INTRODUCTION

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Sporulation is a process of cellular differentiation that is initiated in diploid a/ cells of the yeast Saccharomyces cerevisiae in response to nitrogen starvation in the presence of a nonfermentable carbon source. As a cell progresses through the events of meiosis and spore wall formation, a coordinated series of genetic and morphological events generates a tetrad of haploid spores within an ascus (reviewed in references 9 and 30). The sporulation program begins when starved cells exit the mitotic cell cycle and undergo premeiotic DNA replication. This is followed by a lengthy prophase in which homologous chromosomes pair, the synaptonemal complex (SC) is elaborated, and a high level of genetic recombination occurs. At pachytene, the penultimate stage of prophase and the last stage at which cells can return to mitotic growth, the chromosomes are fully synapsed, and the spindle pole bodies lie side-by-side in the nuclear envelope. As cells exit from pachytene, the SC disassembles, chiasmata appear, and the spindle pole bodies separate to form the meiosis I spindle. The two meiotic divisions occur in rapid succession, leading first to segregation of homologous chromosomes and then to segregation of sister chromatids, giving rise to a four-lobed nucleus. The prospore walls, which initiate as a membranous outgrowth from a modified outer plaque of the spindle pole bodies, expand around each nuclear lobe. Ultimately, a haploid nucleus and a portion of maternal cytoplasmic material is engulfed within each prospore. Maturation of the spore wall into a multilayered structure completes the process of spore formation. The successive events of meiosis and spore formation are dependent on the ordered expression of at least four classes of genes, referred to as early, middle, mid-late, and late based on their time of expression during sporulation (reviewed in references 30 and 39). This temporal pattern of gene expression during sporulation in S. cerevisiae provides a simple model for studying the mechanisms by which morphological and genetic changes might control gene expression during development.

Expression of early meiotic genes, such as HOP1 and DMC1, which are involved in pairing and synapsis of homologous chromosomes and meiotic recombination, is regulated by the transcription factors Ime1p and Ume6p (reviewed in reference 30). Repression of early meiotic genes in vegetative cells is mediated by Ume6p, which binds to an URS1 promoter element and recruits a Sin3p-Rpd3p-containing repression complex to the DNA (23). IME1, a key inducer of meiosis, is rapidly upregulated when diploid cells are transferred to sporulation medium. An Ime1p-Rim11p activation complex, which replaces the Sin3p-Rpd3p repression complex (7, 36, 52, 63), then acts in conjunction with other upstream activation sequence (UAS)-bound proteins, such as Abf1p (15), to promote expression of early meiotic genes. Ime2p kinase, an early meiotic gene product, acts in a second pathway to bring about full activation of early meiotic genes (reviewed in reference 39).

As cells enter the meiotic divisions, expression of early meiotic genes ceases and expression of middle sporulation genes begins. Activation of middle sporulation genes, such as SPS1 and SMK1, which contribute to spore wall formation (14, 29), is dependent on Ime2p kinase activity (40, 62). A sporulation-specific activation element, referred to as the middle sporulation element (MSE), has been defined in the promoters of the SPS4 and SPR3 genes, and sequence inspection reveals that similar sites are present in the 5'-flanking sequence of other middle sporulation genes (19, 43).

The transition from expression of early meiotic genes to expression of middle sporulation genes occurs at about the time that cells exit from prophase and form the meiosis I spindle. Commencement of the meiotic divisions, which is dependent on activation of maturation-promoting factor (MPF), is an important regulatory point in the meiotic cell cycle of many organisms (reviewed in reference 44). MPF refers to the complex of cyclin-dependent kinase with its regulatory partner, a B-type cyclin. Several yeast mutants that are defective at intermediate stages of recombination arrest in pachytene, at the end of prophase (2, 4, 37, 38, 48, 68, 70). During meiotic recombination, recombination complexes provide a signal that activates the meiotic recombination checkpoint, preventing exit from pachytene. This pachytene arrest requires the mitotic DNA damage checkpoint genes RAD17, RAD24, and MEC1 (35) and the meiosis-specific recombination genes RED1 and MEK1 (78). Completion of recombination events removes this signal, allowing entry into meiosis I (4, 35, 78, 79). Exit from pachytene also appears to be regulated by other factors (34, 49, 71); for example, mutation of NDT80 leads to pachytene arrest in the absence of any significant defects in meiotic recombination (79).

In this study, we identified SIN3, RPD3, and NDT80 in a genetic screen for regulators of middle sporulation gene expression. The proposal by Xu and colleagues (79) that Ndt80p might control entry into meiosis by modulating the activity of MPF prompted us to test whether MPF activity itself was required for activation of middle sporulation genes. We found that middle sporulation genes were efficiently expressed in cells of a clb1 clb3 clb4 strain, which are deficient in MPF activity and are unable to enter meiosis (13, 18). Thus, entry into meiosis is not a prerequisite for activation of middle sporulation genes. In contrast, we found that mutation of DMC1, which leads to a defect in the strand exchange step of meiotic recombination and triggers pachytene arrest (4, 78), blocked activation of middle sporulation genes. Preventing pachytene arrest by mutation of the meiotic recombination checkpoint gene RAD17 restored middle sporulation gene expression to dmc1 cells. These results suggest that the meiotic recombination checkpoint regulates both entry into meiosis and activation of middle sporulation gene expression. The recent demonstration that Ndt80p is a transcriptional activator that binds to the MSE element (10) accounts for our isolation of NDT80 in a screen for regulators of middle gene expression.

