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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.

A hallmark of meiosis is the rearrangement of parental alleles to ensure genetic diversity in the gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSBs occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance, Prdm9 has been repeatedly lost across many animal lineages. Both the reason for mouse sterility in the absence of PRDM9 and the mechanism by which Prdm9 can be lost remain unclear. Here, we explore whether mice can tolerate the loss of Prdm9. By generating Prdm9 functional knockouts in an array of genetic backgrounds, we observe a wide range of fertility phenotypes and ultimately demonstrate that PRDM9 is not required for completion of male meiosis. Although DSBs still form at a common subset of functional sites in all mice lacking PRDM9, meiotic outcomes differ substantially. We speculate that DSBs at functional sites are difficult to repair as a crossover and that by increasing the efficiency of crossover formation at these sites, genetic modifiers of recombination rates can allow for meiotic progression. This model implies that species with a sufficiently high recombination rate may lose Prdm9 yet remain fertile.

Sex-specific modulation of a meiotic DNA damage checkpoint limits the requirement for PRDM9 in mammalian fertility., In many mammals, genomic sites for recombination are determined by the histone methyltransferase PRMD9. Some mouse strains lacking PRDM9 are infertile, but instances of fertility or semifertility in the absence of PRDM9 have been reported in mice, canines, and a human female. Such findings raise the question of how the loss of PRDM9 is circumvented to maintain fertility. We show that genetic background and sex-specific modifiers can obviate the requirement for PRDM9 in mice. Specifically, the meiotic DNA damage checkpoint protein CHK2 acts as a modifier allowing female-specific fertility in the absence of PRDM9. We also report that, in the absence of PRDM9, a PRDM9-independent recombination system is compatible with female meiosis and fertility, suggesting sex-specific regulation of meiotic recombination, a finding with implications for speciation.

PRDM9 is a zinc finger protein that binds DNA at specific locations in the genome where it trimethylates histone H3 at lysines 4 and 36 at surrounding nucleosomes. During meiosis in many species, including humans and mice where PRDM9 has been most intensely studied, these actions determine the location of recombination hotspots, where genetic recombination occurs. In addition, PRDM9 facilitates the association of hotspots with the chromosome axis, the site of the programmed DNA double-strand breaks (DSBs) that give rise to genetic exchange between chromosomes. In the absence of PRDM9 DSBs are not properly repaired. Collectively, these actions determine patterns of genetic linkage and the possibilities for chromosome reorganization over successive generations.

Rare gene knockouts in adult humans On average, most people's genomes contain approximately 100 completely nonfunctional genes. These loss-of-function (LOF) mutations tend to be rare and/or occur only as a single copy within individuals. Narasimhan et al. investigated LOF in a Pakistani population with high levels of consanguinity. Examining LOF alleles that were identical by descent, they found, as expected, an absence of homozygote LOF for certain protein-coding genes. However, they also identified many homozygote LOF alleles with no apparent deleterious phenotype, including some that were expected to confer genetic disease. Indeed, one family had lost the recombination-associated gene PRDM9 . Science , this issue p. 474

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.

In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse models to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on PRDM9 hotspot trimethylation, double strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1., Author summary Meiotic recombination increases genetic diversity by ensuring novel combination of alleles passing correctly to the next generation. In most mammals, the meiotic recombination sites are determined by histone methyltransferase PRDM9. These sites are proposed to become associated with the chromosome axis with the participation of additional proteins and undergo double strand breaks, which are repaired by homologous recombination. In budding yeast, Spp1 (ortholog of CXXC1) binds to methylated H3K4 and connects these sites with the chromosome axis promoting DSB formation. However, our data suggest that even though CXXC1 interacts with PRDM9 in male germ cells, it does not play a crucial role in mouse meiotic recombination. These results indicate that, unlike in yeast, a recombination initiation pathway that includes CXXC1 could only serve as a non-essential pathway in mouse meiosis.

