Specialized meiotic cell division is a key process in sexual reproduction. Accurate chromosome pairing, synapsis and recombination during meiotic prophase ensure proper chromosome segregation later on, which is vital in cell division in order to avoid aneuploidy (reviewed in Gerton and Hawley 2005). The key feature of meiosis is two successive chromosome segregations: one homolog segregation (maternal and paternal chromosomes), and another sister chromatid segregation, as in mitosis. Homolog synapsis along chromosomes length involves dynamic movements, which in their turn involve direct association of chromosome ends (reviewed in Koszul 2009). The movement and homolog pairing occur at the early stages of meiotic prophase, and are very important events in meiosis (reviewed in Koszul 2009).
The main event in meiotic prophase is homolog pairing. The way chromosomes pair is still not completely understood, and it has been noticed that different organisms engaged different features to drive pairing mechanism (reviewed in McKee 2004; Ding 2009). The early stages of the process are likely to involve interactions between telomeres and/or centromeres of homologs, which at these stages are not associated with recombination processes (reviewed in Bhalla and Dernburg 2008). The pairing becomes obvious when G2 prophase recombination process takes place (reviewed in Koszul 2009). However, in some organisms meiotic homolog pairing proceeds regardless of whether recombination takes place or not (see Vazquez et al. 2002).
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It is worth noticing that in many reviews 'pairing' is referred to as side-by-side alignment of homologs, which is clearly distinct from the synapsis of chromosomes in many organisms, which, in its turn, is described as specific association of chromosomes in the synaptonemal complex (SC) (reviewed in McKee 2004; Roeder 1997).
In organisms like plants, animals and filamentous fungi, the movement starts from chromosome axes being brought together along their length, so that they could be closely associated through the SC that holds homologs close together. Nevertheless, there are certain organisms in which chromosome pairing is not accompanied by the formation of SC in meiotic prophase, e.g. Schizosccharamyces pomb. However, the close association of homologous chromosomes is necessary in order to avoid their engagement with unrelated chromosomes (Koszul 2009).
These dynamic chromosome movements, pairing and synapsis occur during early stages of meiotic prophase I: chromosome polarisation and orientation during leptotene, chromosome pairing and synaptonemal complex (SC) formation during zygotene, and thickening of paired chromosomes and SC extension along the full length of the chromosomes during pachytene (Koszul 2009).
At these stages of meiosis another characteristic arrangement of chromosomes takes place which is called 'bouquet' formation, during which chromosomes' telomeres are attached to the inner nuclear envelope (NE), so that chromosomes would appear to be clustered together forming a structure that resembles a bouquet of flowers (Cande et al. 2004; Scherthan 2001). This stage of bouquet formation was observed in variety of organisms studied (Scherthan 2001), apart from Caenorhabditis elegans and Drosophila melanogaster, which showed to have a different methods of homology search (McKee 2004). Many research experiments suggested that the bouquet promoted homologous chromosome pairing and synapsis.
Some studies suggested there had to be a motion for the chromosomes to align and to pair, which would play a role of an attracting force for the homologous partners, and also a repulsing force to prevent contacts between non-homologous chromosomes (Ding et al. 2009). Two members of conserved domain protein families of SUN and KASH were identified to be involved in a mechanism for the intranuclear chromosome motion. Sad1 and Kms1 form a protein complex on the nuclear envelope and promote telomere movement (Starr 2009).
Hence, it is seen that a bouquet formation is used by the organisms as a mechanism for the chromosome alignment. Though, it is still not clear how homologs recognise each other. Ding et al. (2009) suggested that heterochromatin blocks could be involved in a specific 'barcode' formation for each chromosome, so that homologous partners could identify each other.
Another factor affecting homolog pairing are meiotic DNA double-stranded breaks (DSBs), which are generated by type II topoisomerase-like enzyme Spo11 (Keeney 1997). It has been proposed that DNA homology search during DSB repair could also allow pairing to occur in such way (Gerton and Hawley 2005). However, it was found that spo11Δ mutants could still express pairing, even though there was a defect in the mechanism of DSB formation and DNA recombination (see Weiner and Kleckner 1994). Same worked for the other genes involved in DSB formation: mutations in RAD50, MEI4 and REC102 reduced pairing frequency, but did not eliminate pairing completely.
