Pch2 is a widely conserved protein that is required in baker' s yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Δ mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Δ mutants show defects in crossover interference. Third, crossovers observed in pch2Δ require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Δ mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover.
Meiosis generates haploid gametes from diploid progenitor cells. The reduction in ploidy results from the segregation of homologous chromosome pairs in the first meiotic division (MI, [1]). Prior to MI, each chromosome is joined to its homolog at chiasmata, which serve to tether homologs to each other. This interaction promotes the tension between homologs needed to form a bipolar spindle that facilitates homolog segregation. Homologous chromosome pairs lacking chiasmata connections often fail to segregate properly at MI. Chromosome nondisjunction can also result if chiasmata are present, but not properly placed on chromosomes, or if sister chromatid cohesion is disrupted [2]–[5]. Regardless of the cause, chromosome missegregation produces aneuploid gametes that lead to infertility or conditions like Down syndrome in humans [6]. Chiasmata form at sites where programmed Spo11-catalyzed DNA double-stranded breaks (DSBs), induced early in meiotic prophase, are repaired to form crossovers [1]. In baker' s yeast, crossovers (COs) are formed via two main pathways. The first pathway, by which the majority of COs are made, involves Msh4-Msh5 and Mlh1-Mlh3 [7]–[15]. In this pathway, DSBs are processed and acted upon by strand exchange enzymes to form single-end invasion intermediates (SEIs) that are converted into double Holliday junctions (dHJs). The latter are resolved into crossovers which display interference; the COs are more uniformly spaced than if placed at random (see below; [16]–[22]). The COs formed via the second major pathway, which require Mms4-Mus81, are not subject to CO interference [10], [11], [23]. Little is known about the intermediates that form in this latter pathway. The recombination steps that lead to CO formation occur in meiotic prophase. In leptotene, when meiotic DSB formation initiates recombination, an axial element containing Hop1 and Red1 proteins assembles along each pair of sister chromatids. In zygotene, when SEIs are detected, mature tripartite synaptonemal complex (SC) starts to form when the Zip1-containing central element connects the axial elements, which are now termed “lateral elements. ” Mature SC initiation begins at centromeres and later at CO-designated sites. These SC initiation events then spread outward until synapsis is completed in pachytene [24], [25]. Hop1/Red1 and Zip1 are enriched in separate domains on the mature SC. This organization is Pch2-dependent because in pch2Δ mutants, Zip1 and Hop1 appear to be more uniformly distributed along the chromosome axes [26], [27]. At the end of pachytene, recombination intermediates are resolved (reviewed in [28]). In yeast, ∼40% of the ∼140–170 meiotic DSBs are repaired to generate noncrossover (NCO) products [29], [30]. These NCO products are thought to form by a synthesis-dependent strand annealing mechanism (SDSA, [31]), separate from the interfering CO mechanism, and do not result in MI disjunction-promoting chiasmata. Martini et al. [32] found that when meiotic programmed DSBs are decreased in spo11 hypomorphic strains, COs are favored at the expense of NCOs [24]. This CO homeostasis phenomenon may be an additional manifestation of CO interference [32], [33]. The above studies indicate that DSBs are subject to a CO vs. NCO decision step, which is regulated by interference. Interference regulates this decision by ensuring that CO designation for a given DSB inhibits nearby DSBs from receiving this designation, thereby relegating them to a NCO fate. It is not clear whether non-interfering COs are formed through such a decision process; these