Molecular Mechanisms of Homologous Chromosome Pairing and Segregation in Plants

In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division （MI） with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division （MII） with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation.     

Dynamics of rye chromosome 1R regions with high or low crossover frequency in homology search and synapsis development

In many organisms, homologous pairing and synapsis depend on the meiotic recombination machinery that repairs double-strand DNA breaks (DSBs) produced at the onset of meiosis. The culmination of recombination via crossover gives rise to chiasmata, which locate distally in many plant species such as rye, Secale cereale. Although, synapsis initiates close to the chromosome ends, a direct effect of regions with high crossover frequency on partner identification and synapsis initiation has not been demonstrated. Here, we analyze the dynamics of distal and proximal regions of a rye chromosome introgressed into wheat to define their role on meiotic homology search and synapsis. We have used lines with a pair of two-armed chromosome 1R of rye, or a pair of telocentrics of its long arm (1RL), which were homozygous for the standard 1RL structure, homozygous for an inversion of 1RL that changes chiasma location from distal to proximal, or heterozygous for the inversion. Physical mapping of recombination produced in the ditelocentric heterozygote (1RL/1RL(inv)) showed that 70% of crossovers in the arm were confined to a terminal segment representing 10% of the 1RL length. The dynamics of the arms 1RL and 1RL(inv) during zygotene demonstrates that crossover-rich regions are more active in recognizing the homologous partner and developing synapsis than crossover-poor regions. When the crossover-rich regions are positioned in the vicinity of chromosome ends, their association is facilitated by telomere clustering; when they are positioned centrally in one of the two-armed chromosomes and distally in the homolog, their association is probably derived from chromosome elongation. On the other hand, chromosome movements that disassemble the bouquet may facilitate chromosome pairing correction by dissolution of improper chromosome associations. Taken together, these data support that repair of DSBs via crossover is essential in both the search of the homologous partner and consolidation of homologous synapsis.     

Unexpected behavior of an inverted rye chromosome arm in wheat

Distal location of chiasmata in chromosome arms is thought to be a consequence of the distal initiation of synapsis. Observations of meiotic behavior of a rye chromosome with an inverted arm show that patterns of chiasma distribution and frequency are also inverted; therefore, the patterns of synapsis and chiasma distribution are independent, and recombination frequency along a chromosome is position-independent and segment-specific. Since cases of random distribution of chiasmata and recombination are known in rye, a genetic mechanism must be present that licenses specific chromosome regions for recombination. Large differences in the metaphase I pairing of the inversion in various combinations of two armed and telocentric chromosomes confirm the major role of the telomere bouquet in early homologue recognition. However, occasional synapsis and chiasmate pairing of the distal regions of normal arms with the proximal regions of the inversion suggest that an alternative mechanism for juxtaposing of homologues must also be present. Synapsis in inversion heterozygotes was mostly complete but in the antiparallel orientation, hence defying homology, but non-homologues never synapsed. Instances of synapsis strictly limited to the chiasma-capable segments of the arm suggest that, in rye, both recombination-dependent and recombination-independent mechanisms for homologue recognition must be present.     

Improper chromosome synapsis is associated with elongated RAD51 structures in the maize<Emphasis Type="Italic"> desynaptic2</Emphasis> mutant

The RecA homolog, RAD51, performs a central role in catalyzing the DNA strand exchange event of meiotic recombination. During meiosis, RAD51 complexes develop on pairing chromosomes and then most disappear upon synapsis. In the maize meiotic mutant desynaptic2 (dsy2), homologous chromosome pairing and recombination are reduced by ~70% in male meiosis. Fluorescent in situ hybridization studies demonstrate that a normal telomere bouquet develops but the pairing of a representative gene locus is still only 25%. Chromosome synapsis is aberrant as exemplified by unsynapsed regions of the chromosomes. In the mutant, we observed unusual RAD51 structures during chromosome pairing. Instead of spherical single and double RAD51 structures, we saw long thin filaments that extended along or around a single chromosome or stretched between two widely separated chromosomes. Mapping with simple sequence repeat (SSR) markers places the dsy2 gene to near the centromere on chromosome 5, therefore it is not an allele of rad51. Thus, the normal dsy2 gene product is required for both homologous chromosome synapsis and proper RAD51 filament behavior when chromosomes pair. Edited by: P. Moens     

The initiation of homologous chromosome synapsis in mouse fetal oocytes is not directly driven by centromere and telomere clustering in the bouquet

