Meiotic crossover patterns: Obligatory crossover,interference and homeostasis in a single process

During meiosis, crossover recombination is tightly regulated. A spatial patterning phenomenon known as interference ensures that crossovers are well-spaced along the chromosomes. Additionally, every pair of homologs acquires at least one crossover. A third feature, crossover homeostasis, buffers the system such that the number of crossovers remains steady despite decreases or increases in the number of earlier recombinational interactions. Here we summarize recent work from our laboratory supporting the idea that all 3 of these aspects are intrinsic consequences of a single basic process and suggesting that the underlying logic of this process corresponds to that embodied in a particular (beam-film) model.     

Meiotic crossover patterns: Obligatory crossover,interference and homeostasis in a single process

During meiosis, crossover recombination is tightly regulated. A spatial patterning phenomenon known as interference ensures that crossovers are well-spaced along the chromosomes. Additionally, every pair of homologs acquires at least one crossover. A third feature, crossover homeostasis, buffers the system such that the number of crossovers remains steady despite decreases or increases in the number of earlier recombinational interactions. Here we summarize recent work from our laboratory supporting the idea that all 3 of these aspects are intrinsic consequences of a single basic process and suggesting that the underlying logic of this process corresponds to that embodied in a particular (beam-film) model.     

Does crossover interference count in Saccharomyces cerevisiae?

We previously proposed a "counting model" for meiotic crossover interference, in which double-strand breaks occur independently and a fixed number of noncrossovers occur between neighboring crossovers. Whereas in some organisms (group I) this simple model alone describes the crossover distribution, in other organisms (group II) an additional assumption--that some crossovers lack interference--improves the fit. Other differences exist between the groups: Group II needs double-strand breaks and some repair functions to achieve synapsis, while repair in group I generally occurs after synapsis is achieved; group II, but not group I, has recombination proteins Dmc1, Mnd1, and Hop2. Here we report experiments in msh4 mutants that are designed to test predictions of the revised model in a group II organism. Further, we interpret these experiments, the above-mentioned differences between group I and II meiosis, and other data to yield the following proposal: Group II organisms use the repair of leptotene breaks to promote synapsis by generating double-Holliday-junction intermediates that lock homologs together (pairing pathway). The possible crossover or noncrossover resolution products of these structures lack interference. In contrast, for both group I and group II, repair during pachytene (disjunction pathway) is associated with interference and generates only two resolution types, whose structures suggest that the Holliday junctions of the repair intermediates are unligated. A crossover arises when such an intermediate is stabilized by a protein that prevents its default resolution to a noncrossover. The protein-binding pattern required for interference depends on clustering of sites that have received, or are normally about to receive, meiotic double-strand breaks.     

Characterization of human crossover interference

We present an analysis of crossover interference over the entire human genome, on the basis of genotype data from more than 8,000 polymorphisms in eight CEPH families. Overwhelming evidence was found for strong positive crossover interference, with average strength lying between the levels of interference implied by the Kosambi and Carter-Falconer map functions. Five mathematical models of interference were evaluated: the gamma model and four versions of the count-location model. The gamma model fit the data far better than did any of the other four models. Analysis of intercrossover distances was greatly superior to the analysis of crossover counts, in both demonstrating interference and distinguishing between the five models. In contrast to earlier suggestions, interference was found to continue uninterrupted across the centromeres. No convincing differences in the levels of interference were found between the sexes or among chromosomes; however, we did detect possible individual variation in interference among the eight mothers. Finally, we present an equation that provides the probability of the occurrence of a double crossover between two nonrecombinant, informative polymorphisms.     

Pch2 Links Chromosome Axis Remodeling at Future Crossover Sites and Crossover Distribution during Yeast Meiosis

Segregation of homologous chromosomes during meiosis I depends on appropriately positioned crossovers/chiasmata. Crossover assurance ensures at least one crossover per homolog pair, while interference reduces double crossovers. Here, we have investigated the interplay between chromosome axis morphogenesis and non-random crossover placement. We demonstrate that chromosome axes are structurally modified at future crossover sites as indicated by correspondence between crossover designation marker Zip3 and domains enriched for axis ensemble Hop1/Red1. This association is first detected at the zygotene stage, persists until double Holliday junction resolution, and is controlled by the conserved AAA+ ATPase Pch2. Pch2 further mediates crossover interference, although it is dispensable for crossover formation at normal levels. Thus, interference appears to be superimposed on underlying mechanisms of crossover formation. When recombination-initiating DSBs are reduced, Pch2 is also required for viable spore formation, consistent with further functions in chiasma formation. pch2Δ mutant defects in crossover interference and spore viability at reduced DSB levels are oppositely modulated by temperature, suggesting contributions of two separable pathways to crossover control. Roles of Pch2 in controlling both chromosome axis morphogenesis and crossover placement suggest linkage between these processes. Pch2 is proposed to reorganize chromosome axes into a tiling array of long-range crossover control modules, resulting in chiasma formation at minimum levels and with maximum spacing.     