COs are thought to form through a parallel pathway [10], [23]. For this paper, the CO vs. NCO decision refers solely to COs that are subject to interference. The interference-regulated CO vs. NCO decision likely occurs very early in recombination, roughly at the time of SEI formation (late leptotene-early zygotene) and does not appear to be controlled by domains or sequences contained within the chromosome [17], [18], [20], [21], [34], [35]. CO interference is strongest near a CO event and weakens with distance along the chromosome, although interference can act over large distances, up to ∼150 kb in yeast and ∼60 Mb in mice [30], [33], [36], [37]. In addition, interference between COs appears stronger on longer chromosomes compared to shorter chromosomes [35], [38]–[40], but see [41]. However, smaller chromosomes have relatively high DSB density and may also have a higher density of non-interfering COs [39], [41], [42]. The mechanisms underlying interference regulation of the CO vs. NCO decision are unknown, despite the fact that numerous mutants showing defects in CO interference have been identified in baker' s yeast. For at least a subset of these mutants, the defects in CO interference likely reflect problems in CO formation and not in the early CO vs. NCO decision (reviewed in [28]). For example, mutants defective in either the SC central element protein Zip1 or the CO-promoting factor Msh4 have reduced CO levels and the remaining COs show reduced or no interference [7], [33], [43]–[45]. However, Zip2 foci, which mark the early CO designated sites, still display interference in zip1 and msh4 mutants [35]. This result, combined with the fact that NCOs form in zip1Δ and msh4Δ, suggests that interference regulation of the CO vs. NCO decision requires neither these factors nor the mature SC [20], [22], [35], [46]. Rather, Zip1 and Msh4 are needed downstream of the decision to ensure CO formation [22], [35]. It is likely that these results are applicable to other members of the ZMM (Zip, Msh, Mer) class of proteins. We examined the PCH2 gene for a role in interference regulation of the CO vs. NCO decision. PCH2 is a putative AAA-ATPase widely conserved in organisms that construct a synaptonemal complex in meiosis [26], [47]. PCH2 was first identified in S. cerevisiae as a meiotic checkpoint factor due to the ability of pch2Δ to suppress the meiotic arrest of zip1Δ mutants [26]. This observation was extended by Wu and Burgess [47]; they proposed that Pch2 and Rad17 comprise separate branches of a checkpoint that ensures proper timing of the MI division, with the Pch2-dependent branch monitoring synaptonemal complex formation and the Rad17-dependent branch monitoring recombination events. Other checkpoint roles for Pch2 were reported in C. elegans, where it is required for apoptosis in response to unsynapsed pairing centers and in Drosophila, where it is required to delay meiotic progression in certain CO formation mutants [48], [49]. Recent studies indicate that PCH2 is not solely a checkpoint factor; it is essential for proper meiotic axis organization and timely meiotic progression in baker' s yeast, and complete DSB repair and fertility in mice [27], [47], [50]. Here we report that pch2 mutants display increased meiotic CO levels on larger chromosomes and are defective in CO interference. We also show that mutation of PCH2 reduces spore viability in spo11 hypomorphic strains. These data support an early role for Pch2 in DSB repair and a model in which Pch2-promoted meiotic axis organization controls CO levels and their distribution.