We investigated the behaviour of centromeres and distal telomeres during the initial phases of female meiosis in mice. In particular, we wished to determine whether clustering of centromeres and telomeres (bouquet formation) played the same crucial role in homologous chromosome pairing in female meiosis as it does in the male. We found that synapsis (intimate homologous chromosome pairing) is most frequently initiated in the interstitial regions of homologous chromosomes, apparently ahead of the distal regions. The proximal ends of the chromosomes appear to be disfavoured for synaptic initiation. Moreover, initiation of synapsis occurred in oocytes that showed little or no evidence of bouquet formation. A bouquet was present in a substantial proportion of cells at mid to late zygotene, and was still present in some pachytene oocytes. This pattern of bouquet formation and pairing initiation is in stark contrast to that previously described in the male mouse. We propose that although dynamic movements of centromeres and telomeres to form clusters may facilitate alignment of homologues or homologous chromosome segments during zygotene, in the female mouse positional control of synaptic initiation is dependent on some other mechanism.     

Coordinating the events of the meiotic prophase

Meiosis is a specialized type of cell division leading to the production of gametes. During meiotic prophase I, homologous chromosomes interact with each other and form bivalents (pairs of homologous chromosomes). Three major meiotic processes--chromosome pairing, synapsis and recombination--are involved in the formation of bivalents. Many recent reports have uncovered complex networks of interactions between these processes. Chromosome pairing is largely dependent on the initiation and progression of recombination in fungi, mammals and plants, but not in Caenorhabditis elegans or Drosophila. Synapsis and recombination are also tightly linked. Understanding the coordination between chromosome pairing, synapsis and recombination lends insight into many poorly explained aspects of meiosis, such as the nature of chromosome homology recognition.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.     

Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis

A polarized chromosomal arrangement with clustered telomeres in a meiotic prophase nucleus is often called bouquet and is thought to be important for the pairing of homologous chromosomes. Fluorescence in situ hybridization in fission yeast indicated that chromosomal loci are positioned in an ordered manner as anticipated from the bouquet arrangement. Blocking the formation of the telomere cluster with the kms1 mutation created a disorganized chromosomal arrangement, not only for the regions proximal to the telomere but also for interstitial regions. The kms1 mutation also affected the positioning of a linear minichromosome. Consistent with this cytological observation, the frequency of ectopic homologous recombination between a linear minichromosome and a normal chromosome increased in the kms1 background. Intragenic recombination between allelic loci is reduced in the kms1 mutant, but those between non-allelic loci are unaffected or slightly increased. Thus, telomere-led chromosome organization facilitates homologous pairing and also restricts irregular chromosome pairing during meiosis.     

Quantitative Dynamics of Telomere Bouquet Formation

The mechanism by which homologous chromosomes pair during meiosis, as a prelude to recombination, has long been mysterious. At meiosis, the telomeres in many organisms attach to the nuclear envelope and move together to form the telomere bouquet, perhaps to facilitate the homologous search. It is believed that diffusion alone is not sufficient to account for the formation of the bouquet, and that some directed movement is also required. Here we consider the formation of the telomere bouquet in a wheat-rye hybrid both experimentally and using mathematical modelling. The large size of the wheat nucleus and wheat''s commercial importance make chromosomal pairing in wheat a particularly interesting and important process, which may well shed light on pairing in other organisms. We show that, prior to bouquet formation, sister chromatid telomeres are always attached to a hemisphere of the nuclear membrane and tend to associate in pairs. We study a mutant lacking the Ph1 locus, a locus ensuring correct homologous chromosome pairing, and discover that bouquet formation is delayed in the wild type compared to the mutant. Further, we develop a mathematical model of bouquet formation involving diffusion and directed movement, where we show that directed movement alone is sufficient to explain bouquet formation dynamics.     