Cost-benefit analysis of recombination and its application for understanding of chiasma interference.

A cost-benefit analysis of recombination was undertaken. The beneficial effects of crossing-over are proportional to the frequency of recombinant offspring, while its harmful effects (errors of crossing-over leading to mutations) are proportional to the number of crossover exchanges. An equilibrium point should exist where the beneficial effects of crossing-over are balanced by its harmful effects. It is suggested that natural selection sustains a number of crossover exchanges per meiosis at the level that provides highest benefit-cost difference. Chiasma interference prevents the arising of closely located exchanges which are less effective in the production of recombinants than exchanges separated by some "interference distance". Computer simulation shows that chiasma interference increases the recombination effectiveness of the multiple crossover exchanges as compared to the case without interference.     

Plant genetics: when not to interfere

New evidence suggests that the model plant Arabidopsis has two biochemically distinct pathways that produce genetic crossovers. Studies in several organisms have revealed that one kind of crossover regulation - crossover interference - is applied differently from species to species. Arabidopsis appears to use an interference system similar to that of budding yeast.     

Detection of Genetic Interference: Simulation Studies and Mouse Data

Genetic chiasma interference occurs when the occurrence of one crossover (or chiasma) influences the probability of another crossover occurring nearby. We investigated, by simulation studies, the power of three statistical methods to detect interference. Neither the traditional three-locus method nor a multiplicative model approach are very powerful, while a multilocus-feasible map function approach is more powerful, particularly as the number of loci increases. We show that the power to detect interference is quite sensitive to the underlying type of interference. When we tested for interference in two mouse data sets (from chromosomes 1 and 12), we found significant evidence of positive interference.     

Statistical Analysis of Chromatid Interference

The nonrandom occurrence of crossovers along a single strand during meiosis can be caused by either chromatid interference, crossover interference or both. Although crossover interference has been consistently observed in almost all organisms since the time of the first linkage studies, chromatid interference has not been as thoroughly discussed in the literature, and the evidence provided for it is inconsistent. In this paper with virtually no restrictions on the nature of crossover interference, we describe the constraints that follow from the assumption of no chromatid interference for single spore data. These constraints are necessary consequences of the assumption of no chromatid interference, but their satisfaction is not sufficient to guarantee no chromatid interference. Models can be constructed in which chromatid interference clearly exists but is not detectable with single spore data. We then extend our analysis to cover tetrad data, which permits more powerful tests of no chromatid interference. We note that the traditional test of no chromatid interference based on tetrad data does not make full use of the information provided by the data, and we offer a statistical procedure for testing the no chromatid interference constraints that does make full use of the data. The procedure is then applied to data from several organisms. Although no strong evidence of chromatid interference is found, we do observe an excess of two-strand double recombinations, i.e., negative chromatid interference.     

Heterozygous insertions alter crossover distribution but allow crossover interference in Caenorhabditis elegans

The normal distribution of crossover events on meiotic bivalents depends on homolog recognition, alignment, and interference. We developed a method for precisely locating all crossovers on Caenorhabditis elegans chromosomes and demonstrated that wild-type animals have essentially complete interference, with each bivalent receiving one and only one crossover. A physical break in one homolog has previously been shown to disrupt interference, suggesting that some aspect of bivalent structure is required for interference. We measured the distribution of crossovers in animals heterozygous for a large insertion to determine whether a break in sequence homology would have the same effect as a physical break. Insertions disrupt crossing over locally. However, every bivalent still experiences essentially one and only one crossover, suggesting that interference can act across a large gap in homology. Although insertions did not affect crossover number, they did have an effect on crossover distribution. Crossing over was consistently higher on the side of the chromosome bearing the homolog recognition region and lower on the other side of the chromosome. We suggest that nonhomologous sequences cause heterosynapsis, which disrupts crossovers along the distal chromosome, even when those regions contain sequences that could otherwise align. However, because crossovers are not completely eliminated distal to insertions, we propose that alignment can be reestablished after a megabase-scale gap in sequence homology.     

