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.     

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.     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

A test of the CoHR motif associated with meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is not random along chromosomes; rather, there are preferred regions for initiation called hotspots. Although the general properties of meiotic hotspots are known, the requirements at the DNA sequence level for the determination of hotspot activity are still unclear. The sequence of six known hotspots in Saccharomyces cerevisiae was compared to identify a common homology region (CoHR). They reported that the locations of CoHR sequences correspond to mapped double-strand break (DSB) sites along three chromosomes (I, III, VI). We report here that a deletion of CoHR at HIS2, a hotspot used to identify the motif, has no significant effect on recombination. In the absence of CoHR, DSB formation occurs at a high frequency and at the same sequences as in wild-type strains. In cases where the deletion of sequences containing the CoHR motif has been shown to reduce recombination, we propose that it may be a reflection of the location of the deletion, rather than the loss of CoHR, per se.     

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 ^+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.     

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.     

Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot

In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases) called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.     

A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes

Meiotic recombination is a fundamental process in all eukaryotes. Among organisms in which recombination initiates prior to synapsis, recombination preferentially occurs in short 1-to 2-kb regions, known as recombination hotspots. Among mammals, genotyping sperm DNA has provided a means of monitoring recombination events at specific hotspots in male meiosis. To complement these current techniques, we developed an assay for amplifying all copies of a hotspot from the DNA of male and female germ cells, cloning the products into Escherichia coli, and SNP genotyping the resulting colonies using fluorescence technology. This approach examines the molecular details of crossover and noncrossover events of individual meioses directly at active hotspots while retaining the simplicity of using pooled DNA. Using this technique, we analyzed recombination events at the Hlx1 hotspot located on mouse chromosome 1, finding that the results agree well with a prior genetic characterization of 3026 male and 3002 female meioses.     

High recombination rates and hotspots in a Plasmodium falciparum genetic cross

Background

The human malaria parasite Plasmodium falciparum survives pressures from the host immune system and antimalarial drugs by modifying its genome. Genetic recombination and nucleotide substitution are the two major mechanisms that the parasite employs to generate genome diversity. A better understanding of these mechanisms may provide important information for studying parasite evolution, immune evasion and drug resistance.

Results

Here, we used a high-density tiling array to estimate the genetic recombination rate among 32 progeny of a P. falciparum genetic cross (7G8 × GB4). We detected 638 recombination events and constructed a high-resolution genetic map. Comparing genetic and physical maps, we obtained an overall recombination rate of 9.6 kb per centimorgan and identified 54 candidate recombination hotspots. Similar to centromeres in other organisms, the sequences of P. falciparum centromeres are found in chromosome regions largely devoid of recombination activity. Motifs enriched in hotspots were also identified, including a 12-bp G/C-rich motif with 3-bp periodicity that may interact with a protein containing 11 predicted zinc finger arrays.

Conclusions

These results show that the P. falciparum genome has a high recombination rate, although it also follows the overall rule of meiosis in eukaryotes with an average of approximately one crossover per chromosome per meiosis. GC-rich repetitive motifs identified in the hotspot sequences may play a role in the high recombination rate observed. The lack of recombination activity in centromeric regions is consistent with the observations of reduced recombination near the centromeres of other organisms.     

Redirecting meiotic DNA break hotspot determinant proteins alters localized spatial control of DNA break formation and repair

During meiosis, DNA double-strand breaks (DSBs) are formed at high frequency at special chromosomal sites, called DSB hotspots, to generate crossovers that aid proper chromosome segregation. Multiple chromosomal features affect hotspot formation. In the fission yeast S. pombe the linear element proteins Rec25, Rec27 and Mug20 are hotspot determinants – they bind hotspots with high specificity and are necessary for nearly all DSBs at hotspots. To assess whether they are also sufficient for hotspot determination, we localized each linear element protein to a novel chromosomal site (ade6 with lacO substitutions) by fusion to the Escherichia coli LacI repressor. The Mug20-LacI plus lacO combination, but not the two separate lac elements, produced a strong ade6 DSB hotspot, comparable to strong endogenous DSB hotspots. This hotspot had unexpectedly low ade6 recombinant frequency and negligible DSB hotspot competition, although like endogenous hotspots it manifested DSB interference. We infer that linear element proteins must be properly placed by endogenous functions to impose hotspot competition and proper partner choice for DSB repair. Our results support and expand our previously proposed DSB hotspot-clustering model for local control of meiotic recombination.     