Previous work indicated that pch2Δ mutants show delays in meiotic DSB repair; thus, a time course comparison of DSB levels in meiotic prophase between pch2Δ and wild-type could be misleading [27], [47], [61]. Wu and Burgess [47] assayed DSB formation at the well-characterized HIS4LEU2 hotspot in wild-type and pch2Δ in a sae2Δ strain background where DSBs are formed but not resected or repaired. They reported that wild-type and pch2Δ strains displayed similar DSB levels. More recently, the Hochwagen group, using microarray analysis, observed increases in DSB formation in pch2Δ surrounding the rDNA on chromosome XII, but nowhere else in the genome (A. Hochwagen personal communication). We assayed DSB formation in pch2Δ mutants at the YCR048W hotspot on chromosome III and near the centromere on chromosome XV [42], [62], [63]. These experiments were performed in a dmc1Δ background where DSBs are formed at wild-type levels and resected (eventually hyperresected), but not repaired [29], [42], [64]. This approach allowed us to assay total DSB at loci other than HIS4LEU2, where DSBs are thought to occur at saturating levels, and avoid the use of the sae2Δ background where maximal DSB levels may not be reached [29], [32], [42], [65]. One concern with performing this analysis in the dmc1Δ background is that two reports [26], [66] indicated that the checkpoint arrest seen in dmc1 mutants is bypassed in pch2 dmc1 strains; however, a more recent report [61] indicated that it is not. Our pch2Δ dmc1Δ mutants displayed a meiotic arrest as measured by a failure to form spores (< 0. 6% spore formation for pch2Δ dmc1Δ vs. ∼90% for wild-type at T = 24 hrs). However as shown below, we observed a significant bypass of the dmc1 arrest in pch2Δ spo11-HA dmc1Δ strains. Quantification of DSB levels in the dmc1Δ background is difficult due to the extensive resection of the breaks. We therefore analyzed five independent cultures of dmc1Δ and pch2Δ dmc1Δ strains. Similar to previous work ([47]; A. Hochwagen personal communication), we saw no difference in DSB levels (% of total DNA) between dmc1Δ and pch2Δ dmc1Δ strains at the YCR048W (5 and 6 kb DSB bands; 19±6% for dmc1Δ, 18±5% for pch2Δ dmc1Δ) and CEN15 (8 kb DSB band; 4. 7±1. 2% for dmc1Δ, 4. 5±1. 0% for pch2Δ dmc1Δ) hotspots (Figure 5A and 5B; T = 7 hrs in meiosis). It is important to note that Hochwagen et al. [61] reported that pch2Δ dmc1Δ mutants do not resect DSB ends as rapidly as dmc1Δ; however, such a difference in resection rate could only result in an overestimation of the level of DSBs in pch2Δ dmc1Δ. These data, together with previous work, suggest that the pch2 mutation does not disrupt DSB levels in a SPO11 background. As shown above, the pch2Δ mutation severely compromised the spore viability of spo11 hypomorph strains. Because some spo11 mutations confer semi-dominant and conditional phenotypes, as well as alter DSB patterns [60], we assayed DSB levels at YCR048W in spo11-HA dmc1Δ strains in the presence or absence of the pch2Δ mutation (Figure 5C). At T = 3. 5 hrs in meiosis, similar DSB levels were observed in pch2Δ spo11-HA dmc1Δ (16%) and spo11-HA dmc1Δ (15%) strains. However, at T = 7 hrs, lower levels were observed in pch2Δ spo11-HA dmc1Δ (13±6 %; seven independent cultures) compared to spo11-HA dmc1Δ (18±6%; seven independent cultures). In time courses performed side by side, pch2Δ spo11-HA dmc1Δ strains displayed 30 to 90% of the spo11-HA dmc1Δ levels at T = 7 hrs. Such variability was not observed in side-by-side experiments involving pch2Δ dmc1Δ and dmc1Δ strains. As shown below and analyzed in the Discussion, we attribute the variability in DSB levels to the bypass of the dmc1Δ arrest in pch2Δ spo11-HA dmc1Δ. This was determined by measuring the completion of the MI division in spo11-HA dmc1Δ and pch2Δ spo11-HA dmc1Δ strains. At T = 28 hrs in meiosis, only 1-2% of spo11-HA dmc1Δ strains completed MI; this indicates that the dmc1Δ arrest is maintained in these strains. For pch2Δ spo11-HA dmc1Δ, at T = 4. 5 hrs, no cells (n>200) had completed the MI division. However, at T = 6. 5 hrs, 8 to 30% of the cells completed MI, and these values increased to 54 to 60% (with similar spore formation levels) at T = 28 hrs. As predicted for a dmc1Δ mutant, the spores produced by pch2Δ spo11-HA dmc1Δ were inviable.