Crossover interference in the mouse

We present an analysis of crossover interference in the mouse genome, on the basis of high-density genotype data from two reciprocal interspecific backcrosses, comprising 188 meioses. Overwhelming evidence was found for strong positive crossover interference with average strength greater than that implied by the Carter-Falconer map function. There was some evidence for interchromosomal variation in the level of interference, with smaller chromosomes exhibiting stronger interference. We further compared the observed numbers of crossovers to previous cytological observations on the numbers of chiasmata and evaluated evidence for the obligate chiasma hypothesis.     

Crossover interference in Saccharomyces cerevisiae requires a TID1/RDH54- and DMC1-dependent pathway

Two RecA-like recombinases, Rad51 and Dmc1, function together during double-strand break (DSB)-mediated meiotic recombination to promote homologous strand invasion in the budding yeast Saccharomyces cerevisiae. Two partially redundant proteins, Rad54 and Tid1/Rdh54, act as recombinase accessory factors. Here, tetrad analysis shows that mutants lacking Tid1 form four-viable-spore tetrads with levels of interhomolog crossover (CO) and noncrossover recombination similar to, or slightly greater than, those in wild type. Importantly, tid1 mutants show a marked defect in crossover interference, a mechanism that distributes crossover events nonrandomly along chromosomes during meiosis. Previous work showed that dmc1Delta mutants are strongly defective in strand invasion and meiotic progression and that these defects can be partially suppressed by increasing the copy number of RAD54. Tetrad analysis is used to show that meiotic recombination in RAD54-suppressed dmc1Delta cells is similar to that in tid1; the frequency of COs and gene conversions is near normal, but crossover interference is defective. These results support the proposal that crossover interference acts at the strand invasion stage of recombination.     

Chromosome-wide control of meiotic crossing over in C. elegans

A central event in sexual reproduction is the reduction in chromosome number that occurs at the meiosis I division. Most eukaryotes rely on crossing over between homologs, and the resulting chiasmata, to direct meiosis I chromosome segregation, yet make very few crossovers per chromosome pair. This indicates that meiotic recombination must be tightly regulated to ensure that each chromosome pair enjoys the crossover necessary to ensure correct segregation. Here, we investigate control of meiotic crossing over in Caenorhabditis elegans, which averages only one crossover per chromosome pair per meiosis, by constructing genetic maps of end-to-end fusions of whole chromosomes. Fusion of chromosomes removes the requirement for a crossover in each component chromosome segment and thereby reveals a propensity to restrict the number of crossovers such that pairs of fusion chromosomes composed of two or even three whole chromosomes enjoy but a single crossover in the majority of meioses. This regulation can operate over physical distances encompassing half the genome. The meiotic behavior of heterozygous fusion chromosomes further suggests that continuous meiotic chromosome axes, or structures that depend on properly assembled axes, may be important for crossover regulation.     

Negative crossover interference in maize translocation heterozygotes.

Negative interference describes a situation where two genetic regions have more double crossovers than would be expected considering the crossover rate of each region. We detected negative crossover interference while attempting to genetically map translocation breakpoints in maize. In an attempt to find precedent examples we determined there was negative interference among previously published translocation breakpoint mapping data in maize. It appears that negative interference was greater when the combined map length of the adjacent regions was smaller. Even positive interference appears to have been reduced when the combined lengths of adjacent regions were below 40 cM. Both phenomena can be explained by a reduction in crossovers near the breakpoints or, more specifically, by a failure of regions near breakpoints to become competent for crossovers. A mathematical explanation is provided.     

Evidence That Masking of Synapsis Imperfections Counterbalances Quality Control to Promote Efficient Meiosis

Reduction in ploidy to generate haploid gametes during sexual reproduction is accomplished by the specialized cell division program of meiosis. Pairing between homologous chromosomes and assembly of the synaptonemal complex at their interface (synapsis) represent intermediate steps in the meiotic program that are essential to form crossover recombination-based linkages between homologs, which in turn enable segregation of the homologs to opposite poles at the meiosis I division. Here, we challenge the mechanisms of pairing and synapsis during C. elegans meiosis by disrupting the normal 1∶1 correspondence between homologs through karyotype manipulation. Using a combination of cytological tools, including S-phase labeling to specifically identify X chromosome territories in highly synchronous cohorts of nuclei and 3D rendering to visualize meiotic chromosome structures and organization, our analysis of trisomic (triplo-X) and polyploid meiosis provides insight into the principles governing pairing and synapsis and how the meiotic program is “wired” to maximize successful sexual reproduction. We show that chromosomes sort into homologous groups regardless of chromosome number, then preferentially achieve pairwise synapsis during a period of active chromosome mobilization. Further, comparisons of synapsis configurations in triplo-X germ cells that are proficient or defective for initiating recombination suggest a role for recombination in restricting chromosomal interactions to a pairwise state. Increased numbers of homologs prolong markers of the chromosome mobilization phase and/or boost germline apoptosis, consistent with triggering quality control mechanisms that promote resolution of synapsis problems and/or cull meiocytes containing synapsis defects. However, we also uncover evidence for the existence of mechanisms that “mask” defects, thus allowing resumption of prophase progression and survival of germ cells despite some asynapsis. We propose that coupling of saturable masking mechanisms with stringent quality controls maximizes meiotic success by making progression and survival dependent on achieving a level of synapsis sufficient for crossover formation without requiring perfect synapsis.     

