Hotspots of meiotic recombination in the mouse major histocompatibility complex

Meiotic recombination is not random in the proximal region of the mouse major histocompatibility complex (MHC). It is clustered at four restricted positions, so-called hotspots. Some of the MHC haplotypes derived from Asian wild mice enhance recombination at the hotspots in genetic crosses with standard MHC haplotypes of laboratory mouse strains. In particular, the wm7 haplotype derived from Japanese wild mouse indicated an approximately 2% recombination frequency within a 1.2 kb fragment of DNA in the interval between the Pb and Ob genes. Interestingly, this enhancement of recombination was observed only in female meiosis but not in male meiosis. Mating experiments demonstrated that the wm7 haplotype carries a genetic factor in the region proximal to the hotspot, which instigates recombination. In addition, the wm7 haplotype has a genetic factor located in the region distal to the hotspot, which suppresses recombination. From the molecular characterization of the two hotspots located in the Eb gene and the Pb-Ob interval, it appeared that there are several common molecular elements, the consensus of the middle repetitive MT-family, TCTG or CCTG tetramer repeats, and the solitary long terminal repeat (LTR) of mouse retrovirus.     

Genetic control of sex-dependent meiotic recombination in the major histocompatibility complex of the mouse.

Meiotic recombination within the proximal region of the major histocompatibility complex (MHC) of the mouse is not random but occurs in clusters at certain restricted sites, so-called recombinational hotspots. The wm7 haplotype of the MHC, derived from the wild mouse, enhances recombination specifically during female meiosis within a fragment of 1.3 kb of DNA located between the A beta 3 and A beta 2 genes in genetic crosses with laboratory haplotypes. Previous studies revealed no significant strain differences in nucleotide sequences around the hotspot, irrespective of the ability of the strain to enhance the recombination. It appeared that a distant genetic element might, therefore, control the rate of recombination. In the present study, original recombinants whose breakpoints were defined by direct sequencing of PCR-amplified DNAs were tested for the rate of secondary recombination in the crosses with laboratory strains in order to determine the location of such a genetic element. The results clearly demonstrated that the chromosomal segment proximal to the hotspot is essential for enhancement of recombination. Moreover, the male recombination is suppressed by a segment distal to the hotspot.     

Hotspots of homologous recombination in mouse meiosis

The molecular mapping of recombinational breakpoints in the proximal region of the mouse MHC has revealed four hotspots at which breakpoints are clustered. A direct comparison of the nucleotide sequences of two independent hotspots revealed common molecular elements: a consensus sequence of the middle-repetitive MT-family, a repeat of tetramer sequences and a sequence homologous to a solitary LTR of mouse retroviruses. Extremely high frequency of recombination is observed at these hotspots when particular MHC haplotypes are used in genetic crosses. Wild mouse-derived wm7 haplotype instigates recombination at the hotspot located at the 3′-end of the Lmp-2 gene only during female meiosis. Fine genetic analysis demonstrated that the wm7 haplotype carries a genetic factor to instigate recombination and another factor to suppress recombination specifically during male meiosis. In addition, there is no dose effect of the hotspot on frequency of recombination. Finally, we described an attempt to establish an efficient in vitro assay system for monitoring recombination using plasmid DNAs that contain the Lmp-2 hotspot and nuclear extracts prepared from mouse testis.     

No dosage effect of recombinational hotspots in the mouse major histocompatibility complex

The sites of meiotic recombination in the proximal region of the mouse major histocompatibility complex (MHC) are clustered at hotspots. Some MHC haplotypes derived from Asian wild mice increase the frequency of recombination at such hotspots when heterozygous with standard laboratory haplotypes. The wm7 and cas3 haplotypes, have a hotspot close to the Lmp-2 gene (Lmp-2 hotspot), and the cas4 haplotype has a hotspot about 100 kilobase (kb) proximal, close to the Pb gene (Pb hotspot). To examine the effect of a double dose of hotspots, we estimated the rate of recombination and determined the location of the breakpoints in crosses of wm7/cas3 and wm7/cas4. In 3570 backcross progeny we identified 29 new recombinants in the H-2K to Ab interval, at a frequency of 0.81%. This frequency is 40-fold higher than in crosses between laboratory haplotypes and very similar to those previously obtained in crosses between these wild and standard laboratory haplotypes. Thus, a double dose of hotspots has no additive effect on the frequency of meiotic recombination. The site-specificity of recombination was also conserved. Twenty-three breakpoints were confined within 5.4 kb in the Lmp-2 hotspot, and six breakpoints from the cas4 cross were located in the Pb hotspot, which we have now confined to a 15 kb segment. Correspondence to: T. Shiroishi.     