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In this review the genes that are involved in chromosomes movement and pairing are described for yeast, plants and animals. Particular attention is paid to chromosome paring mechanism, trying to identify its key features.
Pairing in Yeast
Weiner and Kleckner (1994) analyzed chromosome pairing in spread yeast nuclei, where short regions were probed by FISH. Pairing was represented as physical interaction between two homologous chromosomes in the probed region or very close to it. The average frequency of pairing for the wild type or meiosis-specific mutant spo11Δ was ~0.45. It was seen that each pair of homologs exhibited independent of each other pairing. They also found that pairing interaction frequency was ~190 interactions per yeast genome of 12,500 kb. It was suggested that genes involved in DSB repair mechanisms could also be candidates for the functions in chromosome pairing, since homology search must be carried out in order to repair chromosomes with DSBs. Hence, the chosen candidates were RAD50, RAD51 and RAD52. It was found that null mutations of RAD50 and RAD51, and a partial deletion of RAD52 caused reduction in the pairing level. Moreover, when cell progressed into meiosis, chromosome pairing started to decrease, which, as was presumed, happened because of the disruption in meiotic DNA replication. Weiner and Kleckner have identified seven mutants that were specific to meiotic prophase and exhibited defects in chromosome pairing at different ranges from slight to severe. These were hop1Δ, mer1Δ, dmc1Δ rad51Δ, dmc1Δ, rad50S, rad50Δ, spo11Δ. Spo11Δ appeared to be the most defective one. There was no SC (synaptonemal complex) detected in hop1Δ and mer1Δ mutants that exhibited wild-type chromosome pairing levels, which suggested that chromosome pairing is independent of SC. Also, hop1Δ mutants showed very low levels of meiosis specific DSBs, which indicated that they were not required for individual pairing interactions. Nonetheless, the phenotypes of Spo11 and Rad50 showed that there was a link between pairing and DSB repair mechanisms.
Several main conclusions were made according to the results: first, chromosomes were paired in yeast cells about to enter meiosis; second, pairing involved multiple interactions, one in approx. 65 kb, which was almost the same as meiotic recombination frequency; third, pairing disappeared during meiotic S phase; forth, pairing interactions were restored during meiotic prophase stage independently of SC; fifth, DSBs specific for meiosis were not required to establish individual pairing interactions; sixth, mutants that were found to be defective at different stages during meiotic recombination, were also defective in chromosome pairing.
The observed multiple pairing interactions were guided by homologous recognition at DNA level. Weiner and Kleckner suggested a single pathway model for chromosomal interactions based on the observations from the experiments. The pathway involved establishment of pairing interactions, disruptions of those interactions during DNA replication, their reformation and stabilization at the same sites, where they were converted into recombinational interactions, and was supported by the results from the experiments with the mutants.
Scherthan et al. (1994), on the other hand, reported a high association level of homologs at the sporulation stage in budding yeast Saccharomyces cerevisiae, however these associations were not the result of meiotic pairing because synaptonemal complex and its structures were missing. It was concluded that signals resulted from the association of homologs were due to general clustering. In the condensed nuclei a significant homologue pairing was observed even for the meiotic mutants. Nevertheless, pairing efficiency was much lower for the mutants comparing to the wild type. There was also a clear inhibition of SCs in all the mutants apart from rad50S, which despite the absence of SC showed some level of homologous chromosome pairing, suggesting that the process of homolog recognition is not dependent on the presence of mature SC. As it was thought, DSBs facilitate homology searching during the early stages of meiosis, but in rad50 and spo11 mutant homology recognition still existed despite the very low amount of meiotic DSBs, indicating that DSBs are not the part of the searching mechanism.