Martinez-Perez [70] recently reported a link between meiotic axis protein organization and CO interference in C. elegans. They analyzed the distribution patterns of the central element protein SYP-1 and the axial element proteins HTP-1 and HTP-2, which, like Hop1, are HORMA domain proteins. Analogous to observations made for Hop1 and Zip1 in yeast, Martinez-Perez et al. [70] found that the HTP axial element and the SYP-1 central element proteins sort into reciprocal domains on late pachytene chromosome axes. Based on the above, the finding that HTP1/2 is depleted at COs, the fact that Spo11 and Msh5 are required for domain formation, and the correlation seen between HTP1/2 depletion sites and chiasmata, Martinez-Perez et al. [70] suggest that HTP/SYP-1 domain boundaries mark CO sites. This information suggests that Hop1/Zip1 boundaries indicate where the CO/NCO decision marks subsequent CO sites. Such a model takes into account the finding that C. elegans displays only one domain of each type whereas S. cerevisiae contains a large number of alternating Zip1/Hop1 domains. This pattern is consistent with the fact that each chromosome pair in C. elegans typically enjoys a single CO whereas chromosome pairs in S. cerevisiae enjoy multiple COs (∼80–90 total COs in S. cerevisiae [30], [35] vs. six in C. elegans [21], [70]). Based on observations presented in Martinez-Perez et al. [70] we suggest that the altered pattern of Hop1 and Zip1 localization on the chromosome axis seen in pch2Δ mutants results from, but is not the cause of, the increase in COs. In this interpretation, the defect in CO control in pch2Δ mutants leads to additional COs, reflected by a greater number of domains, thus making the axis distribution of Hop1 and Zip1 appear more uniform. This model fits with respect to the known timing of the CO vs. NCO decision [17], [18], [20], [21], [34], [35], and the finding that early Hop1 organization appears normal in pch2Δ mutants [27]. Testing such a model will require an examination of Hop1 and Zip1 localization patterns in strains (e. g. pch2Δ spo11 hypomorphs) containing decreased levels of DSBs; our model predicts that the Hop1 and Zip1 domains would become more distinct due to fewer COs, although not completely like wild-type due to defects in CO interference. The wild-type spore viability seen in pch2Δ mutants suggests that Pch2-mediated CO control is not required to maintain the viability of yeast grown in lab conditions. We offer two explanations for this finding: 1) COs are present in excess (∼80–90 per cell) of the number needed for all homologs to receive an obligate CO (16 per cell). 2) The reduction in interference in pch2Δ is accompanied by, and likely causes, an increase in the overall number of COs. This increase in crossing over could compensate for distribution failures that jeopardized obligate CO formation ([30], [33]; Figure 3, Figure 4; Table 4, Table 5, Table S2). Our results and those of Martini et al. [32] demonstrate a buffered system in baker' s yeast in which excess DSBs and COs lessen the need for interference to ensure obligate CO formation. Because of this buffer, obligate CO formation can be maintained if interference or DSBs are reduced, but not both (Figure 3; [32]). Such buffering may exist because the consequences of having too many COs are less severe than too few. For example, pch2Δ mutants have dramatic increases in CO levels, but show wild-type spore viability, whereas mutants that significantly decrease CO levels like mlh3Δ, have reduced spore viabilities due to MI nondisjunction [9], [11]. Future searches for mutants that disrupt the CO vs. NCO decision must be broadened to include genes with high spore viability or synthetic phenotypes with spo11 hypomorphs. Although the role of Pch2 in limiting CO levels, after the requisite number required for ensuring obligate CO formation is reached, is not required, it is likely to be advantageous. Too many COs, especially closely spaced ones, have been suggested to disrupt the sister chromatid cohesion required to create tension on the MI spindles and ensure proper homolog disjunction at MI [71], [72]. In addition, our data suggests that the CO limiting role of Pch2 also promotes timely meiotic progression, which could also be advantageous to cells (Figure 6). What causes the loss in spore viability seen in pch2Δ spo11 hypomorphs? pch2Δ/pch2Δ spo11-HA/spo11-HA strains displayed an excess of tetrads with 4,2, and 0 viable spores, a high percentage of two-spore viable tetrads containing sisters, and an increased frequency of chromosome III nondisjunction. Our data are consistent with MI chromosome nondisjunction being a major component of the spore death phenotype, perhaps due to a failure to ensure obligate CO formation on all chromosomes. In such a model, when DSBs become limiting, the proper distribution of COs becomes even more critical to ensure obligate CO formation. Similar DSB levels were seen at YCRO48W at 3. 