SNP microarray analysis for genome-wide detection of crossover regions

There is a great deal of interest in understanding the non-random distribution of recombination events over the human genome, because it has important implications for using linkage disequilibrium (LD) to identify human disease genes. So far, only a few recombination hotspots in the human genome have been characterised and the identification of new crossover hotspots will contribute to a better understanding of the mechanisms that govern their formation and distribution. This study shows that high-density single nucleotide polymorphism (SNP) arrays, together with the presented analysis method, are an appropriate tool for generating a whole-genome recombination pattern and for detecting new crossover regions with enhanced recombination frequency. Based on the genotype data of 16 members of a Caucasian three-generation family, we identified 825 crossover regions. The average recombination frequency of females and males was 0.77 and 0.56 cM/Mb, respectively. We detected 24 crossover regions showing elevated recombination activity, which comprised known hotspots, like the MHC II region, confirming the non-random distribution of recombination events along the genome. Interestingly, 29.2% of the identified crossover hotspot regions overlapped with regions flanked by segmental duplications published by Bailey et al. (Science 297:1003–1007, 2002) suggesting that segmental duplications and crossover hotspot regions are mechanistically linked. By extrapolating the results of the present study, we conclude that it might be feasible, at least in part, to estimate to what extent the block-like pattern of LD exactly relies on the genome-wide crossover pattern using the next generation high-density SNP microarrays.Electronic Supplementary Material Supplementary material is available for this article at     

Patterns of recombination activity on mouse chromosome 11 revealed by high resolution mapping

The success of high resolution genetic mapping of disease predisposition and quantitative trait loci in humans and experimental animals depends on the positions of key crossover events around the gene of interest. In mammals, the majority of recombination occurs at highly delimited 1-2 kb long sites known as recombination hotspots, whose locations and activities are distributed unevenly along the chromosomes and are tightly regulated in a sex specific manner. The factors determining the location of hotspots started to emerge with the finding of PRDM9 as a major hotspot regulator in mammals, however, additional factors modulating hotspot activity and sex specificity are yet to be defined. To address this limitation, we have collected and mapped the locations of 4829 crossover events occurring on mouse chromosome 11 in 5858 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. This chromosome was chosen for its medium size and high gene density and provided a comparison with our previous analysis of recombination on the longest mouse chromosome 1. Crossovers were mapped to an average resolution of 127 kb, and thirteen hotspots were mapped to <8 kb. Most crossovers occurred in a small number of the most active hotspots. Females had higher recombination rate than males as a consequence of differences in crossover interference and regional variation of sex specific rates along the chromosome. Comparison with chromosome 1 showed that recombination events tend to be positioned in similar fashion along the centromere-telomere axis but independently of the local gene density. It appears that mammalian recombination is regulated on at least three levels, chromosome-wide, regional, and at individual hotspots, and these regulation levels are influenced by sex and genetic background but not by gene content.     

The Red Queen theory of recombination hotspots

Recombination hotspots are small chromosomal regions, where meiotic crossover events happen with high frequency. Recombination is initiated by a double‐strand break (DSB) that requires the intervention of the molecular repair mechanism. The DSB repair mechanism may result in the exchange of homologous chromosomes (crossover) and the conversion of the allelic sequence that breaks into the one that does not break (biased gene conversion). Biased gene conversion results in a transmission advantage for the allele that does not break, thus preventing recombination and rendering recombination hotspots transient. How is it possible that recombination hotspots persist over evolutionary time (maintaining the average chromosomal crossover rate) when they are self‐destructive? This fundamental question is known as the recombination hotspot paradox and has attracted much attention in recent years. Yet, that attention has not translated into a fully satisfactory answer. No existing model adequately explains all aspects of the recombination hotspot paradox. Here, we formulate an intragenomic conflict model resulting in Red Queen dynamics that fully accounts for all empirical observations regarding the molecular mechanisms of recombination hotspots, the nonrandom targeting of the recombination machinery to hotspots and the evolutionary dynamics of hotspot turnover.     