Rockmill and Roeder (1998) speculated that homolog pairing is promoted by telomere-mediated chromosome movements in S.cerevisiae. Their data showed that there was a delay in meiosis in disomic yeast strains compared to the haploids, and that this delay was caused by the presence of homologous chromosomes. The observations showed that yeast cells that proceeded through meiosis on time were those where chromosomes were failing in homolog recognition, whereas cells that were delayed in meiosis were those with chromosomes going through the homolog pairing stage. It was stated that homolog recognition was independent of chromosome recombination and synapsis, since mutants tested still underwent homolog pairing, even though in some of them (spo11, mer2, rec104) meiosis-specific DSBs initiating meiotic recombination were avoided, and the formation of SC failed (spo11, mer2). The data also indicated that telomeres promoted pairing of homologs in disomic strains, since it was demonstrated that in 25% of the cases homologs paired in disomic strains carrying linear chromosomes, and they paired only in 8% when one of the chromosomes was circular. It was shown that two homologs will recognise each other if they are in close proximity, even if the telomeres are absent as in the case of circular chromosomes, indicating that telomeres are not required in this case. Moreover, it was found that meiosis-specific Ndj1 protein that was found to be localized specifically to the ends of meiotic chromosomes, was promoting homolog recognition so causing the delay in sporulation in disomes, and also promoting pairing by mediating telomere-dependent movements of the chromosomes. It was suggested that telomeres promoted homolog recognition by clustering together in a bouquet on the nuclear membrane.
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In addition, Csm4 was identified as a protein required for meiotic telomere dynamics and dependent on Ndj1-mediated telomere/NE association (Wanat 2008). Telomeres tend to co-localize in a bouquet at zygotene stage with less frequency in csm4Δ mutants.
S.cerevisiae hop2 mutants showed high level of SC formation between non-homologous chromosomes (Leu 1998). This was thought to be resulted from the defect in pairing mechanism. Further studies discovered MND1 gene, which when overexpressed could suppress hop2 defective phenotype (Tsubouchi and Roeder 2002). Mnd1 and Hop2 proteins were demonstrated to form a complex and work together to facilitate homologous chromosome pairing and meiotic DSB repair, since both of them were found to resemble Rad51 and Dmc1 proteins, known as key players in the repair of DSBs (Tsubouchi and Roeder 2002).
Studies of homolog pairing in fission yeast Schizosaccharomyces pombe revealed Meu13p protein that was required for proper homologous chromosome pairing and recombination (Nabeshima 2001). S.cerevisiae Spo11 homologue Rec12p, which is known to be involved in recombination process in meiosis, was also found to mediate pairing process in S.pombe. However, Rec12p was not essential for proper functioning of Meu13p, since meu13p mutants showed to have pairing defect regardless whether rec12p was absent or not, suggesting that pairing is not dependent on recombination in S.pombe.
Pairing in Plants
In 1957 Okamoto discovered a single locus called Ph1, which was believed to control chromosome pairing and recombination in wheat by affecting the specificity of centromere associations that initiate homologous pairing (Moore 2010). Luo et al. (1996) in their study showed that Ph1 was instead processing homology along the chromosome lengths. It was also reported that Ph1 eliminated recombination between homeologous chromosome pairs, and even in homeologous segments where centromeres and both telomeres were homologous, whereas in the absence of Ph1 recombination could occur properly in homologous and related chromosome pairs (Luo et al. 1996). These observations suggested the role of Ph1 in recombination rather than in pairing mechanism. Mikhailova et al. (1998) on the other hand suggested that Ph1 was directly involved in chromosome pairing, since it was observed that the deletion of ph1b allele resulted in disruption of premiotic chromosome association. The new role of Ph1 was also emphasised: chromosome condensation appeared to be disturbed in ph1bph1b mutants, which, as was suggested by the authors, could be related to the disturbed homeologous chromosome pairing, possibly by preventing the ordered movement of chromosomes and so increasing the probability of homeologous collisions (Mikhailova et al. 1998). Nevertheless, the results from Corredor et al. (2007) studies indicated that in no way Ph1 locus was responsible for controlling bivalent pairing through the centromeres in wheat, since when the constitution of centromeres was modified in homozygotes and heterozygotes it did not have any effect on meiosis I chiasmata associations of chromosomes neither in wild-type nor in Ph1 mutants. Other studies by Boden et al. (2008) showed that ASY1 homologue TaASY1 in bread wheat, which is required for chromosome synapsis and promotion of homologous chromosome pairing during meiosis I, is affected by Ph1 that regulates the levels of TaASY1 expression during meiosis. The absence of Ph1 also affected localisation of TaASY1 (Boden 2008). Their findings suggested that ASY1 was involved in Ph1-dependent control of homologue pairing.