While much is known about the enzymes that deposit and remove epigenetic modifications, less is known about the trans-acting system that determines when and where these modifications occur. By looking at four diverse... The epigenetic landscape varies greatly among cell types. Although a variety of writers, readers, and erasers of epigenetic features are known, we have little information about the underlying regulatory systems controlling the establishment and maintenance of these features. Here, we have explored how natural genetic variation affects the epigenome in mice. Studying levels of H3K4me3, a histone modification at sites such as promoters, enhancers, and recombination hotspots, we found tissue-specific trans-regulation of H3K4me3 levels in four highly diverse cell types: male germ cells, embryonic stem cells, hepatocytes, and cardiomyocytes. To identify the genetic loci involved, we measured H3K4me3 levels in male germ cells in a mapping population of 59 BXD recombinant inbred lines. We found extensive trans-regulation of H3K4me3 peaks, including six major histone quantitative trait loci (QTL). These chromatin regulatory loci act dominantly to suppress H3K4me3, which at hotspots reduces the likelihood of subsequent DNA double-strand breaks. QTL locations do not correspond with genes encoding enzymes known to metabolize chromatin features. Instead their locations match clusters of zinc finger genes, making these possible candidates that explain the dominant suppression of H3K4me3. Collectively, these data describe an extensive, set of chromatin regulatory loci that control the epigenetic landscape.

Meiotic recombination is required for correct segregation of chromosomes to gametes and to generate genetic diversity. In mice and humans, DNA double-strand breaks (DSBs) are initiated by SPO11 at recombination hotspots activated by PRDM9-catalyzed histone modifications on open chromatin. However, the DSB-initiating and repair proteins are associated with a linear proteinaceous scaffold called the chromosome axis, the core of which is composed of cohesin proteins. STAG3 is a stromalin subunit common to all meiosis-specific cohesin complexes. Mutations of meiotic cohesin proteins, especially STAG3, perturb both axis formation and recombination in the mouse, prompting determination of how the processes are mechanistically related. Protein interaction and genetic analyses revealed that PRDM9 interacts with STAG3 and REC8 in cooperative relationships that promote normal levels of meiotic DSBs at recombination hotspots in spermatocytes. The efficacy of the Prdm9-Stag3 genetic interaction in promoting DSB formation depends on PRDM9-mediated histone methyltransferase activity. Moreover, STAG3 deficiency has a major effect on DSB number even in the absence of PRDM9, showing that its role is not restricted to canonical PRDM9-activated hotspots. STAG3 and REC8 promote axis localization of the DSB-promoting proteins HORMAD1, IHO1, and MEI4, as well as SPO11 activity. These results establish that PRDM9 and axis-associated cohesin complexes together coordinate and facilitate meiotic recombination by recruiting key proteins for initiation of DSBs, thereby associating activated hotspots with DSB-initiating complexes on the axis.

In mammals, meiotic recombination occurs at 1-2 kb genomic regions termed hotspots, whose positions and activities are determined by PRDM9, a DNA-binding histone methyltransferase. We now show that the KRAB domain of PRDM9 forms complexes with additional proteins to allow hotspots to proceed into the next phase of recombination. By a combination of yeast-two hybrid assay, in vitro binding, and co-immunoprecipitation from mouse spermatocytes, we identified four proteins that directly interact with PRDM9’s KRAB domain, namely CXXC1, EWSR1, EHMT2, and CDYL. These proteins are co-expressed in spermatocytes at the early stages of meiotic prophase I, the limited period when PRDM9 is expressed. We also detected association of PRDM9-bound complexes with the meiotic cohesin REC8 and the synaptonemal complex proteins SYCP3 and SYCP1. Our results suggest a model in which PRDM9-bound hotspot DNA is brought to the chromosomal axis by the action of these proteins, ensuring the proper chromatin and spatial environment for subsequent recombination events.

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis., Author Summary Genetic recombination is the meiotic process by which novel combinations of alleles are passed on to the next generation. This process accelerates evolution by creating genetic diversity, and is also essential for successful meiosis. In mammals, the enzyme PRDM9 initiates recombination and determines the subset of sites within the genome—called recombination hotspots—that recombine. PRDM9 does this by binding DNA at hotspots and placing epigenetic marks. Previously, PRDM9 was only known to place the H3K4me3 mark at hotspots in living cells. Here, we show that PRDM9 places the H3K36me3 mark at hotspots, and that H3K4me3 and H3K36me3 coincide only at regions of recombination in germ cells. We prove that this coincidence is driven almost entirely by PRDM9; there is dramatically less coincidence between H3K4me3 and H3K36me3 when PRDM9 is absent. These results reveal a new enzymatic function for PRDM9, and a new epigenetic signature at hotspots that may restrict recombination to these sites. Since aberrant recombination can cause aneuploidy resulting in fetal loss, and abnormal genome rearrangements that underlie many congenital syndromes and some cancers, a thorough understanding of this fundamental process has potentially far-reaching implications.