Telomeres act autonomously in maize to organize the meiotic bouquet from a semipolarized chromosome orientation

During meiosis, chromosomes undergo large-scale reorganization to allow pairing between homologues, which is necessary for recombination and segregation. In many organisms, pairing of homologous chromosomes is accompanied, and possibly facilitated, by the bouquet, the clustering of telomeres in a small region of the nuclear periphery. Taking advantage of the cytological accessibility of meiosis in maize, we have characterized the organization of centromeres and telomeres throughout meiotic prophase. Our results demonstrate that meiotic centromeres are polarized prior to the bouquet stage, but that this polarization does not contribute to bouquet formation. By examining telocentric and ring chromosomes, we have tested the cis-acting requirements for participation in the bouquet. We find that: (a) the healed ends of broken chromosomes, which contain telomere repeats, can enter the bouquet; (b) ring chromosomes enter the bouquet, indicating that terminal position on a chromosome is not necessary for telomere sequences to localize to the bouquet; and (c) beginning at zygotene, the behavior of telomeres is dominant over any centromere-mediated chromosome behavior. The results of this study indicate that specific chromosome regions are acted upon to determine the organization of meiotic chromosomes, enabling the bouquet to form despite large-scale changes in chromosome architecture.     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

The desynaptic (dy) and desynaptic1 (dsy1) mutations in maize (Zea mays L) cause distinct telomere-misplacement phenotypes during meiotic prophase

During meiotic prophase, telomeres actively attach themselves to the nuclear envelope and cluster in an arrangement called the bouquet. The bouquet is unique to meiosis, highly conserved, and thought to facilitate homologous chromosome synapsis. Analy sis of three-dimensional fluorescence in situ hybridization (3-D FISH) image data has been employed to characterize the bouquet in fixed pollen mother cells of maize (Zea mays L.). In order to examine the function of the bouquet further, several meiotic mutants were screened for telomeric defects using 3-D FISH as an assay. Two mutants, desynaptic (dy) and desynaptic1 (dsy1), were found to exhibit novel telomere-misplacement phenotypes. In both cases, the telomere-associated mutant phenotypes occurred prior to what was previously reported as the earliest affected stage. Three alleles of the desynaptic1 mutation (dsy1-1, dsy1-9101, and dsy1-9307) resulted in a partial bouquet phenotype at the zygotene stage of meiotic prophase. By contrast, dy nuclei contained apparently normal bouquets, but then resulted in a premature intranuclear localization of telomeres at the pachytene stage, when telomeres normally disperse but remain attached to the nuclear envelope. The dsy1 mutation is known to impair the fidelity and progression of homologous synapsis, whereas the dy mutation is known to reduce recombination rates. If the telomere misplacements are primary defects of these mutants, then these data would be consistent with the hypothesis that meiotic telomeres have at least two separable functions, one involving proper homologous chromosome synapsis at the bouquet stage and another involving post-bouquet cross-over control.     

The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.)

The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events.     

Meiotic chromosome pairing is promoted by telomere-led chromosome movements independent of bouquet formation

Chromosome pairing in meiotic prophase is a prerequisite for the high fidelity of chromosome segregation that haploidizes the genome prior to gamete formation. In the budding yeast Saccharomyces cerevisiae, as in most multicellular eukaryotes, homologous pairing at the cytological level reflects the contemporaneous search for homology at the molecular level, where DNA double-strand broken ends find and interact with templates for repair on homologous chromosomes. Synapsis (synaptonemal complex formation) stabilizes pairing and supports DNA repair. The bouquet stage, where telomeres have formed a transient single cluster early in meiotic prophase, and telomere-promoted rapid meiotic prophase chromosome movements (RPMs) are prominent temporal correlates of pairing and synapsis. The bouquet has long been thought to contribute to the kinetics of pairing, but the individual roles of bouquet and RPMs are difficult to assess because of common dependencies. For example, in budding yeast RPMs and bouquet both require the broadly conserved SUN protein Mps3 as well as Ndj1 and Csm4, which link telomeres to the cytoskeleton through the intact nuclear envelope. We find that mutants in these genes provide a graded series of RPM activity: wild-type>mps3-dCC>mps3-dAR>ndj1Δ>mps3-dNT = csm4Δ. Pairing rates are directly correlated with RPM activity even though only wild-type forms a bouquet, suggesting that RPMs promote homologous pairing directly while the bouquet plays at most a minor role in Saccharomyces cerevisiae. A new collision trap assay demonstrates that RPMs generate homologous and heterologous chromosome collisions in or before the earliest stages of prophase, suggesting that RPMs contribute to pairing by stirring the nuclear contents to aid the recombination-mediated homology search.     