	MATERIALS AND METHODS

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Yeast strains and genetic procedures. Table 1 lists the S. cerevisiae strains used in this study. DKB414-1 is a derivative of the MATa spo13 strain DKB414 (provided by D. Bishop) that contained the plasmid pRS315-MAT and was used to screen for mutants. We found that an SK1-derived strain was more proficient than our standard W303-derived laboratory strain at carrying out haploid meiosis and at promoting a high level of expression of a UAS^SPS4-lacZ reporter gene during sporulation. However, cells from an SK1 strain background lost the ability to grow on acetate more frequently than did cells from a W303 strain background; since growth on nonfermentable carbon is a prerequisite for sporulation, we routinely checked putative mutants for their ability to grow on acetate-containing medium. NKY2296 (79) (provided by N. Kleckner) was transformed with pNKY1212, a plasmid that contains NDT80 (79), and sporulated to obtain the haploid MAT ndt80 strain NKY2296B. The wild-type diploid strain DKB98 and its isogenic dmc1/dmc1 and dmc1/dmc1 rad17/rad17 derivatives, DKB608 and DKB1170, respectively, were provided by D. Bishop (35). Construction of ime2/ime2 (YKE2) (19) and clb1/clb1 clb3/clb3 clb4/clb4 (CD140; provided by B. Futcher) (13) derivatives of W303 have been described previously.

Media and growth conditions. SD medium is a minimal medium (2% glucose, 0.7% yeast nitrogen base without amino acids) supplemented with 40 µg of adenine sulfate per ml, 20 µg of L-arginine per ml, 20 µg of L-histidine per ml, 60 µg of L-leucine per ml, 30 µg of L-lysine (mono-HCl) per ml, 20 µg of L-methionine per ml, 50 µg of L-phenylalanine per ml, 200 µg of L-threonine per ml, 40 µg of L-tryptophan per ml, 30 µg of L-tyrosine per ml, and 20 µg of uracil per ml. SDX medium refers to SD medium that lacks supplement X. Rich medium (YEPD), presporulation medium (YEPA), and sporulation medium (SPO) were as described previously (19) with the exception that for SK1-derived strains YEPA contained 2% potassium acetate, 2% Bacto Peptone, and 1% yeast extract and SPO medium contained 2% potassium acetate supplemented with adenine sulfate, histidine, leucine, lysine, tryptophan, and uracil at the same concentrations as those in SD medium. All yeast cultures were grown at 30°C.

Plasmids. The multicopy CYC1-lacZ-containing plasmids pLG312(Bgl), pLG312SSS(Bgl), and pCYC1-SPS4-lacZ [previously referred to as 29×4/pLG312(Bgl)], have been described previously (19). pSPS4-lacZ, which contains four copies of fragment 29 (nucleotides 145 to 116 of the SPS4 gene) adjacent to a CYC1-lacZ gene that lacks a UAS, was constructed as follows. A SalI-SalI fragment recovered from pCYC1-SPS4-lacZ was cloned into the BglII site of pLG312SSS(Bgl) after the restriction endonuclease-generated ends of the vector and the insert had been filled in with the Klenow form of DNA polymerase. Forward orientation of the insert was confirmed by dideoxy sequence analysis. pRS315-MAT was constructed by cloning a 4.2-kb MAT-containing HindIII fragment from pJM9 (provided by A. Mitchell) into the HindIII site of pRS315 (61). pAV79B contains a HOP1-lacZ translational fusion gene (provided by A. Vershon) (72). The YCp50-based plasmids pMV1 and pMV34 contain RPD1 and RPD3, respectively (provided by R. Gaber) (73, 74). p(SPO13)8 is a YCp50-based plasmid which contains the SPO13 gene (provided by C. Giroux) (8). All plasmids were amplified in Escherichia coli DH5.

Assay for -galactosidase activity. -Galactosidase expression from lacZ reporter genes was monitored by use of a colony overlay assay (3). Ten milliliters of X-Gal-containing agar (0.5% agar, 0.5 M potassium phosphate [pH 7.0], 6% dimethyl formamide, 0.1% sodium dodecyl sulfate, 0.1 to 0.4 mg of 5-bromo-4-chloro-3-indoyl--D-galactopyranoside [X-Gal] per ml) was poured over colonies on plates, and the plates were incubated at 30°C until blue color development was evident.