Pawlowski et al. (2004) identified poor homologous synapsis1 (phs1) gene in maize that was regulating homologous chromosome pairing and was required to prevent synapsis between non-homologous chromosomes. A very low level of synapsis was observed for phs1 mutants at zygonema stage of meiosis I prophase, and those structures that synapsed showed improper alignment. Maize also expresses telomere bouquet formation, which is known to promote chromosome pairing, and was found to be little affected in phs1 mutants (Pawlowski 2004). phs1 mutants also expressed a defect in recombination that resulted in the reduction in the number of RAD51 foci. These mutant phenotypes suggested that the phs1 gene could be involved in the mechanism of homology search and the process of coordinating pairing, recombination and synapsis.
Budding yeast protein Hop2 has a similar role to Phs1 protein in maize (Pawlowski 2004; Leu 1998).
Studies in rice identified PAIR3 which localised to the core of the chromosomes appearing as foci in preleptotene (shown by immunological experiments), and showed to be essential for telomere clustering and bouquet formation, homologous chromosome pairing, SC assembly and normal chromosome recombination during meiosis (Wang 2011; Yuan 2009). Moreover, PAIR3 appeared to be required for proper chromosome localization of PAIR2, which is associated with chromosome axes and its mutation causes elimination of homologous paring and synapsis (Nonomura 2004).
As was already mentioned (see introduction), telomere clustering in a bouquet arrangement facilitates early stages of homologous chromosome pairing in many organisms. However, in Arabidopsis thaliana telomeres do not form a bouquet, but instead a nucleolus-associated cluster is formed in early leptotene (Armstrong 2001). GFP-labelling experiments in Arabidopsis diploid guard cells indicated to the significant level of pairing, but showed no contribution to meiotic pairing in centromere associations in somatic cells (Kato and Lam 2003).
In male Drosophila pairing occurs in the absence of recombination.
In C.elegans homologous chromosome recognition, pairing and synapsis occur independently of DSB formation and recombination.
Similar mechanisms may drive the meiotic telomere movements during prophase I for the variety of species (reviewed in Osman et al. 2011).
A role of cohesins in chromosome pairing was revealed in Coprinus cinereus, which involved Rad9 protein acting in Mre11-dependent repair of DNA (Cummings 2002). It was found that the cohesion was lost in rad9-1 mutant nuclei which also failed at homolog pairing. Furthermore, msh5-22 mutation, resulting in the defect in premeiotic DNA replication and failure to make sister chromatids, seemed to partially suppress the rad9-1 mutant phenotype in homolog pairing, still exhibiting quite noticeable decrease in the level of pairing, suggesting that pairing defects in rad9-1 mutant could be affected by poor interactions of sister chromatids (Cummingns 2002).
Other studies described Mer3, Msh4 and Mlh1 recombination proteins essential for normal meiotic chromosome pairing in filamentous fungus Sordaria macrospora (Storlazzi 2002). Mer3 is involved in stabilizing recombinational interactions, and Msh4 and Mlh1 are acting as DNA mismatch repair proteins (Nakagawa 2002; Snowden 2004; Argueso 2003). All three mutants: mer3Δ, msh4Δ and mlh1Δ, showed to have defects in pairing and synapsis, as well as in bouquet formation (Storlazzi 2002).
Pairing in Animals
In S.cerevisiae recombination is required for SC formation and chromosome segregation, whereas if there is a mutation affecting SC formation, it does not prevent recombination (Vazquez 2002). In contrast, in Drosopphila and C.elegans the formation of SC and chromosome segregation occur even in the absence of recombination.
In the study by Vazquez et al. (2002) pairing in male Drosophila was analysed. They have concluded that Drosophila chromosomes were already paired when they entered meiosis and that the level of chromosome pairing was not dependent upon synapsis and recombination (reviewed in McKee 2004). It was found that homologous pairing was taking place in spermatogonial nuclei during interphase, and that homologs remained paired in G1 and S phases. The observations suggested that in young spermatocytes pairing occurred strictly at euchromatic regions and it was non-specific at heterochromatic regions (Vazquez 2002). In addition, earlier studies using fluorescent in situ hybridization (FISH) revealed that pairing between Drosophila chromosomes was achieved at separated individual loci, rather than through continuous interaction of the sites along the length of the chromosomes (Fung 1998). Tomkiel et al. (2001) discovered a teflon (tef) gene which was suggested to be responsible for establishing and/or regulating pairing of all autosomal bivalents in meiosis I in male Drosophila. Mutation in tef did not affect univalent chromosome transmission in meiosis, which indicated to its particular role in pairing-dependent processes. Furthermore, it was speculated that tef defect was likely to impact maintenance of homolog pairing rather than the initiation, since no difference was noticed between wild-type and mutant spermatocytes until late prophase (Tomkiel 2001).