Molecular characterization of a meiotic recombinational hotspot enhancing homologous equal crossing-over.

We have cloned and sequenced a meiotic recombinational hotspot between the A beta 3 and A beta 2 genes in the major histocompatibility complex (MHC) of the mouse. This recombinational hotspot in the Mus musculus castaneus cas3 haplotype was previously localized to a region of 9.5 kb of DNA in which five independent crossing-over events occurred at the unusually high frequency of 0.6%. Aside from cas3, the hotspot appears to be absent in many other MHC haplotypes. We have now confined the five recombinational breakpoints to a stretch of 3.5 kb of DNA. From the nucleotide sequence around the recombinational breakpoints, determined in the parental cas3 and b haplotypes as well as for two recombinant haplotypes, we show that the two recombinant haplotypes were generated by homologous equal crossing-over and place the breakpoints within two non-overlapping stretches of 10 and 36 bp, respectively. Comparison of the DNA sequences of the hotspot-positive cas3 and the hotspot-negative b haplotypes reveals a number of differences, in particular, a CAGA-repeat sequence which is present in CAS3 in six, but only four copies in C57BL/6 DNA. This repeat sequence is reminiscent of one in a previously characterized hotspot in the E beta gene.     

Live hot, die young: transmission distortion in recombination hotspots

There is strong evidence that hotspots of meiotic recombination in humans are transient features of the genome. For example, hotspot locations are not shared between human and chimpanzee. Biased gene conversion in favor of alleles that locally disrupt hotspots is a possible explanation of the short lifespan of hotspots. We investigate the implications of such a bias on human hotspots and their evolution. Our results demonstrate that gene conversion bias is a sufficiently strong force to produce the observed lack of sharing of intense hotspots between species, although sharing may be much more common for weaker hotspots. We investigate models of how hotspots arise, and find that only models in which hotspot alleles do not initially experience drive are consistent with observations of rather hot hotspots in the human genome. Mutations acting against drive cannot successfully introduce such hotspots into the population, even if there is direct selection for higher recombination rates, such as to ensure correct segregation during meiosis. We explore the impact of hotspot alleles on patterns of haplotype variation, and show that such alleles mask their presence in population genetic data, making them difficult to detect.     

Localized breakdown in linkage disequilibrium does not always predict sperm crossover hot spots in the human MHC class II region

To investigate the relationship between meiotic crossover hot spots and block-like linkage disequilibrium (LD), we have extended our high-resolution studies of the human MHC class II region to a 90-kb segment upstream of the HLA-DOA gene. LD blocks in this region are not as well defined as in the neighboring 210-kb DNA segment but do show two regions of LD breakdown in which coalescent analysis indicates substantial historical recombination. Sperm crossover analysis of one region revealed a novel localized hot spot similar in intensity and morphology to most other MHC hot spots. Crossovers at this hot spot are not obviously affected by a large insertion/deletion polymorphism near the hot spot. The second region of LD breakdown, within the DPB1 gene, shows an extremely low level of sperm crossover activity and does not contain a sperm crossover hot spot. These results highlight the complexity of LD patterns and the importance of experimentally verifying crossover hot spots.     

A high-resolution linkage-disequilibrium map of the human major histocompatibility complex and first generation of tag single-nucleotide polymorphisms

