (DN1568); , hta1-htb1::URA3 (DN1570); , hta1-htb1::URA3 pGAL-IME1 (DN1569); , bub2::KanMX hta1-htb1::URA3 (DN1623); asterisks, bub2::KanMX hta1-htb1::URA3 pGAL-IME1 (DN1622).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The hta1-htb1 mutation significantly reduces the ability of diploid cells to go through the process of meiosis and complete sporulation (TSUI et al. 1997 ). Since we were unable to identify any differences in the expression patterns and dosage-compensation properties of the two H2A-H2B loci in mitosis vs. meiosis, we concluded that a unique feature of meiosis is that it is particularly sensitive to low levels of histone dimers. We hypothesized two potential models to explain the arrest of the histone mutant. Our first model, the transcription model, suggested that depletion of the histone dimer alters the expression of key meiotic genes, thus preventing the completion of meiosis. The second model, the checkpoint model, suggested that altered chromatin structure, caused by depletion of histone dimers, triggers a meiotic checkpoint, resulting in the observed arrest. In this report we show that both models are in part correct.

Transcriptional regulation:
Our data show that a major defect in the sporulation pathway of the hta1-htb1 mutant is the failure to fully induce both early and middle meiotic genes. Although the level of expression of the early genes is only moderately reduced, the timing of induction is significantly altered compared to wild-type cells. In wild-type cells, the induction of HOP1 and IME2 begins at 3 hr into sporulation and these genes are almost fully off by 9 hr. In contrast, induction in the histone mutant begins at 6 hr and the cells are still expressing these genes after 24 hr. This prolonged expression may be a signal that some early events are not fully completed in the hta1-htb1 mutant, thereby blocking further progression of the pathway. In support of the hypothesis that reduced expression of early genes contributes to the arrest of the histone mutant, we have shown that ectopically expressing IME1 results in an increase in the expression of meiosis-specific genes and partially suppresses the meiotic defect of the histone mutant. These results, along with our Northern blot data and previous results, show that at least one effect of histone depletion is to lower the level of IME1 expression (TSUI et al. 1997 ).

Previous work has shown that histone depletion does result in decreased expression of some genes, and, in fact, a microarray study showed that the expression of nearly 10% of all genes is reduced by depletion of histone H4 (WYRICK et al. 1999 ). In contrast to most promoters, the IME1 promoter is very large (>2 kb) and its regulation is quite complex, with at least 10 distinct regulatory elements (SAGEE et al. 1998 ). It is possible that the depletion of histone dimers alters the chromatin structure and in turn the regulation of IME1, resulting in the observed decrease in the level of expression of IME1. On the basis of the size and complexity of the IME1 promoter, it is reasonable to think that chromatin structure may play a significant role in its regulation. Indeed, it has been suggested that Rme1 interacts with the IME1 promoter 2 kb upstream of the start site and recruits Sin4 and Rgr1 to alter the chromatin structure of IME1 and repress its transcription (COVITZ et al. 1994 ; SHIMIZU et al. 1997 ). Thus, depletion of the H2A-H2B dimer may cause reduced IME1 expression by preventing the complete activation of its promoter. Although we did not observe gross rearrangements of nucleosome positioning or chromatin structure at the IME1 promoter, it may be that this promoter is particularly sensitive to alterations in global chromatin structure. Depletion of the H2A-H2B dimers in the hta1-htb1 mutant shifts the transcriptional start sites at some promoters (CLARK-ADAMS et al. 1988 ), bypasses the requirement for the Swi/Snf general transcription complex at other promoters (HIRSCHHORN et al. 1992 ), and induces transcription of heat-shock genes (NORRIS and OSLEY 1987 ). Depletion of the H2A-H2B dimer may alter IME1 expression through similar mechanisms. In addition, the H2A homolog H2A.Z has been shown to be specifically required for recruitment of RNA polymerase II to the GAL1-10 promoter (ADAM et al. 2001 ). High levels of the H2A-H2B dimer may similarly be required for recruitment of the transcription machinery or of a regulatory factor to the IME1 promoter.

The spindle checkpoint:
The observed reduction in middle sporulation gene expression in the histone mutant is due in part to a checkpoint-mediated arrest. We have demonstrated that deletion of genes encoding checkpoint proteins can partially bypass the meiotic arrest of the histone mutant and that this bypass is accompanied by an increase in middle sporulation gene expression. Interestingly, the genes that show this phenotype, BUB2 and BFA1, are components of one branch of the spindle checkpoint pathway. Deletion of components in the pachytene checkpoint pathway or in the other branch of the spindle checkpoint pathway failed to show the same bypass. These results illustrate that, as in mitosis, two distinct branches of the spindle checkpoint exist in meiosis and that only one of the two branches of the spindle checkpoint is involved in the arrest of the hta1-htb1 mutant.