Screens for mutants defective in expression of SPS4. One screen, aimed at the identification of mutants that failed to activate a UAS^SPS4-lacZ reporter gene during sporulation, was carried out with strain DKB414-1 containing pSPS4-lacZ. Cells that had been mutagenized to ~50% survival by exposure to ethyl methanesulfonate (EMS) in accordance with the procedure of Lawrence (33) were plated on SDLeuUra medium at 200 to 500 cells per plate and incubated for 4 to 5 days. About 32,000 colonies were patched onto SDLeuUra medium, incubated for 1 to 2 days, and then replica plated to SPO medium. The SPO plates were incubated for 14 to 18 h, and the patches were then overlaid with X-Gal-containing agar (see above). We recovered 258 mutants that failed to form blue patches in this assay and that retained the ability to grow on acetate-containing YEPA medium. To perform secondary tests, these mutant strains were grown on SD medium containing 5-FOA to select for cells that had lost pSPS4-lacZ (5). Each mutant was then cotransformed in duplicate with pRS315-MAT and either pSPS4-lacZ or pAV79B, which contains HOP1-lacZ. The transformants that grew readily on YEPA medium were identified, patched onto SDLeuUra medium, grown for 1 to 2 days, and then patched in duplicate onto SPO medium to retest for expression of UAS^SPS4-lacZ and to test for expression of HOP1-lacZ. Most mutants were discarded because they expressed UAS^SPS4-lacZ or because they showed a reduced or undetectable level of HOP1-lacZ expression on transfer to sporulation medium. Three mutant strains, m51, m312, and m320, that had the desired phenotype were identified; they expressed HOP1-lacZ at a high level and UAS^SPS4-lacZ at a reduced or undetectable level. These strains were found subsequently to contain the mutant alleles das1-1, das3-1, and das1-2, respectively. The gene designation refers to defective in activation of SPS4. We found that DAS1 was identical to NDT80 and that DAS3 was identical to SIN3.

Genetic analysis. Mutants were placed into complementation groups by using standard techniques (57). Allelism with IME2, SIN3, RPD3, and NDT80 was determined by mating das mutants with YKE2, SRH416B, SRH415B, and NKY2296B, respectively, and testing the resultant diploids for spore formation.

Cloning DAS4 by complementation. An a/ das4-1/das4-1 strain that contained pSPS4-lacZ was transformed with a pSB32-based (CEN ARS LEU2) library that consisted of a partial Sau3A digest of genomic DNA (constructed by P. Hieter and provided by B. Andrews) (50). Transformants were selected on SDLeuUra medium and then replica plated to SPO medium. After incubation for 14 to 16 h, colonies that expressed a high level of -galactosidase were identified by an overlay assay. These strains were retested for expression of UAS^SPS4-lacZ and examined for spore formation. After complemented strains had been grown on medium containing 5-FOA to select for loss of pSPS4-lacZ, library plasmids were recovered by introducing total yeast genomic DNA into E. coli. Purified library plasmids were retested for their ability to complement the Spo phenotype of the das4-1/das4-1 strain. Complementing plasmids were subjected to partial dideoxy sequence analysis with primers that flanked the site of insertion of genomic yeast DNA into pSB32. The resultant sequence was used in a BLAST search of the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces) to identify the chromosomal region that was present in the complementing plasmid.

Transposon disruption mutagenesis. pDAS4-32, which contained yeast genomic DNA that complemented the sporulation defect of an a/ das4-1/das4-1 strain, was mutagenized by transposon-mediated disruption with a HIS3-tagged version of Tn1000 () as described previously (41, 56). Donor and recipient E. coli strains were provided by M. Donoviel and B. Andrews. The site of transposon insertion in a plasmid that was unable to complement the a/ das4-1/das4-1 mutant was determined by dideoxy sequence analysis with primers that annealed to the ends of the transposon (41, 56). The resultant sequence was used in a BLAST search of the Saccharomyces Genome Database (see above).

FACS analysis. The relative DNA content of propidium iodide-stained cells was determined by fluorescence-activated cell sorter (FACS) analysis as previously described (14). For each experiment, 10,000 gated events were analyzed with LYSYS II software.

DAPI staining and microscopy. Cells to be stained with 4',6-diamidino-2-phenylindole (DAPI) were harvested from about 1 ml of culture, fixed by resuspension in 70% ethanol, and stored at 4°C. Just prior to use, cells were pelleted by centrifugation and resuspended in 50 µl of water. Three microliters of this cell suspension was placed on a glass slide and mixed with 3 µl of a solution that contained 0.6 mg of DAPI per ml of mounting medium (90% glycerol and 10% antiquenching solution [10 mg of p-phenylenediamine per ml of phosphate-buffered saline, pH 9.0]). To monitor the meiotic divisions, the cells were then examined with a Nikon Microphot FXA microscope with Nomarski and fluorescence optics.

RNA isolation and Northern analysis. RNA was prepared from yeast as described previously (46), except for the experiment shown in Fig. 6. In that case, cells harvested from 50-ml cultures were disrupted by a freeze-thaw procedure (55) and RNA was purified as described previously (55) with the addition of a lithium chloride and an ethanol precipitation of RNA. Northern blot analysis was performed as previously described (19). Gene-specific probes were prepared with the following templates: SMK1, an 800-bp StyI-StyI fragment from pLAKK40 (29); NDT80, a 1.2-kb Eco47III-BamHI fragment from pNKY1212 (79); SPS100, a 750-bp BamHI-NcoI fragment from pE18-B8a (32); and pC4, an uncharacterized control gene (32). Other templates were as described previously (19).