SUN1 is required for telomere attachment to nuclear envelope and gametogenesis in mice

Prior to the pairing and recombination between homologous chromosomes during meiosis, telomeres attach to the nuclear envelope and form a transient cluster. However, the protein factors mediating meiotic telomere attachment to the nuclear envelope and the requirement of this attachment for homolog pairing and synapsis have not been determined in animals. Here we show that the inner nuclear membrane protein SUN1 specifically associates with telomeres between the leptotene and diplotene stages during meiotic prophase I. Disruption of Sun1 in mice prevents telomere attachment to the nuclear envelope, efficient homolog pairing, and synapsis formation in meiosis. Massive apoptotic events are induced in the mutant gonads, leading to the abolishment of both spermatogenesis and oogenesis. This study provides genetic evidence that SUN1-telomere interaction is essential for telomere dynamic movement and is required for efficient homologous chromosome pairing/synapsis during mammalian gametogenesis.     

Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing

The preconditions and early steps of meiotic chromosome pairing were studied by fluorescence in situ hybridization (FISH) with chromosome- specific DNA probes to mouse and human testis tissue sections. Premeiotic pairing of homologous chromosomes was not detected in spermatogonia of the two species. FISH with centromere- and telomere- specific DNA probes in combination with immunostaining (IS) of synaptonemal complex (SC) proteins to testis sections of prepuberal mice at days 4-12 post partum was performed to study sequentially the meiotic pairing process. Movements of centromeres and then telomeres to the nuclear envelope, and of telomeres along the nuclear envelope leading to the formation of a chromosomal bouquet were detected during mouse prophase. At the bouquet stage, pairing of a mouse chromosome-8- specific probe was observed. SC-IS and simultaneous telomere FISH revealed that axial element proteins appear as large aggregates in mouse meiocytes when telomeres are attached to the nuclear envelope. Axial element formation initiates during tight telomere clustering and transverse filament-IS indicated the initiation of synapsis during this stage. Comparison of telomere and centromere distribution patterns of mouse and human meiocytes revealed movements of centromeres and then telomeres to the nuclear envelope and subsequent bouquet formation as conserved motifs of the pairing process. Chromosome painting in human spermatogonia revealed compacted, largely mutually exclusive chromosome territories. The territories developed into long, thin threads at the onset of meiotic prophase. Based on these results a unified model of the pairing process is proposed.     

From early homologue recognition to synaptonemal complex formation

This review focuses on various aspects of chromosome homology searching and their relationship to meiotic and vegetative pairing and to the silencing of unpaired copies of genes. Chromosome recognition and pairing is a prominent characteristic of meiosis; however, for some organisms, this association (complete or partial) is also a normal part of nuclear organization. The multiple mechanisms suggested to contribute to homologous pairing are analyzed. Recognition of DNA/DNA homology also plays an important role in detecting DNA segments that are present in inappropriate number of copies before and during meiosis. In this context, the mechanisms of methylation induced premeiotically, repeat-induced point mutation, meiotic silencing by unpaired DNA, and meiotic sex chromosome inactivation will be discussed. Homologue juxtaposition during meiotic prophase can be divided into three mechanistically distinct steps, namely, recognition, presynaptic alignment, and synapsis by the synaptonemal complex (SC). In most organisms, these three steps are distinguished by their dependence on DNA double-strand breaks (DSBs). The coupling of SC initiation to (and downstream effects of) DSB formation and the exceptions to this dependency are discussed. Finally, this review addresses the specific factors that appear to promote chromosome movement at various stages of meiotic prophase, most particularly at the bouquet stage, and on their significance for homologue pairing and/or achieving a final pachytene configuration.The synaptonemal complex - 50 years     

A mammalian KASH domain protein coupling meiotic chromosomes to the cytoskeleton

Chromosome pairing is an essential meiotic event that ensures faithful haploidization and recombination of the genome. Pairing of homologous chromosomes is facilitated by telomere-led chromosome movements and formation of a meiotic bouquet, where telomeres cluster to one pole of the nucleus. In metazoans, telomere clustering is dynein and microtubule dependent and requires Sun1, an inner nuclear membrane protein. Here we provide a functional analysis of KASH5, a mammalian dynein-binding protein of the outer nuclear membrane that forms a meiotic complex with Sun1. This protein is related to zebrafish futile cycle (Fue), a nuclear envelope (NE) constituent required for pronuclear migration. Mice deficient in this Fue homologue are infertile. Males display meiotic arrest in which pairing of homologous chromosomes fails. These findings demonstrate that telomere attachment to the NE is insufficient to promote pairing and that telomere attachment sites must be coupled to cytoplasmic dynein and the microtubule system to ensure meiotic progression.     

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.