5 hours in meiosis in spo11-HA dmc1Δ and pch2Δ spo11-HA dmc1Δ; however, by 7 hrs, fewer breaks were observed in the triple mutant (Figure 5C and 5D). Our DSB level measurements are not definitive due to the checkpoint bypass observed in the triple mutant. We provide two explanations for the triple mutant phenotype. In one scenario, early forming DSBs appear at wild-type levels while later-forming DSBs form at lower levels that are insufficient for sustained recombination checkpoint activation. In a second scenario, DSBs form normally, but undergo some level of Dmc1-independent, possibly intersister, repair that permits a bypass of the checkpoint. Such repair would not lead to MI disjunction-promoting chiasmata. Both of these scenarios are sufficient to explain the spore inviability seen in pch2Δ spo11 hypomorphs (Figure 3). Future experiments to distinguish these hypotheses should include an analysis of meiotic Rad51foci in spo11-HA dmc1Δ and pch2Δ spo11-HA dmc1Δ strains [73]. We cannot rule out that other cellular defects contribute to the MI non-disjunction phenotype seen in pch2Δ spo11 mutants. For example, both pch2Δ and spo11 hypomorphs have SC defects, which could lead to CO control-independent synthetic phenotypes in the double mutants [24], [27]. It is also possible that Pch2 promotes MI disjunction by regulating sister chromatid cohesion establishment and/or removal, or by preventing/resolving chromosome entanglements [2]–[4], [68], or that some spore death in pch2Δ spo11 hypomorphs is independent of MI non-disjunction. Interference mutants have been proposed to act downstream of the CO vs. NCO decision (e. g. zip1, msh4; Introduction; [22], [35]), or display an apparent defect in interference due to an increase in non-interfering COs (ndj1, csm4; [15], [35]). The only other yeast interference mutants that appear similar to pch2Δ are tid1Δ and dmc1Δ-2µRad54 [34; but see 58]. We will focus on tid1Δ, because its CO phenotype is better characterized. Tid1/Rdh54 is a member of the Swi2/Snf2 family, and thus may act in meiotic chromatin axis remodeling, though this has yet to be tested [74]. tid1Δ mutants display moderate levels of spore viability (58% 4-spore viable tetrads), and Tid1 has been shown to be involved in the strand exchange step of recombination [57]. Similar to pch2Δ, tid1Δ mutants display a defect in interference and increased gene conversion. Also, like pch2Δ, CO levels in tid1Δ appear similar to wild-type on a small chromosome (III). On a medium-sized chromosome (V), tid1Δ mutants displayed wild-type CO levels in two intervals, but a significant (2. 4-fold) increase in a third [34]. These data suggest that tid1Δ and pch2Δ have similar CO patterns. We are eager to test this hypothesis in the strain sets used in this study. Furthermore, we are intrigued by the idea that strand exchange and meiotic chromatin axis components are both required/involved in interference-regulation of the CO vs. NCO decision.
Yeast strains are listed in Table S1. All strains were grown at 30°C on standard YPD (yeast peptone dextrose; [75]). The sporulation media was described previously [11], [68]. For tetrad genotyping, synthetic minimal selective media, synthetic complete media with 5 µM Cu, and YPD supplemented with complete amino acid mix and 3 mg/L cycloheximide were used [75]. When required, Geneticin (Invitrogen), nourseothricin (Hans-Knoll Institute fur Naturstoff-Forschung), and hygromycin B (Calbiochem) were added to YPD media as described [76], [77]. The EAY1108/EAY1112 SK1 congenic strain set is described in Argueso et al. [11], and the NHY942/NHY943 SK1 isogenic strain set is described in de los Santos et al. [10]. The spo11 hypomorphic mutants were described by Diaz et al. [60] and Henderson and Keeney [24] although the NHY942/NHY943 strains containing these alleles, which are used in this work, are described in Martini et al. [32]. As in Martini et al. [32], we refer to spo11-HA3His6 as spo11-HA, spo11 (D290A) -HA3His6 as spo11da-HA, and spo11 (Y135F) -HA3His6 as spo11yf-HA. Strains EAY2562-2565 are derivatives of a cross between EAY2260 and SKY633. The msh5Δ, mms4Δ, and dmc1Δ alleles used in this work were all complete open reading frame (ORF) deletions. The pch2Δ allele contains a deletion of amino acids 17–587 (in the 603 amino acid ORF). All deleted regions were replaced with HPHMX4, KANMX4, or NATMX4 as shown in Table S1 [76], [77]. The deletion cassettes were made via PCR and integrated into the genome using standard techniques [78]. Details on strain construction and primer sequences are available on request. Diploids