Complete gene knockouts are highly informative about gene function. We exome sequenced 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous likely loss of function (rhLOF) genotypes predicted to disrupt (knockout) 781 genes. Based on depletion of rhLOF genotypes, we estimate that 13.6% of knockouts are incompatible with adult life, finding on average 1.6 heterozygous recessive lethal LOF variants per adult. Linking to lifelong health records, we observed no association of rhLOF genotypes with prescription- or doctor-consultation rate, and no disease-related phenotypes in 33 of 42 individuals with rhLOF genotypes in recessive Mendelian disease genes. Phased genome sequencing of a healthy PRDM9 knockout mother, her child and controls, showed meiotic recombination sites localised away from PRDM9-dependent hotspots, demonstrating PRDM9 redundancy in humans.

Significant departures from expected Mendelian inheritance ratios (transmission ratio distortion, TRD) are frequently observed in both experimental crosses and natural populations. TRD on mouse Chromosome (Chr) 2 has been reported in multiple experimental crosses, including the Collaborative Cross (CC). Among the eight CC founder inbred strains, we found that Chr 2 TRD was exclusive to females that were heterozygous for the WSB/EiJ allele within a 9.3 Mb region (Chr 2 76.9 – 86.2 Mb). A copy number gain of a 127 kb-long DNA segment (designated as responder to drive, R2d) emerged as the strongest candidate for the causative allele. We mapped R2d sequences to two loci within the candidate interval. R2d1 is located near the proximal boundary, and contains a single copy of R2d in all strains tested. R2d2 maps to a 900 kb interval, and the number of R2d copies varies from zero in classical strains (including the mouse reference genome) to more than 30 in wild-derived strains. Using real-time PCR assays for the copy number, we identified a mutation (R2d2WSBdel1) that eliminates the majority of the R2d2WSB copies without apparent alterations of the surrounding WSB/EiJ haplotype. In a three-generation pedigree segregating for R2d2WSBdel1, the mutation is transmitted to the progeny and Mendelian segregation is restored in females heterozygous for R2d2WSBdel1, thus providing direct evidence that the copy number gain is causal for maternal TRD. We found that transmission ratios in R2d2WSB heterozygous females vary between Mendelian segregation and complete distortion depending on the genetic background, and that TRD is under genetic control of unlinked distorter loci. Although the R2d2WSB transmission ratio was inversely correlated with average litter size, several independent lines of evidence support the contention that female meiotic drive is the cause of the distortion. We discuss the implications and potential applications of this novel meiotic drive system., Author Summary One of the strongest expectations in genetics is that chromosomes segregate randomly during meiosis. However, genetic loci that exhibit transmission ratio distortion (TRD) are sometimes observed in offspring of F1 hybrids. Meiotic drive is a type of non-Mendelian inheritance in which a “selfish” genetic element exploits asymmetric female meiotic cell division to promote its preferential inclusion in ova. We previously reported TRD on Chr 2 in the CC, a mouse recombinant inbred panel with contributions from three Mus musculus subspecies. Here we show that maternal TRD consistent with a novel meiotic drive system is caused by a copy number gain. This mutation is similar in size and structure to other known meiotic drive responders, such as the knobs of maize. A deletion of most of the copies is sufficient to restore Mendelian segregation, proving that the copy number variant is causative of the observed TRD. In the CC, and also the related DO population, the transmission frequency of the favored allele varies dependent on genetic background, demonstrating that this system is under genetic control. In conclusion, we describe a novel wild-derived meiotic drive locus on mouse Chr 2 that exploits female meiosis asymmetry to violate the Laws of Mendelian inheritance.