	RESULTS

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Isolation of mutants that are defective in expression of the SPS4 gene. We used the haploid meiosis strain DKB414-1, which is a spo13 derivative of SK1, to screen for mutations in potential regulators of expression of SPS4, a middle sporulation gene. We previously defined a 15-bp MSE, which we refer to as UAS^SPS4, in the promoter region of this gene that is sufficient to direct sporulation-specific gene expression (19). The presence of a MAT-containing plasmid in the MATa strain DKB414-1 enables cells to initiate meiosis in response to nutrient starvation. The spo13 mutation causes the cells to bypass what would be a lethal meiosis I division and to enter directly into meiosis II, a mitosis-like division in which sister chromatids are segregated (reviewed in reference 30). Subsequent formation of spore walls generates a dyad of viable haploid spores within each ascus. Strain DKB414-1, therefore, allows identification of recessive mutations affecting sporulation-specific events.

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Mutant strains express a HOP1-lacZ reporter gene but not an SPS4-lacZ reporter gene. Duplicate patches of cells from the wild-type strain (DKB414-1) and from mutant m51, m83, m96, m312, and m320 strains were incubated for 18 h on sporulation medium and then overlaid with X-Gal-containing agar to monitor -galactosidase expression (see Materials and Methods). As indicated at the top of the figure, one set of cells harbored pAV79B, which contains a HOP1-lacZ reporter gene, and another set harbored pSPS4-lacZ, which contains a UASSPS4-lacZ reporter gene.

Three complementation groups of DAS genes. The five mutants selected for further study fell into three complementation groups, which we designated DAS1, DAS3, and DAS4 to indicate that the mutants were defective in activation of SPS4 (see Materials and Methods). Two strains, m51 and m320, contained mutant alleles of DAS1, referred to as das1-1 and das1-2, respectively; m312 contained a mutant allele of DAS3 referred to as das3-1; and m83 and m96 contained mutant alleles of DAS4, referred to as das4-1 and das4-2, respectively. We next constructed and sporulated a/ spo13/SPO13 das/DAS diploid strains. The spore progeny from each of 10 tetrads analyzed for each mutant gave 2:2 segregation for a Spo⁺:Spo phenotype on mating with the original das strain or its MATa derivative (see Materials and Methods). This suggests that the phenotype of each das strain was caused by a single mutation.

das mutants complete premeiotic DNA synthesis but remain mononucleate. We next monitored the ability of the das mutants to undergo premeiotic DNA synthesis and to complete meiosis. Prior studies have shown that premeiotic DNA synthesis is required for expression of meiotic genes (25, 31). DNA synthesis was assessed by flow cytometric determination of the relative DNA content of cells that had been stained with propidium iodide. For this analysis, we compared the wild-type diploid strain (a/ spo13/spo13 DAS/DAS) with homozygous mutant diploid strains (a/ spo13/spo13 das/das). The FACS scans of cells of the wild-type and mutant strains during vegetative growth gave the expected distribution of cells in the G₁ (2N) and G₂ (4N) stages of the cell cycle (Fig. 2A, panels VEG). The FACS scans of cells that had been incubated in sporulation medium for 8 h revealed that most cells of the wild-type strain and of the mutant das1-1/das1-1, das3-1/das3-1, and das4-1/das4-1 strains had a 4N DNA content, indicating that they had completed premeiotic DNA synthesis (Fig. 2A, panels SPO).

View larger version (79K): [in this window] [in a new window]   FIG. 2.   Mutant strains complete premeiotic DNA synthesis but do not segregate their chromosomes. (A) FACS analysis of wild-type and mutant cells during vegetative growth and after transfer to sporulation medium. Aliquots of wild-type DAS/DAS spo13/spo13 cells (DKB414C) and mutant das1-1/das1-1 spo13/spo13, das3-1/das3-1 spo13/spo13, and das4-1/das4-1 spo13/spo13 cells were harvested during logarithmic growth in YEPA medium (VEG) and after incubation for 8 h in sporulation medium (SPO). Cells were fixed and stained with propidium iodide prior to analysis in a Becton-Dickinson FACScan. The peaks marked 2N and 4N represent cells in G1 and G2 portions of the cell cycle, respectively. Relative DNA content (x axis) is plotted versus cell number (y axis). (B) Microscopic appearance of wild-type and mutant cells on transfer to sporulation medium. Photographs are shown of wild-type DAS/DAS spo13/spo13 cells (DKB414C) and mutant das1-1/das1-1 spo13/spo13, das3-1/das3-1 spo13/spo13, and das4-1/das4-1 spo13/spo13 cells that had been incubated in sporulation medium for 24 h, fixed, and then stained with DAPI (see Materials and Methods). Cells were viewed with Nomarski (Nomarski) or fluorescence (DAPI) optics.

das mutants are defective in activation of middle sporulation genes. We next tested the hypothesis that the failure of the mutant strains to express the UAS^SPS4-lacZ reporter gene and to form spores reflected a general defect in middle sporulation gene expression. We monitored transcript accumulation by Northern blot analysis of RNA from wild-type diploid cells (a/ DAS/DAS spo13/spo13) and from mutant das1-1/das1-1, das3-1/das3-1, and das4-1/das4-1 cells during vegetative growth and at various times after transfer to sporulation medium. Blots were probed for the presence of transcripts from the early meiotic genes IME1, IME2, and HOP1, the middle sporulation genes SPS1, SPS4, and SMK1, and the late sporulation gene SPS100.