Crossing over during Caenorhabditis elegans meiosis requires a conserved MutS-based pathway that is partially dispensable in budding yeast.

Formation of crossovers between homologous chromosomes during Caenorhabditis elegans meiosis requires the him-14 gene. Loss of him-14 function severely reduces crossing over, resulting in lack of chiasmata between homologs and consequent missegregation. Cytological analysis showing that homologs are paired and aligned in him-14 pachytene nuclei, together with temperature-shift experiments showing that him-14 functions during the pachytene stage, indicate that him-14 is not needed to establish pairing or synapsis and likely has a more direct role in crossover formation. him-14 encodes a germline-specific member of the MutS family of DNA mismatch repair (MMR) proteins. him-14 has no apparent role in MMR, but like its Saccharomyces cerevisiae ortholog MSH4, has a specialized role in promoting crossing over during meiosis. Despite this conservation, worms and yeast differ significantly in their reliance on this pathway: whereas worms use this pathway to generate most, if not all, crossovers, yeast still form 30-50% of their normal number of crossovers when this pathway is absent. This differential reliance may reflect differential stability of crossover-competent recombination intermediates, or alternatively, the presence of two different pathways for crossover formation in yeast, only one of which predominates during nematode meiosis. We discuss a model in which HIM-14 promotes crossing over by interfering with Holliday junction branch migration.     

Three homologous genes encoding sn-glycerol-3-phosphate acyltransferase 4 exhibit different expression patterns and functional divergence in Brassica napus

Brassica napus is an allotetraploid (AACC) formed from the fusion of two diploid progenitors, Brassica rapa (AA) and Brassica oleracea (CC). Polyploidy and genome-wide rearrangement during the evolution process have resulted in genes that are present as multiple homologs in the B. napus genome. In this study, three B. napus homologous genes encoding endoplasmic reticulum-bound sn-glycerol-3-phosphate acyltransferase 4 (GPAT4) were identified and characterized. Although the three GPAT4 homologs share a high sequence similarity, they exhibit different expression patterns and altered epigenetic features. Heterologous expression in yeast further revealed that the three BnGPAT4 homologs encoded functional GPAT enzymes but with different levels of polypeptide accumulation. Complementation of the Arabidopsis (Arabidopsis thaliana) gpat4 gpat8 double mutant line with individual BnGPAT4 homologs suggested their physiological roles in cuticle formation. Analysis of gpat4 RNA interference lines of B. napus revealed that the BnGPAT4 deficiency resulted in reduced cutin content and altered stomatal structures in leaves. Our results revealed that the BnGPAT4 homologs have evolved into functionally divergent forms and play important roles in cutin synthesis and stomatal development.     

Crossover homeostasis in yeast meiosis

Crossovers produced by homologous recombination promote accurate chromosome segregation in meiosis and are controlled such that at least one forms per chromosome pair and multiple crossovers are widely spaced. Recombination initiates with an excess number of double-strand breaks made by Spo11 protein. Thus, crossover control involves a decision by which some breaks give crossovers while others follow a predominantly noncrossover pathway(s). To understand this decision, we examined recombination when breaks are reduced in yeast spo11 hypomorphs. We find that crossover levels tend to be maintained at the expense of noncrossovers and that genomic loci differ in expression of this "crossover homeostasis." These findings define a previously unsuspected manifestation of crossover control, i.e., that the crossover/noncrossover ratio can change to maintain crossovers. Our results distinguish between existing models of crossover control and support the hypothesis that an obligate crossover is a genetically programmed event tied to crossover interference.     

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not

BACKGROUND: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae. RESULTS: To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. CONCLUSIONS: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.     

The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover Interference

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.