Autoimmune, inflammatory, and infectious diseases present a major burden to human health and are frequently associated with loci in the human major histocompatibility complex (MHC). Here, we report a high-resolution (1.9 kb) linkage-disequilibrium (LD) map of a 4.46-Mb fragment containing the MHC in U.S. pedigrees with northern and western European ancestry collected by the Centre d'Etude du Polymorphisme Humain (CEPH) and the first generation of haplotype tag single-nucleotide polymorphisms (tagSNPs) that provide up to a fivefold increase in genotyping efficiency for all future MHC-linked disease-association studies. The data confirm previously identified recombination hotspots in the class II region and allow the prediction of numerous novel hotspots in the class I and class III regions. The region of longest LD maps outside the classic MHC to the extended class I region spanning the MHC-linked olfactory-receptor gene cluster. The extended haplotype homozygosity analysis for recent positive selection shows that all 14 outlying haplotype variants map to a single extended haplotype, which most commonly bears HLA-DRB1*1501. The SNP data, haplotype blocks, and tagSNPs analysis reported here have been entered into a multidimensional Web-based database (GLOVAR), where they can be accessed and viewed in the context of relevant genome annotation. This LD map allowed us to give coordinates for the extremely variable LD structure underlying the MHC.     

Trans-Regulation of Mouse Meiotic Recombination Hotspots by Rcr1

Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1–2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) × CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74–17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.     

A combination of cis and trans control can solve the hotspot conversion paradox

There is growing evidence that in a variety of organisms the majority of meiotic recombination events occur at a relatively small fraction of loci, known as recombination hotspots. If hotspot activity results from the DNA sequence at or near the hotspot itself (in cis), these hotspots are expected to be rapidly lost due to biased gene conversion, unless there is strong selection in favor of the hotspot itself. This phenomenon makes it very difficult to maintain existing hotspots and even more difficult for new hotspots to evolve; it has therefore come to be known as the "hotspot conversion paradox." I develop an analytical framework for exploring the evolution of recombination hotspots under the forces of selection, mutation, and conversion. I derive the general conditions under which cis- and trans-controlled hotspots can be maintained, as well as those under which new hotspots controlled by both a cis and a trans locus can invade a population. I show that the conditions for maintenance of and invasion by trans- or cis-plus-trans-controlled hotspots are broader than for those controlled entirely in cis. Finally, I show that a combination of cis and trans control may allow for long-lived polymorphisms in hotspot activity, the patterns of which may explain some recently observed features of recombination hotspots.     

Persistence and loss of meiotic recombination hotspots

The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This "hotspot paradox" has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activity and its evolutionary consequences. With this model, unopposed hotspot activity could drive strong hotspots from 50% representation to extinction within 70 generations. Although the crossing over that hotspots cause can increase population fitness, this benefit was always too small to slow the loss of hotspots. Hotspots could not be maintained by plausible rates of de novo mutation, nor by crossover interference, which alters the frequency and/or spacing of crossovers. Competition among hotspots for activity-limiting factors also did not prevent their extinction, although the rate of hotspot loss was slowed. Key factors were the probability that the initiating hotspot allele is destroyed and the nonmeiotic contributions hotspots make to fitness. Experimental investigation of these deserves high priority, because until the paradox is resolved all components of the mechanism are open to doubt.     

Application of coalescent methods to reveal fine-scale rate variation and recombination hotspots

There has been considerable recent interest in understanding the way in which recombination rates vary over small physical distances, and the extent of recombination hotspots, in various genomes. Here we adapt, apply, and assess the power of recently developed coalescent-based approaches to estimating recombination rates from sequence polymorphism data. We apply full-likelihood estimation to study rate variation in and around a well-characterized recombination hotspot in humans, in the beta-globin gene cluster, and show that it provides similar estimates, consistent with those from sperm studies, from two populations deliberately chosen to have different demographic and selectional histories. We also demonstrate how approximate-likelihood methods can be used to detect local recombination hotspots from genomic-scale SNP data. In a simulation study based on 80 100-kb regions, these methods detect 43 out of 60 hotspots (ranging from 1 to 2 kb in size), with only two false positives out of 2000 subregions that were tested for the presence of a hotspot. Our study suggests that new computational tools for sophisticated analysis of population diversity data are valuable for hotspot detection and fine-scale mapping of local recombination rates.     

The M26 hotspot of Schizosaccharomyces pombe stimulates meiotic ectopic recombination and chromosomal rearrangements.