View larger version (60K): [in this window] [in a new window]   FIG. 3.   Expression of sporulation-specific genes in wild-type and das strains. RNA was purified from cells of wild-type strain DKB414C (WT) and mutant das1-1/das1-1 (das1), das3-1/das3-1 (das3), and das4-1/das4-1 (das4) strains, as indicated to the left of the panels. Cells were harvested during logarithmic growth in YEPA medium (0 h) and at various times after transfer to sporulation medium, as indicated (in hours) below the panels. Northern blots of these RNAs were sequentially hybridized with radioactively labeled gene-specific probes (see Materials and Methods), as indicated at the top of the panels. One set of filters was hybridized with probes for transcripts from SPS4, IME2, HOP1, SPS1, IME1, and the control gene. The control probe was prepared with pC4. Two HOP1-hybridizing transcripts have been described previously (20). A duplicate set of filters was used for hybridization with probes for transcripts from SMK1 and SPS100. Only the portions of the autoradiograms that revealed hybridization with the probes are shown.

DAS1 is NDT80, DAS3 is SIN3, and DAS4 is RPD3. Having confirmed that the das1-1, das3-1, and das4-1 mutant strains were defective in activation of middle sporulation genes, we proceeded to identify the wild-type DAS genes. We first tested two candidate genes, IME2 and NDT80, for allelism with the das genes. We tested IME2 because it is known to be essential for middle sporulation gene expression (40, 62), including expression of SPS4 (19). We tested NDT80, a key regulatory gene required for the transition from pachytene into the meiosis I division (79), because we had found that the transcriptional defect of the das mutants correlated with a failure to complete meiosis.

das1-1/ime2

das3-1/ime2

das4-1/ime2

das1-1/ndt80::LEU2

das3-1/sin3::URA3

das4-1/rpd3::URA3

The B-type cyclins Clb1p, Clb3p, and Clb4p are not required for activation of middle sporulation gene expression. The middle sporulation genes SPS1 and SMK1 have been characterized in detail and have been shown to play an essential role in spore wall morphogenesis (14, 29). Because spore wall maturation is a postmeiotic event, with the initial prospore membranes emanating from the spindle pole bodies and growing around the four lobes of the postmeiotic nucleus (42; reviewed in reference 30), we considered that the sequence of events during sporulation could be as follows: DNA replication  meiotic recombination  meiotic divisions  middle sporulation genes  spore wall formation. In this cascade, entry into meiosis I would provide a regulatory signal for activation of middle sporulation genes. However, the fact that the mutants that we isolated as defective in expression of middle sporulation genes were also defective in the meiotic divisions suggested that a common regulatory event might promote both entry into meiosis I and the activation of middle genes. To distinguish between these possibilities, we tested whether middle genes were expressed in a strain that arrests at pachytene and does not enter meiosis I due to a reduction in the activity of MPF.

View larger version (73K): [in this window] [in a new window]   FIG. 4.   Middle sporulation genes are expressed in a clb1 clb3 clb4 strain. RNA was purified from cells of the wild-type strain (LP112) (WT) and from its isogenic clb1/clb1 clb3/clb3 clb4/clb4 derivative (CD140), as indicated to the right of the figure. Northern blot analysis was performed as described in the legend to Fig. 3. The times at which cells were harvested are indicated (in hours) at the top of the figure; the hybridization probes are indicated to the left of the figure; the control transcript was from the PYK1 gene. Only the portions of the autoradiograms that revealed hybridization with the probes are shown.

Mutation of DMC1 prevents middle sporulation gene expression. Our experiments indicated that middle sporulation genes were not expressed in cells that arrested in pachytene and failed to enter the meiotic divisions due to mutation of NDT80, a putative regulator of MPF activity (79). In contrast, we found that middle sporulation genes were expressed in clb1 clb3 clb4 cells that failed to enter the meiotic divisions due to reduced MPF activity. Thus, MPF activity and entry into the meiotic divisions are not required for activation of middle sporulation genes. This suggested that pachytene arrest itself might prevent activation of middle sporulation genes. To test this idea, we examined gene expression in a dmc1 strain, which also arrests in pachytene. Whereas pachytene arrest occurs in the das1-1 (ndt80) strain in the absence of any significant defect in meiotic recombination (79), in a dmc1 strain a defect in meiotic recombination results in accumulation of DNA double-strand breaks that triggers the meiotic recombination checkpoint and subsequent pachytene arrest (4).