In mitosis, the Bfa1/Bub2 GTPase-activating protein (GAP) activates the spindle checkpoint by keeping Tem1 in its inactive GDP-bound form (FRASCHINI et al. 1999 ; LI 1999 ). Inactivating Tem1, a component of the mitotic exit network, prevents cells from exiting mitosis, reduplicating their DNA, and rebudding in the presence of microtubule damage (MORGAN 1999 ). Interestingly, it has also been shown that Bub2 and Bfa1 are required to prevent exit from mitosis in response to misoriented spindles and DNA damage (WANG et al. 2000 ; HU et al. 2001 ). Also, it has been demonstrated that the Bub2/Bfa1 branch of the pathway is active in each cell cycle and that disruption of the spindle seems to prolong the normal activity of the GAP complex (LEE et al. 2001 ). Taken together, these results show that the Bub2/Bfa1 GAP likely serves both as a checkpoint to arrest cells in the presence of the microtubule or spindle pole body disruption and as a coordinator of normal mitotic events, linking the completion of anaphase with mitotic exit. Such a mechanism may be necessary in meiosis to coordinate the completion of the first meiotic division with the initiation of meiosis II.

We have previously shown by electron microscopy that microtubule structure in the histone mutant appears to be normal (TSUI et al. 1997 ). However, because the positioning of the nucleosomes surrounding the centromeres is highly ordered and histone H2A has previously been shown to be important for this positioning (PINTO and WINSTON 2000 ), depletion of H2A-H2B dimers may alter the chromatin surrounding the centromere, thus activating a branch of the spindle checkpoint. Although we did not observe gross changes of the chromatin structure at the CEN3 region, it is possible that subtle changes may activate the Bub2/Bfa1 branch of the spindle checkpoint pathway. This checkpoint is not activated in mitotic hta1-htb1 cells, indicating that meiosis contains a more robust surveillance mechanism or is more sensitive to altered chromatin structure.

An alternative explanation for the activation of the spindle checkpoint is that depletion of the H2A-H2B histone dimer results in reduced expression of a meiosis-specific component of the centromere or kinetochore. Under normal conditions, the expression of this component would have to be induced early in meiosis, prior to activation of the spindle checkpoint. The expression of this component may be regulated in a manner similar to IME1, making it sensitive to histone depletion. If reduced expression of a centromere or kinetochore component triggers the meiotic arrest of the histone mutant, then ectopic expression of this component would be expected to partially bypass the arrest of the hta1-htb1 mutant.

We have also shown that the checkpoint bypass caused by deleting BUB2 or BFA1 is accompanied by an increase in the expression of the middle-meiotic gene NDT80 and its targets CLB1, SMK1, and SPS1 (Fig 7). It has been shown that mutations that alter meiotic recombination or formation of the SC induce the pachytene arrest pathway, which prevents accumulation and activation of the Ndt80 protein (CHU and HERSKOWITZ 1998 ; HEPWORTH et al. 1998 ; TUNG et al. 2000 ). Our results indicate that the spindle checkpoint pathway also plays a role in the regulation of NDT80 expression.

On the basis of our observations, we have concluded that depletion of histones affects both major forms of meiosis regulation. Impaired transcription of the meiotic regulator IME1 prevents full induction and the proper regulation of early meiotic genes such as HOP1 and IME2. This results in slowing down of the sporulation pathway as the cells wait for the expression of these components to rise to the proper level to carry out their function. Additionally, activation of the BUB2- and BFA1-dependent branch of the spindle checkpoint prevents the accumulation of NDT80 and of its targets CLB1, SMK1, and SPS1. Finally, we showed that a strain that contains both bub2 and pGAL-IME1 shows a higher level of bypass than strains that contain either bub2 or pGAL-IME1 alone. Thus, both altered transcription and an activated checkpoint contribute to the meiotic arrest of the hta1-htb1 mutant. These results also indicate that depletion of the H2A-H2B histone dimer does not trigger meiotic arrest by a global disruption of chromatin structure during sporulation. Instead, it appears that depletion affects specific aspects of sporulation.

	FOOTNOTES

¹ Present address: PAREXEL International, Bedminster, NJ 07921.

	ACKNOWLEDGMENTS

We thank L. Neigeborn and K. Tsui for providing plasmids and yeast strains. This work was supported by National Institutes of Health grant GM-57058 to D.N. and GM-58762 to A.K.V.

Manuscript received January 15, 2003; Accepted for publication April 21, 2003.

	LITERATURE CITED

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