Control of formation of crossovers for proper chromosome segregation is governed by the crossover - non crossover ratio i.e., crossovers are formed at the expense of non crossovers; better known as the crossover homeostasis.
Subject Terms: Meiosis, Crossover Control, Crossover Interference, Crossover Homeostasis
Cell division is inevitable for the proper growth and development of any organism. While Mitosis helps in the somatic cell division, Meiosis acts in the germ cell for gametic division or gametogenesis. Meiosis starts with Prophase - 1 where the formation of crossover takes place. Crossovers are really important as they result into proper segregation and disjunction of chromosomes (Page and Hawley, 2003) and also, they bring about the variation in the species. Lack of crossover formation results into improper segregation and non-disjunction of chromosome and this results into aneuploidy, which can be detrimental for the organism. Thus, formation of crossovers in Meiosis is highly important. Upstream of crossovers are the double strand breaks (DSBs) that lead to the formation of crossovers (Keeney, 2001). The quality of double strand breaks to give rise to guaranteed crossover formation is known as obligate crossover or chiasma (Jones, 1984). For the accurate segregation of chromosomes, it is highly necessary that there should be at least one double strand break hence at least one crossover formation per chromosome pair. Thus, formation of crossover is a tightly controlled and regulated event in Meiosis (Kleckner et al., 2004).
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This control is achieved by two important factors. First is the phenomenon of crossover interference in which the formation of one crossover affects the formation of the other (Muller, 1916 and Sturtevant, 1915) and the second one is the crossover homeostasis that is the formation of crossovers at the expense of non crossovers. These double strand breaks are a result of spo-11 gene that produces topoisomerase like SPO-11 protein resulting into crossover formation (Keeney, 2001). Martini et al. have conducted research on the spo-11 hypomorphic (partial loss of function) allelic series to study about the crossover control in S. cerevisiae (Diaz et al., 2002; Henderson and Keeney, 2004).
The spo-11 hypomorphs have varying magnitudes of inhibitory effect on the formation of double strand breaks. spo-11da-HA produces just 20% of wild-type double strand breaks, spo-11yf-HA produces 30% of wild-type double strand break whereas spo-11-HA produces 80% of wild-type double strand breaks; where spo-11da-HA, spo-11yf-HA and spo-11-HA are different hypomorphic mutant alleles of spo-11. Usually significant amounts of double strand breaks are produced resulting into crossovers and non crossovers and so, reduction in double strand breaks should result in reduction of crossovers as well. But that is not the case. Martini et al. constructed strains carrying heterozygous markers to analyse crossover frequencies in different intervals, and they observed that in spite of reduction in double strand breaks, the crossover numbers are maintained between threshold limits of 80% and 30%. This suggests that there is some other mechanism acting in place for the control of crossover formation.
To look into this, Martini et al. tried to analyse the crossover - non crossover ratio. We know that non crossovers lead to gene conversions, and they used this fact to score for crossovers and gene conversions. They used arg-4 mutants with flanked markers (ura3 and thr1) that cannot grow on arginine lacking medium, and only wild-type with functional arg-4 gene can survive on arginine free medium. The wild-type can be generated only through a gene conversion. They used this property to screen for arg-4+ recombinants. They also measured the fraction of recombinants that had exchanged the flanking markers by growing them on uracil or threonine lacking medium. Through this they observed that the arg-4+ recombinants generated by the spo-11 mutants were less in number than those generated by the spo-11 wild-type i.e., the number of gene conversions was decreasing. These results suggested that instead of having the impact on crossovers, the reduction of double strand breaks was affecting the non crossovers or the gene conversions thus not giving rise to the arg-4+ recombinants. Hence, they arrived at the conclusion that the crossovers are formed at the expense of non crossovers in turn affecting the crossover - non crossover ratio. This phenomenon is better known as the Crossover Homeostasis.
Now, after looking into this, they had to check whether the double strand breaks were affecting the crossover interference or not. For this, they performed tetrad analysis where they looked for the presence of tetra types and non-parental ditype spores and absence of parental ditypes (Malkova et al., 2004). They checked the recombination frequency in the adjacent intervals to find out whether the interference was being affected or not. They observed that the recombination frequency hence the interference remains consistent in all the mutants and the wild-type of spo-11. This in turn proved the fact that the double strand breaks have negligible impact on the crossover interference.