Background Among the three functions of DNA, mammalian replication and transcription can be subject to epigenetic imprinting specified by the parental origin of chromosomes, and although there is suggestive indication that this is also true for meiotic recombination, no definitive evidence has yet been reported. Results We have now obtained such evidence on mouse chromosome 7 by assaying meiotic recombination as it occurs in reciprocal F1 mice. A 166 kb region near the Kcnq1 transcriptionally imprinted domain showed significantly higher recombination activity in the CAST×B6 parental direction (p < 0.03). Characterizing hotspots within this domain revealed a cluster of three hotspots lying within a 100 kb span, among these hotspots, Slc22a18 showed a definitive parent of origin effect on recombination frequency (p < 0.02). Comparing recombination activity in the mouse Kcnq1 and neighboring H19-Igf2 imprinted domains with their human counterparts, we found that elevated recombination activity in these domains is a consequence of their chromosomal position relative to the telomere and not an intrinsic characteristic of transcriptionally imprinted domains as has been previously suggested. Conclusion Similar to replication and transcription, we demonstrate that meiotic recombination can be subjected to epigenetic imprinting and hotspot activity can be influenced by the parental origin of chromosomes. Furthermore, transcriptionally imprinted regions exhibiting elevated recombination activity are likely a consequence of their chromosomal location rather than their transcriptional characteristic.

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 Prdm9Cst arose, and M.m. domesticus, into which Prdm9Cst 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 PRDM9Cst 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., Author Summary Sexually reproducing creatures need to produce germ cells, notably sperm and egg, and do so using a specialized cell division, termed meiosis. A hallmark of meiosis is the process of recombination, in which pieces of maternal and paternal genetic material are exchanged, creating new combinations that are inherited by their progeny. Recombination increases diversity in subsequent generations, facilitating evolution. However, if recombination goes awry, it can lead to genetic disorders and spontaneous abortions in humans. In most mammals the sites of recombination are directed by the enzyme PRDM9 to specific regions on DNA, termed hotspots. For several decades it has been speculated that the process of recombination should lead to the eventual inactivation of hotspots, resulting in the loss of ability to reproduce. The discovery of PRDM9 provided a potential solution to this dilemma when the appearance of new PRDM9 alleles with altered DNA binding specificity would immediately create a new set of hotspots. We have now used the power of mouse genetics and large scale measurements of PRDM9 location and activity to show that this cycle of hotspot loss and recovery does indeed occur over the course of hundreds of thousands of years, and is directed by PRDM9.

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., Author Summary Recombination is an essential aspect of meiosis, ensuring proper contact and exchange of genetic material between homologous parental chromosomes, as well as their subsequent segregation to produce haploid gametes. In humans and mice, recombination events are located at preferential sites termed hotspots, whose placement and activity are tightly regulated. We have now identified a hotspot-regulating locus in mammals, Rcr1, that simultaneously controls the locations of multiple hotspots. The discovery of Rcr1 indicates the existence of a newly emerging class of genes important in the recombination processes. Gaining further insights into their function may contribute to a better understanding of genetic factors underlying human fertility and evolution., Rcr1 is identified as atrans-regulator of meiotic recombination hotspots that appears to act at the initiation of recombination and that maps to a 5.3-megabase region on mouse Chromosome 17.

Evidence from inbred strains of mice indicates that a quarter or more of the mammalian genome consists of chromosome regions containing clusters of functionally related genes. The intense selection pressures during inbreeding favor the coinheritance of optimal sets of alleles among these genetically linked, functionally related genes, resulting in extensive domains of linkage disequilibrium (LD) among a set of 60 genetically diverse inbred strains. Recombination that disrupts the preferred combinations of alleles reduces the ability of offspring to survive further inbreeding. LD is also seen between markers on separate chromosomes, forming networks with scale-free architecture. Combining LD data with pathway and genome annotation databases, we have been able to identify the biological functions underlying several domains and networks. Given the strong conservation of gene order among mammals, the domains and networks we find in mice probably characterize all mammals, including humans., Synopsis The arrangement of genes along chromosomes affects their function as well as the likelihood that particular combinations of genes will be inherited together, and evolution has had many millions of years to optimize these arrangements. Because the arrangements are nearly identical in all mammals, one can use the powerful techniques of mouse genetics to explore their roles in our own genomes. The authors find that genes that cooperate in bringing about various cellular and physiological functions, such as immune responses, are often clustered together on chromosomes, and that detailed maps of these relationships can be built. The new techniques have proven so powerful that they can identify functional interactions among genes that are not even on the same chromosome. Beyond illuminating the evolutionary pressures that brought them about, mapping these arrangements will be of great utility in the ongoing searches in many laboratories for the genes underlying our common diseases, such as cancer, heart disease, and diabetes.