Homologous recombination is increased during meiosis between DNA sequences at the same chromosomal position (allelic recombination) and at different chromosomal positions (ectopic recombination). Recombination hotspots are important elements in controlling meiotic allelic recombination. We have used artificially dispersed copies of the ade6 gene in Schizosaccharomyces pombe to study hotspot activity in meiotic ectopic recombination. Ectopic recombination was reduced 10-1000-fold relative to allelic recombination, and was similar to the low frequency of ectopic recombination between naturally repeated sequences in S. pombe. The M26 hotspot was active in ectopic recombination in some, but not all, integration sites, with the same pattern of activity and inactivity in ectopic and allelic recombination. Crossing over in ectopic recombination, resulting in chromosomal rearrangements, was associated with 35-60% of recombination events and was stimulated 12-fold by M26. These results suggest overlap in the mechanisms of ectopic and allelic recombination and indicate that hotspots can stimulate chromosomal rearrangements.     

Recombination locations and rates in beef cattle assessed from parent-offspring pairs

Background

Recombination events tend to occur in hotspots and vary in number among individuals. The presence of recombination influences the accuracy of haplotype phasing and the imputation of missing genotypes. Genes that influence genome-wide recombination rate have been discovered in mammals, yeast, and plants. Our aim was to investigate the influence of recombination on haplotype phasing, locate recombination hotspots, scan the genome for Quantitative Trait Loci (QTL) and identify candidate genes that influence recombination, and quantify the impact of recombination on the accuracy of genotype imputation in beef cattle.

Methods

2775 Angus and 1485 Limousin parent-verified sire/offspring pairs were genotyped with the Illumina BovineSNP50 chip. Haplotype phasing was performed with DAGPHASE and BEAGLE using UMD3.1 assembly SNP (single nucleotide polymorphism) coordinates. Recombination events were detected by comparing the two reconstructed chromosomal haplotypes inherited by each offspring with those of their sires. Expected crossover probabilities were estimated assuming no interference and a binomial distribution for the frequency of crossovers. The BayesB approach for genome-wide association analysis implemented in the GenSel software was used to identify genomic regions harboring QTL with large effects on recombination. BEAGLE was used to impute Angus genotypes from a 7K subset to the 50K chip.

Results

DAGPHASE was superior to BEAGLE in haplotype phasing, which indicates that linkage information from relatives can improve its accuracy. The estimated genetic length of the 29 bovine autosomes was 3097 cM, with a genome-wide recombination distance averaging 1.23 cM/Mb. 427 and 348 windows containing recombination hotspots were detected in Angus and Limousin, respectively, of which 166 were in common. Several significant SNPs and candidate genes, which influence genome-wide recombination were localized in QTL regions detected in the two breeds. High-recombination rates hinder the accuracy of haplotype phasing and genotype imputation.

Conclusions

Small population sizes, inadequate half-sib family sizes, recombination, gene conversion, genotyping errors, and map errors reduce the accuracy of haplotype phasing and genotype imputation. Candidate regions associated with recombination were identified in both breeds. Recombination analysis may improve the accuracy of haplotype phasing and genotype imputation from low- to high-density SNP panels.     

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.     

The E beta hot spot of recombination in wild-derived natural recombinant MHC haplotypes. Cross-over site mapping and the identification of a 1.0-kb E beta deletion in the p and w14 haplotypes

Detailed molecular analysis of three wild-derived MHC haplotypes provided evidence for an important role of the E beta recombinational hot spot in the recent evolution of the mouse I region. Examination of RFLP and restriction maps of cloned DNA permitted the mapping of the natural cross-over events in the haplotypes carried by strains B10.GAA37 (w21) and B10.KPB128 (w19) to a fragment of DNA not exceeding 4.1 kb, which lies almost entirely within the intron separating the beta 1 and beta 2 exons of the E beta gene. In the w14 haplotype (strain B10.STC77), which appears to be a natural recombinant between a p-like parental haplotype and another wild-derived haplotype, the site of crossing over can be mapped to a segment between the beta 2 exon of the E beta gene (left border) and the E beta 2 gene (right border). This segment containing the cross-over site in the w14 haplotype includes the E beta hot spot. In addition, the w14 haplotype as well as the standard p haplotype contain a deletion of approximately 1.0 kb in the second intron of the E beta gene, which may represent the product of an unequal cross-over event in a E beta recombinational hot spot.