Another protein called SUN1 was described to be associated with telomeres during prophase in meiosis I. Results from the studies in mice showed that the disruption of Sun1 gene led to a prevention of telomere attachment to the nuclear envelop (NE), followed by the prevention of meiotic homolog pairing and meiotic synapsis (Ding 2007). Sun1-/- mutants showed dramatic decrease in pairing levels for chromosomes 8 and 19 analyzed in the study. Moreover, because of the impairment of telomere attachment to the NE and failure to form a bouquet, the work of pairing and synapsis mechanisms was inhibited, but could still be proceeded if the formation of DSBs was initiated, as was shown in the study by Ding et al. (2007). This demonstrated the important role of telomere attachment to the NE in pairing and synapsis mechanisms for homologous chromosomes in mammals.
Mammalian SUN1 and SUN2 belong to the SUN-domain protein family, which is conserved from yeast to mammals (Hodzic 2004). Both proteins are identified as homologs of UNC-84 protein in C.elegans required for nuclear migration and positioning (Hodzic 2004; Schmitt 2007; Ding 2007). As Sun1, Sun2 was also found to be associated with NE attachment sites of meiotic chromosome telomeres (Schmitt 2007). Hence, because of the Sun2 involvement in telomere clustering, which, as was previously established, affects homologous chromosome pairing during meiosis, it could be speculated that Sun2 also contributes to the functioning of the pairing mechanism.
SUN1 in C.elegans acts in a complex with matefin at leptotene and zygotene stages of meiosis I. SUN1/matefin protein was proposed to move chromosome ends bringing them together, so they could then shuffled and could find the right partner for synapsis (Baudrimont 2010). Phosphorylation of SUN1 was said to regulate homolog pairing and recombination in C.elegans (Baudrimont 2010).
S.pombe Sad1p and mammalian Sun2 were suggested to have a common telomere clustering and bouquet formation mechanism which is conserved in eukaryotes (Schmitt 2007).
Earlier studies of Caenorhabditis elegans proposed the existence of homolog recognition regions (HRR) at one end of the chromosome, which apparently defined proper pairing and recombination of homologs (McKim 1993). It was noticed that these regions had to be positioned in cis for meiotic homologous chromosome recombination and segregation (Phillips 2009). Mutant analysis using duplications and translocations revealed unc-54 region on chromosome I, which when disrupted caused failure in homolog pairing (McKim 1993). It was noticed that homologs only paired and recombined if the HRR was present (reviewed in McKim 2007).
DSBs are not required for the initiation of synapsis in C.elegans and Drosophila (McKim 2007).
Homolog recognition regions, also called pairing centres (PC), were found to be required for initiation of synapsis and stabilisation of pairing, but either mechanism could function without the presence of one another (MacQueen 2005). However, different studies showed that these meiotic events, synapsis and pairing, did not necessarily require PCs to be able to take place, but still synapsis failed when the PC was deleted (MacQueen 2005).
A gene called him-8 was discovered to encode zinc-finger protein C2H2 that localised to a PC on X chromosome in C.elegans (Phillips 2005). Mutation in the gene resulted in the defect in stabilization of homolog pairing and initiation of synapsis, hence, suggesting its role in both mechanisms.
HIM-8 paralogs ZIM-1,2 and 3 were also found to be localised to PCs during early stages of meiosis, and to be required for meiotic pairing and synapsis (Phillips 2006; reviewed in McKim 2007). All four are known to be associated with nuclear envelop, which indicates their role in a similar to a bouquet formation mechanism (Phillips 2006; reviewed in Hawley and Gilliland 2009). Moreover, HIM-8/ZIM proteins work together to ensure proper binding of each of these proteins to a specific short sequence domain on the every chromosome (Phillips 2009). The short sequences were found in pairing centres of the chromosomes, and were suggested to be specifically required for the recognition by HIM-8/ZIM proteins, and proper subsequent homolog pairing, synapsis, recombination and segregation (Phillips 2009).