for tetrad analysis were all made using the zero growth mating protocol [79]. The haploid parental strains were patched together on YPD for 4 hours and then spread on sporulation plates. The plates were incubated at 30°C for 2 days, after which tetrads were dissected. Tetrads from the EAY1108/EAY1112 strain background were dissected on synthetic complete media, whereas tetrads from the NHY942/NHY943 strain background were dissected on YPD media supplemented with complete amino acids. All tetrads were incubated 3–4 days at 30°C and then replica-plated to various selective media. The replica plates were scored after one day of incubation at 30°C. In the EAY strain background, the data for wild-type, mms4Δ, and msh5Δ were originally published in Argueso et al. [11]. In the NHY strain background, a subset of the wild-type data was originally published in Wanat et al. [68]. The distributions of each tetrad type were calculated using RANA software [11]. Genetic map distances±the standard error were calculated using the Stahl Laboratory Online Tools (http: //www. molbio. uoregon. edu/~fstahl/) which utilizes the formula of Perkins [54]. The G-test spreadsheet, available from The Online Handbook of Biological Statistics (http: //udel. edu/~mcdonald/statintro. html), was used to compare tetrad distribution patterns between strains. The Dunn-Sidak correction (p value of 0. 05/ number of comparisons) was applied when multiple comparisons per data set were performed [80]. Recombination frequencies from spore data were calculated as described previously (RANA software; [11]), with p-values determined as above (http: //udel. edu/~mcdonald/statintro. html). Three different analyses were performed to measure interference. The NPD ratio (Table 4) was determined using the “Better Way” calculator (http: //www. molbio. uoregon. edu/~fstahl/). This method compares the number of each tetrad type observed to the numbers expected if CO distribution was random and calculates a chi square value, which was converted to a p value using VassarStats (http: //faculty. vassar. edu/lowry/VassarStats. html). Coefficients of coincidence (Table 5) were determined as described previously [11], [68]. Tetrads were sorted using Mactetrad 6. 9 software to calculate interference via the Malkova et al. method ([37], Figure 4; Table S2). For all time courses, a saturated YPD overnight culture from each strain to be analyzed was diluted in 200 ml YPA (2% potassium acetate) and grown for 17 hours. The YPA culture was then spun down, washed once in 1% potassium acetate and resuspended in 100 ml 1% potassium acetate (similar to [81]). All strains were grown in the same batches of media and treated identically. DAPI staining to analyze progression past MI (MI + MII) was performed as described [81]. Cells were visualized using an Olympus BX60 microscope and at least 200 cells were counted for each time point. DNA was isolated from meiotic cultures as described [29]. Southern blotting was performed using standard techniques [82]. The percent of DSB formation for four to six independent time courses (% of hybridizing bands±standard deviation, SD) was calculated using Image Quant software.
During meiosis, cells that ultimately become gametes (such as eggs or sperm) undergo a single round of DNA replication followed by two consecutive divisions. In most organisms, the segregation of chromosomes at the first meiotic division is dependent upon genetic exchange, or crossing over, at homologous sites along chromosomes. Crossing over must therefore be regulated to ensure that every pair of matched chromosomes receives at least one crossover. Matched chromosomes that do not receive a crossover frequently undergo missegregation at the first meiotic division, yielding gametes that do not contain the normal chromosome number. Such missegregation events have been linked to human infertility syndromes. We used a genetic approach to study meiotic crossover control in baker' s yeast. Our work suggests that Pch2 is required in crossover control during meiosis; mutants lacking Pch2 display altered crossover levels and distribution. Furthermore, pch2 mutations cause enhanced gamete inviability in strains that are mildly defective in initiating recombination. Based on these observations, we hypothesize that Pch2 acts early in crossover control, in steps that occur prior to those proposed for previously characterized crossover-promoting factors.
cell biology/nuclear structure and function
molecular biology/recombination
genetics and genomics/nuclear structure and function
molecular biology/chromosome structure
genetics and genomics/chromosome biology
molecular biology/chromatin structure
molecular biology/dna repair
The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover Interference