View larger version (85K): [in this window] [in a new window]   FIG. 5.   Sporulation-specific gene expression in wild-type (WT), dmc1/dmc1 (dmc1), and dmc1/dmc1 rad17/rad17 (dmc1 rad17) strains. RNA was purified from cells of the wild-type strain (DKB98) and its isogenic dmc1/dmc1 (DKB608) and dmc1/dmc1 rad17/rad17 (DKB1170) derivatives. Northern blot analysis was performed as described in the legend to Fig. 3. The times at which cells were harvested are indicated (in hours) above the panels. One filter was sequentially hybridized with radioactivity labeled probes for IME1 and IME2 transcripts, and a duplicate filter was sequentially hybridized with probes for SPS1, SPS4, HOP1, NDT80, SMK1, and a control transcript. The control probe was prepared with pC4. Only the portions of the autoradiograms that revealed hybridization with the probes are shown.

Meiotic arrest of sin3 and rpd3 strains is not suppressed by mutation of SPO11 or RAD17. We tested whether mutation of SPO11 would overcome the apparent arrest that was observed in das3 (sin3) and das4 (rpd3) strains. Mutation of SPO11 prevents initiation of recombination, thus bypassing defects at intermediate stages of recombination that normally lead to pachytene arrest (2, 4, 27, 37, 38, 48, 68, 70); for example, a spo11/spo11 dmc1/dmc1 strain is able to form spores, albeit inviable ones (4). Whereas cells of a DAS/DAS spo11/spo11 spo13/spo13 strain formed ~90% dyads on transfer to sporulation medium, cells of isogenic das3-1/das3-1 spo11/spo11 spo13/spo13 and das4-1/das4-1 spo11/spo11 spo13/spo13 strains were unable to form spores (data not shown). This suggests that mutation of SIN3 and mutation of RPD3 prevent the meiotic divisions by a defect other than, or in addition to, a defect in meiotic recombination. Consistent with this, abrogation of the meiotic recombination checkpoint by mutation of the checkpoint gene RAD17 did not allow efficient meiosis to occur in either the das mutants or a ndt80 strain (data not shown).

Unique regulation of expression of NDT80 and SMK1. Close examination of the gene expression patterns that we obtained during the course of this study suggested that NDT80 and SMK1 belong to a temporally distinct class of sporulation genes. Activation of these genes occurs after the early meiotic genes are turned on and before middle sporulation genes are first expressed. Additionally, NDT80 and SMK1 are regulated in a unique manner.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Expression of NDT80, a pre-middle sporulation-specific gene, depends on IME2. (A) NDT80 is expressed prior to middle sporulation-specific genes. A set of filters from the experiment shown in Fig. 3, containing RNAs purified from cells of the wild-type strain (DKB414C) (WT) and from mutant das1-1/das1-1 [das1 (ndt80)], das3-1/das3-1 [das3 (sin3)], and das4-1/das4-1 [das4 (rpd3)] strains, was stripped after hybridization with the probe for SMK1-derived transcripts and hybridized with a probe for NDT80-derived transcripts. Northern blot analysis was performed as described in the legend to Fig. 3. The autoradiograms depicting accumulation of IME2, SPS2, and SPS4 transcripts in the wild-type strain are from Fig. 3. The times at which cells were harvested are indicated (in hours) at the top and bottom of the figure. (B) IME2 is required for expression of NDT80 in sporulating cells. RNAs that had been purified previously (19) from cells of the wild-type strain (LP112) (WT) and its isogenic ime2/ime2 derivative (YKE2) (ime2/ime2), as indicated above the panels, were used to prepare filters for Northern blot analysis. The times at which cells were harvested are indicated (in hours) above the panels. The filters were sequentially hybridized with probes for detection of HOP1-, NDT80-, and control PYK1-derived transcripts. Only the portions of the autoradiograms that revealed hybridization with the probes are shown. (C) UME6 is not required for repression of NDT80 in vegetative cells. RNAs that had been purified from cells of the wild-type strain (LP112) (WT) and its isogenic ume6/ume6 derivative (YSH2) (ume6/ume6) growing in YEPD medium, as indicated above the panel, were subjected to Northern blot analysis (19). The autoradiogram of the filter that was hybridized with a probe for HOP1-derived transcripts is from the work of Hepworth et al. (19). A duplicate filter was sequentially hybridized with probes for NDT80- and control PYK1-derived transcripts. Only the portions of the autoradiograms that revealed hybridization with the probes are shown.

	DISCUSSION

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The SPS4 gene of S. cerevisiae belongs to a group of middle sporulation-specific genes that are expressed during the meiotic divisions. A 15-bp regulatory element from the promoter region of SPS4, referred to as UAS^SPS4, suffices for activation of a heterologous gene midway through sporulation (19). In this study, we identified mutants that had defects in regulation through UAS^SPS4. The five mutants that we studied in detail defined three complementation groups, referred to as DAS1, DAS3, and DAS4, with similar mutant phenotypes. Cells of the mutant strains completed premeiotic DNA synthesis and expressed early meiotic genes on transfer to sporulation medium but did not complete the meiotic divisions or activate expression of middle sporulation genes. We found that DAS1, DAS3, and DAS4 were identical to the previously characterized genes NDT80, SIN3, and RPD3, respectively.