Unexpected DNA context-dependence identifies a new determinant of Chi recombination hotspots

Homologous recombination occurs especially frequently near special chromosomal sites called hotspots. In Escherichia coli, Chi hotspots control RecBCD enzyme, a protein machine essential for the major pathway of DNA break-repair and recombination. RecBCD generates recombinogenic single-stranded DNA ends by unwinding DNA and cutting it a few nucleotides to the 3′ side of 5′ GCTGGTGG 3′, the sequence historically equated with Chi. To test if sequence context affects Chi activity, we deep-sequenced the products of a DNA library containing 10 random base-pairs on each side of the Chi sequence and cut by purified RecBCD. We found strongly enhanced cutting at Chi with certain preferred sequences, such as A or G at nucleotides 4–7, on the 3′ flank of the Chi octamer. These sequences also strongly increased Chi hotspot activity in E. coli cells. Our combined enzymatic and genetic results redefine the Chi hotspot sequence, implicate the nuclease domain in Chi recognition, indicate that nicking of one strand at Chi is RecBCD''s biologically important reaction in living cells, and enable more precise analysis of Chi''s role in recombination and genome evolution.     

PRDM9 Drives Evolutionary Erosion of Hotspots in Mus musculus through Haplotype-Specific Initiation of Meiotic Recombination

Meiotic recombination generates new genetic variation and assures the proper segregation of chromosomes in gametes. PRDM9, a zinc finger protein with histone methyltransferase activity, initiates meiotic recombination by binding DNA at recombination hotspots and directing the position of DNA double-strand breaks (DSB). The DSB repair mechanism suggests that hotspots should eventually self-destruct, yet genome-wide recombination levels remain constant, a conundrum known as the hotspot paradox. To test if PRDM9 drives this evolutionary erosion, we measured activity of the Prdm9 ^Cst allele in two Mus musculus subspecies, M.m. castaneus, in which Prdm9^Cst arose, and M.m. domesticus, into which Prdm9^Cst was introduced experimentally. Comparing these two strains, we find that haplotype differences at hotspots lead to qualitative and quantitative changes in PRDM9 binding and activity. Using Mus spretus as an outlier, we found most variants affecting PRDM9^Cst binding arose and were fixed in M.m. castaneus, suppressing hotspot activity. Furthermore, M.m. castaneus×M.m. domesticus F1 hybrids exhibit novel hotspots, with large haplotype biases in both PRDM9 binding and chromatin modification. These novel hotspots represent sites of historic evolutionary erosion that become activated in hybrids due to crosstalk between one parent''s Prdm9 allele and the opposite parent''s chromosome. Together these data support a model where haplotype-specific PRDM9 binding directs biased gene conversion at hotspots, ultimately leading to hotspot erosion.     

Sexual preference of meiotic recombination within the H-2 complex

The recombination frequency between the H-2K and H-2D marker loci in male mice was measured using heterozygotes that carry the H-2 ^wm7 haplotype derived from the Japanese wild mouse and common H-2 haplotypes derived from inbred mice. Previous mating experiments in which backcross progeny of heterozygous females were screened demonstrated that the H-2 ^2m7 displays marked enhancement of recombination within the H-2 complex. In contrast to recombination in female mice, no enhancement of recombination was observed during male meiosis in the present study. Thus, it appeared that enhancement of recombination is specific to female mice. A genealogical study of recombination indicated that the postmeiotic stage is not involved in the generation of sexual preference of enhancement of recombination, suggesting that the preference is meiotic-drive and that a female-specific mechanism is involved in meiotic recombination mediated by the H-2 ^wm7 haplotype.     

A Recombination Hotspot in a Schizophrenia-Associated Region of GABRB2

Background

Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the β₂ subunit of GABA_A receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes.

Methodology/Principal Findings

In the present study, the possible occurrence of recombination in this ‘S1–S29’ segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H_d) over mutation rate (θ), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia.

Conclusions/Significance

The co-occurrence of hotspot recombination and positive selection in the S1–S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.     

Prdm9 Polymorphism Unveils Mouse Evolutionary Tracks

PR/SET domain containing 9 (Prdm9) mediates histone modifications such as H3K4me3 and marks hotspots of meiotic recombination. In many mammalian species, the Prdm9 gene is highly polymorphic. Prdm9 polymorphism is assumed to play two critical roles in evolution: to diversify the spectrum of meiotic recombination hotspots and to cause male hybrid sterility, leading to reproductive isolation and speciation. Nevertheless, information about Prdm9 sequences in natural populations is very limited. In this study, we conducted a comprehensive population survey on Prdm9 polymorphism in the house mouse, Mus musculus. Overall M. musculus Prdm9 displays an extraordinarily high level of polymorphism, particularly in regions encoding zinc finger repeats, which recognize recombination hotspots. Prdm9 alleles specific to various M. musculus subspecies dominate in subspecies territories. Moreover, introgression into other subspecies territories was found for highly divergent Prdm9 alleles associated with t-haplotype. The results of our phylogeographical analysis suggest that the requirement for hotspot diversity depends on geographical range and time span in mouse evolution, and that Prdm9 polymorphism has not been maintained by a simple balanced selection in the population of each subspecies.     

A Highlights from MBoC Selection: EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.