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Thus, Martini et al. were able to propound that the crossover control is indeed aided by the crossover interference to have at least one crossover per chromosome pair and, crossover homeostasis acts to regulate the formation of crossovers at the expense of non crossovers and that reduction in double strand breaks in parallel does not have a considerable impact on the formation of crossovers due to the regulatory mechanism.
Martini et al. also devised a two-dimensional electrophoresis technique to analyse both the gene conversions as well as the crossovers in a HIS4LEU2 gene locus. HIS4LEU2 gene locus is a hotspot for double strand breaks and has the LEU2 sequence inserted near the HIS4 sequence (Cao et al., 1990). Martini et al. observed that in HIS4LEU2 locus, the crossover homeostasis was less functional; i.e., in the HIS4LEU2 region, the crossover homeostasis was not very much capable of maintaining or regulating the crossover control. The exact reason for this regional or locus specific variation is still unknown and needs to be determined.
(Martini et al., 2006)
Figure 1: Graph showing the crossover homeostasis i.e. the changes occurring in the crossover/non crossover (crossover/gene conversion) ratio as the Arg+ recombination activity of the spo-11 mutant decreases.
In my opinion, this is a really important paper with overwhelming information and the research carried out by Martini et al. is fabulous in terms of the spectacular results they have got and the conclusions they have drawn from it. Crossover homeostasis has been observed in several other organisms as well; for example in C. elegans where a kind of reverse homeostasis takes place to give rise to a 50cM chromosome after fusion of two 50cM long chromosomes (Hillers and Villeneuve, 2003). Also, crossover homeostasis is responsible for the formation of crossovers in a particular region of some chromosomes like the micro chromosomes in birds (Rahn and Solari, 1986) and the pseudoautosomal regions in the sex chromosomes in mammals (Burgoyne, 1982). Not only this, but this homeostatic mechanism is absent in organisms in which the crossover interference does not play a role, for example S. pombe (Munz, 1994). Similar studies have also been conducted in plants, specifically Arabidopsis thaliana showing the crossover control in meiotic division (Mercier et al., 2007). This makes our understanding of the meiosis more clear and uncovers some useful mechanisms of crossover control. Although the research demonstrates such wonderful results and conclusions, it fails to explain why the crossover homeostasis of meiotic regulation is not completely efficient; as it has been observed that it is not necessary that a single double strand break will always give rise to a crossover. Either this can be due to the limitation of the underlying molecular mechanism of the crossover homeostasis or maybe because there is some other different types of double strand break, some that resolve into crossovers while others that convert into non crossovers (Stahl et al., 2004). The paper also fails to explain the non crossover repair of the double strand breaks. To look into these aspects, a different group of scientists ventured into a different study and observed that it is the SDSA (Synthesis dependent strand annealing) pathway that helps in resolving non crossovers (Bishop et al., 2007). This shows that with the crossover homeostasis and crossover interference, there is a third dimension of control that executes non crossovers for the regulation of crossover control. Recent research carried out in C. elegans has showed that apart from double strand breaks and crossover interference, there is antirecombinase RTEL-1 (Human RTEL1) that also acts to prevent the formation of excess crossovers. This has been demonstrated by using rtel-1 mutants that have increased crossovers. It has been found that crossover interference as well as the crossover homeostasis are both suppressed in these mutants. This proves that RTEL-1 acts as a second step control in regulation of crossovers in C. elegans by promoting non crossovers (Boulton et al., 2010) in a similar fashion as observed in S. cerevisiae. Although these findings enhance our knowledge of the crossover control, however we are far away from deducing an accurate model for the crossover regulation. In spite of all this research, only hypothetical models have been proposed till date and things still remain unclear regarding the molecular aspects of the mechanism of action. These research statistics suggest that the work carried out by Martini et al. has definitely opened up a whole new field for research, and lots of work needs to be done to achieve valuable insight of the regulation of crossover control in meiosis. Several researchers are currently working on to unravel the mystery of the underlying molecular mechanism by which the regulation of crossover control takes place. Also, tremendous work needs to be done to propose the mechanism of crossover homeostasis with regards to crossover interference and the region specific differences arising in the HIS4LEU2 gene locus. Probably by using chromosome engineering we can further our understanding about this.
Figure 2: Flow diagram explaining the control of crossover formation and regulation of meiosis through crossover homeostasis and crossover interference in S. cerevisiae.
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