The roles of these three genes have been well characterized. Mutation of NDT80 has been shown to cause cells to arrest in pachytene in the absence of any defects in meiotic recombination (79). Because the arrest phenotype of an ndt80 mutant is similar to the meiotic phenotype of a cdc28(Ts) mutant (59), Xu and colleagues (79) proposed that NDT80 might encode a regulator of MPF activity. MPF, which consists of Cdc28p kinase and a B-type cyclin as its regulatory partner, controls entry into meiosis I and meiosis II. Indeed, Chu and Herskowitz (10) have recently shown that Ndt80p is a transcriptional activator that induces expression of CLB1, CLB3, and CLB4 during sporulation in addition to activating middle sporulation gene expression.

SIN3 and RPD3 have been identified repeatedly in mutational analyses as modifiers of gene transcription [e.g., as RPD1 (SIN3) and RPD3 (73, 74); as SIN3 (76); as UME4 (SIN3) (65; reviewed in reference 77)]. Recently, it has been shown that Rpd3p, a histone deacetylase, is part of a large multiprotein complex that includes Sin3p (26; reviewed in references 45 and 67). In a mechanism that is conserved among eukaryotes, this complex is recruited to promoters by sequence-specific DNA-binding factors (23). Subsequent histone deacetylation is thought to generate a chromatin structure that represses transcription (24, 54).

Clearly, our screen for regulators of middle sporulation gene expression was not saturating; some genes that might have been identified, such as IME2 and DMC1, were not found. Because expression of the HOP1-lacZ reporter gene is reduced in an ime2 strain (data not shown; see also Fig. 6B), it is possible that any ime2 strain that might have been recovered as a potential das mutant was discarded on the basis of reduced expression of this early meiotic gene. Two of the three genes that we identified, however, were isolated twice. We attribute this bias to the sensitivity of our UAS^SPS4-lacZ reporter gene. It is likely that strains with mutations that gave a leaky phenotype would have escaped detection. Similarly, mutations in genes that contribute to, but are not essential for, activation of SPS4 would not have been detected.

Pachytene arrest blocks activation of middle sporulation genes. The observations presented in this study have allowed us to distinguish between two models that we had considered for coordination of the meiotic divisions and middle sporulation gene expression. The first model postulated that entry into the meiotic divisions was required for activation of middle sporulation genes; in this case, expression of middle sporulation genes would depend on activation of MPF. We tested this model by monitoring gene expression in a clb1 clb3 clb4 strain. This strain, which has almost no MPF activity and fails to complete meiosis (13, 18), was proficient in expression of middle sporulation genes (Fig. 4). We therefore conclude that Clb1p-, Clb3p-, and Clb4p-dependent MPF activity and entry into the meiotic divisions is dispensable for activation of middle sporulation genes. Moreover, the observation that CLB1, which encodes the primary meiotic B-type cyclin (13, 18), is expressed as a middle sporulation gene (10) makes it unlikely that MPF would itself serve in activation of middle genes.

Why does mutation of SIN3 or RPD3 confer a meiosis I block? As mentioned above, SIN3 and RPD3 have been identified repeatedly in mutational analyses as modifiers of gene expression (reviewed in reference 77). Rpd3p, which has histone deacetylase activity, and Sin3p are components of a multiprotein regulatory complex that is recruited to promoters by sequence-specific DNA-binding proteins (26; reviewed in references 45 and 67). Mutation of SIN3 or RPD3 and the resultant histone hyperacetylation could have effects on expression of sporulation genes, on recombination, and on the structure of chromatin and chromosomes. We suggest that a combination of such defects leads to pachytene arrest and that this arrest blocks middle sporulation gene expression. Because neither mutation of SPO11 nor mutation of RAD17 bypassed the arrest caused by mutation of SIN3 or RPD3, we suggest that this arrest is mediated by a mechanism that differs, at least in some aspect, from the meiotic recombination checkpoint (for examples, see references 49 and 71). We note that we found that mutation of SIN3 or RPD3 in some SK1-derived backgrounds led to a delay in meiosis and a reduced efficiency of spore formation rather than a complete block (unpublished observations).

Why does mutation of RPD3 (DAS4) lead to expression of the CYC1-UAS^SPS4-lacZ reporter gene in vegetative cells? We identified RPD3 (DAS4) in a screen that took advantage of our previous observation that the CYC1-UAS^SPS4-lacZ reporter gene, which has four copies of a 29-bp, UAS^SPS4-containing fragment inserted between the UAS^CYC1 and the TATA^CYC1, is not expressed in vegetatively growing cells (19). This 29-bp fragment, which contains UAS^SPS4 and 14 bp of upstream sequence, promotes 10-fold-higher expression during sporulation than does a 15-bp UAS^SPS4-containing fragment (19). We hypothesized that a protein that is present in vegetative cells binds to the 29-bp fragment and prevents UAS^CYC1-bound activators from functioning via an indirect mechanism such as steric hindrance (19). Although the CYC1-UAS^SPS4-lacZ reporter gene is expressed in rpd3 cells, as well as in sin3 (das3) cells (data not shown), that are growing vegetatively, the SPS4 gene is not expressed in these cells (Fig. 3, SPS4 panel, 0 time point). We speculate, therefore, that the 29-bp fragment contains a sequence that allows Sin3p-Rpd3p-dependent repression to be established in vegetative cells and that this repression, although effective on a heterologous UAS in vegetative cells, is not required for repression of SPS4 itself. Indeed, we have no evidence that the SPS4 gene is subject to negative control in vegetative cells (19). It is possible, however, that the putative DNA-binding protein that contributes to Sin3p-Rpd3p-dependent repression in vegetative cells is also responsible for enhancing Ndt80p-dependent activation of SPS4 during sporulation. We note that this putative DNA-binding protein is not Ume6p, since vegetative repression of the CYC1-UAS^SPS4-lacZ reporter gene is maintained in a ume6 strain (data not shown). Similarly, as would be expected, vegetative repression of the CYC1-UAS^SPS4-lacZ reporter gene is maintained in an ndt80 strain (data not shown).

NDT80, a pre-middle sporulation gene. In the course of our studies, we observed that regulation of expression of NDT80 and SMK1 was distinct from that of middle sporulation genes. In some of our time course experiments, we could readily detect transcripts from NDT80 prior to the appearance of transcripts from SPS4 (for an example, see Fig. 6A). A temporal and regulatory distinction was also apparent on analysis of transcript accumulation in dmc1 and dmc1 rad17 strains (Fig. 5). In the pachytene-arrested dmc1 strain, SPS1 and SPS4 were not expressed but a low level of transcripts could be detected from NDT80 and SMK1 (Fig. 5). In the dmc1 rad17 strain, transcripts from NDT80 and SMK1 could be detected prior to the appearance of transcripts from SPS1 and SPS4 (Fig. 5).

Model for coordination of middle sporulation gene expression and the meiotic divisions. We present the following model for the sequence of events that leads to expression of middle sporulation genes (Fig. 7). We propose that Ime1p-dependent activation of early meiotic genes is followed by Ime2p-dependent activation of pre-middle sporulation genes, such as NDT80 and SMK1. Ime2p, a serine-threonine protein kinase, has been shown to serve initially in IME1-independent activation of early meiotic genes and subsequently in activation of middle sporulation genes (reviewed in reference 39). The manner in which Ime2p carries out these temporally distinct functions is not clear; however, the two roles can be separated genetically (60). We suggest that the primary role of the late function of Ime2p is initial activation of pre-middle sporulation genes, such as NDT80. Because a modest level of NDT80 transcripts accumulated in a dmc1 strain, but middle sporulation genes were not expressed in this strain, we hypothesize that the early NDT80 transcripts are either not translated or, if they are translated, that active Ndt80p is not generated until cells have completed those aspects of meiotic recombination (78) that are monitored by the RAD17-dependent meiotic checkpoint (35). The active form of Ndt80p that we speculate is generated once the cells have progressed through the meiotic recombination checkpoint could result from translation of previously nontranslated transcripts, a posttranslational modification of Ndt80p, interaction with a cofactor, or some other sporulation-specific event. Finally, active Ndt80p would lead to upregulation of expression of pre-middle sporulation genes, including NDT80 itself, and activation of expression of middle sporulation genes, including CLB1 (10). Clb1p would then contribute to activation of MPF and subsequent entry into meiosis I. Hence, the meiotic divisions would be dependent on prior activation of middle sporulation genes, and activation of middle sporulation genes would be dependent on the absence of an arrest signal generated by intermediates of meiotic recombination. In this way, Ndt80p would coordinate entry into meiosis with spore wall formation. This model is consistent with the recent demonstration by Chu and Herskowitz (10) that Ndt80p is an MSE-binding transcriptional activator that activates its own expression. These investigators also noted that the activity of Ndt80p might be regulated by the meiotic recombination checkpoint machinery (10). The proposed autoregulatory loop for NDT80 expression (10) is also consistent with the low level of expression of ndt80 in our das1-1 (ndt80) strain (Fig. 6A). Interestingly, the mei4⁺ gene of Schizosaccharomyces pombe, which encodes a meiosis-specific transcription factor that is required for meiosis and sporulation-specific gene expression, also appears to be autoregulated (21).

View larger version (18K): [in this window] [in a new window]   FIG. 7.   Model for the role of Ndt80p in regulation of expression of middle sporulation-specific genes. The long horizontal arrows indicate the relative order of expression of various sporulation-specific genes; curved arrows refer to known or putative regulatory events; boxes indicate gene products. Upon transfer of diploid cells to sporulation medium, Ime1p rapidly activates expression of IME2 and other early meiotic genes. Ime2p has an early role in IME1-independent activation of early meiotic genes and a later role in activation of middle sporulation genes. We propose that the major role of the later function of Ime2p is to promote a low level of expression of pre-middle sporulation genes, such as NDT80. Our results suggest that activation of the RAD17-dependent meiotic recombination checkpoint inhibits the activity of Ndt80p and that an active form of Ndt80p is generated only after cells have completed most prophase events. In particular, meiotic recombination must be completed and any defects in meiotic chromosome metabolism that generate a pachytene arrest signal must be resolved before active Ndt80p is produced. We propose that the active form of Ndt80p is then responsible for upregulation of expression of pre-middle sporulation genes, denoted by a thickening of the horizontal arrows, and for activation of middle sporulation genes, such as SPS1 and SPS4.