Rat PRDM9 shapes recombination landscapes, duration of meiosis, gametogenesis, and age of fertility.

BACKGROUND: Vertebrate meiotic recombination events are concentrated in regions (hotspots) that display open chromatin marks, such as trimethylation of lysines 4 and 36 of histone 3 (H3K4me3 and H3K36me3). Mouse and human PRDM9 proteins catalyze H3K4me3 and H3K36me3 and determine hotspot positions, whereas other vertebrates lacking PRDM9 recombine in regions with chromatin already opened for another function, such as gene promoters. While these other vertebrate species lacking PRDM9 remain fertile, inactivation of the mouse Prdm9 gene, which shifts the hotspots to the functional regions (including promoters), typically causes gross fertility reduction; and the reasons for these species differences are not clear. RESULTS: We introduced Prdm9 deletions into the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, about 40% to positions corresponding to Prdm9-independent mouse hotspots, including promoters. Despite the hotspot relocation and decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced healthy offspring. The percentage of normal pachytene spermatocytes in SHR-Prdm9 mutants was almost double than in the PWD male mouse oligospermic sterile mutants. We previously found a correlation between the crossover rate and sperm presence in mouse Prdm9 mutants. The crossover rate of SHR is more similar to sperm-carrying mutant mice, but it did not fully explain the fertility of the SHR mutants. Besides mild meiotic arrests at rat tubular stages IV (mid-pachytene) and XIV (metaphase), we also detected postmeiotic apoptosis of round spermatids. We found delayed meiosis and age-dependent fertility in both sexes of the SHR mutants. CONCLUSIONS: We hypothesize that the relative increased fertility of rat versus mouse Prdm9 mutants could be ascribed to extended duration of meiotic prophase I. While rat PRDM9 shapes meiotic recombination landscapes, it is unnecessary for recombination. We suggest that PRDM9 has additional roles in spermatogenesis and speciation-spermatid development and reproductive age-that may help to explain male-specific hybrid sterility.

Extensive sex differences at the initiation of genetic recombination.

Meiotic recombination differs between males and females; however, when and how these differences are established is unknown. Here we identify extensive sex differences at the initiation of recombination by mapping hotspots of meiotic DNA double-strand breaks in male and female mice. Contrary to past findings in humans, few hotspots are used uniquely in either sex. Instead, grossly different recombination landscapes result from up to fifteen-fold differences in hotspot usage between males and females. Indeed, most recombination occurs at sex-biased hotspots. Sex-biased hotspots seem to be partly determined by chromosome structure, and DNA methylation, which is absent in females at the onset of meiosis, has a substantial role. Sex differences are also evident later in meiosis as the rate at which meiotic breaks are repaired as crossovers differs between males and females in distal regions. The suppression of distal crossovers may help to minimize age-related aneuploidy that arises owing to cohesion loss during dictyate arrest in females.

Interrogating the Functions of PRDM9 Domains in Meiosis.

Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It occurs predominantly at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three conserved domains typically involved in regulation of transcription; yet, the role of PRDM9 in gene expression control is not clear. Here, we analyze the germline transcriptome of Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the effect of the KRAB, SSXRD, and post-SET zinc finger deletions in a cell culture expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in cell culture, the KRAB domain mutant mice show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that domains typically involved in regulation of gene expression do not serve that role in PRDM9, but are likely involved in setting the proper chromatin environment for initiation and completion of homologous recombination.

Erratum: The evolutionary turnover of recombination hot spots contributes to speciation in mice.

Due to a technical error in processing the figures in the above-mentioned article, Figures 3, A and B; 4B; 5B; and 6, A and C contained errors or missing elements. The errors have been corrected in both the PDF and full-text HTML files online.

The evolutionary turnover of recombination hot spots contributes to speciation in mice.

Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation.

DNA recombination. Recombination initiation maps of individual human genomes.

DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.

Suppression of genetic recombination in the pseudoautosomal region and at subtelomeres in mice with a hypomorphic Spo11 allele.

BACKGROUND: Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. RESULTS: We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes - normally the hottest region in the genome. CONCLUSIONS: Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.

Genetic recombination is directed away from functional genomic elements in mice.

Genetic recombination occurs during meiosis, the key developmental programme of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyltransferase, PR domain containing 9 (PRDM9), the product of the only known speciation-associated gene in mammals. PRDM9 is thought to determine the preferred recombination sites--recombination hotspots--through sequence-specific binding of its highly polymorphic multi-Zn-finger domain. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination. Here we map and analyse the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F(1) hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the exception of the pseudo-autosomal region (PAR)--the only area of the genome that undergoes recombination in 100% of cells. Surprisingly, hotspots are still observed in Prdm9 knockout mice, and as in wild type, these hotspots are found at H3 lysine 4 (H3K4) trimethylation marks. However, in the absence of PRDM9, most recombination is initiated at promoters and at other sites of PRDM9-independent H3K4 trimethylation. Such sites are rarely targeted in wild-type mice, indicating an unexpected role of the PRDM9 protein in sequestering the recombination machinery away from gene-promoter regions and other functional genomic elements.

Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA.

Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.

Genome-wide analysis reveals novel molecular features of mouse recombination hotspots.

Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in refs 1-5). To allow a comprehensive analysis of hotspot-associated DNA and chromatin characteristics, we developed a direct molecular approach for mapping meiotic DNA double-strand breaks that initiate recombination. Here we present the genome-wide distribution of recombination initiation sites in the mouse genome. Hotspot centres are mapped with approximately 200-nucleotide precision, which allows analysis of the fine structural details of the preferred recombination sites. We determine that hotspots share a centrally distributed consensus motif, possess a nucleotide skew that changes polarity at the centres of hotspots and have an intrinsic preference to be occupied by a nucleosome. Furthermore, we find that the vast majority of recombination initiation sites in mouse males are associated with testis-specific trimethylation of lysine 4 on histone H3 that is distinct from histone H3 lysine 4 trimethylation marks associated with transcription. The recombination map presented here has been derived from a homogeneous mouse population with a defined genetic background and therefore lends itself to extensive future experimental exploration. We note that the mapping technique developed here does not depend on the availability of genetic markers and hence can be easily adapted to other species with complex genomes. Our findings uncover several fundamental features of mammalian recombination hotspots and underline the power of the new recombination map for future studies of genetic recombination, genome stability and evolution.

Bipartite stimulatory action of the Hop2-Mnd1 complex on the Rad51 recombinase.

The HOP2 and MND1 genes are indispensable for meiotic recombination. The products of these genes associate to form a stable heterodimeric complex that binds DNA and stimulates the recombinase activity of Rad51 and Dmc1. Here we conduct molecular studies to delineate the action mechanism of the Hop2-Mnd1 complex. We present evidence to implicate Hop2 as the major DNA-binding subunit and Mnd1 as the prominent Rad51 interaction entity. Hop2-Mnd1 stabilizes the Rad51-single-stranded DNA (ssDNA) nucleoprotein filament, the catalytic intermediate in recombination reactions. We also show that Hop2-Mnd1 enhances the ability of the Rad51-ssDNA nucleoprotein filament to capture duplex DNA, an obligatory step in the formation of the synaptic complex critical for DNA joint formation. Thus, our results unveil a bipartite mechanism of Hop2-Mnd1 in homologous DNA pairing: stabilization of the Rad51 presynaptic filament and duplex DNA capture to enhance synaptic complex formation.

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases.

Genetic analysis of fission yeast suggests a role for the spHop2-Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2-Mnd1 binds single-strand DNA ends of 3'-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca(2+) alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.

Molecular activities of meiosis-specific proteins Hop2, Mnd1, and the Hop2-Mnd1 complex.

The mouse Hop2 and Mnd1 proteins, which can form a stable heterodimeric complex, ensure the proper synapsis of homologous chromosomes in meiosis by acting in concert with Rad51 and Dmc1 to promote the strand invasion (D-loop formation) step of homologous recombination. Hop2 alone promotes D-loop formation, but Mnd1 and the Hop2-Mnd1 complex do not. Here we show that only the heterodimer complex, but not the individual proteins, can stimulate strand invasion by Dmc1. Furthermore, we demonstrate that the interaction with Mnd1 provokes changes in Hop2 that are responsible not only for abrogating the recombinase activity of Hop2 but also for generating a new molecular interface able to physically interact with and stimulate Dmc1. We also show that coiled-coil motifs in Hop2 and Mnd1 are essential for their interaction with each other and that a clearly delineated region near the COOH terminus of both proteins is necessary for both the DNA binding and single-strand annealing by the Hop-Mnd1 heterodimer. Finally, we describe a point mutation in Hop2 that dissociates its strand invasion activity from its ability to bind and anneal DNA.

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models.

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination.

During the first meiotic division, homologous chromosomes (homologs) have to separate to opposite poles of the cell to ensure the right complement in the progeny. Homologous recombination provides a mechanism for a genome-wide homology search and physical linkage among the homologs before their orderly segregation. Rad51 and Dmc1 recombinases are the major players in these processes. Disruption of meiosis-specific HOP2 or MND1 genes leads to severe defects in homologous synapsis and an early-stage recombination failure resulting in sterility. Here we show that mouse Hop2 can efficiently form D-loops, the first recombination intermediates, but this activity is abrogated upon association with Mnd1. Furthermore, the Hop2-Mnd1 heterodimer physically interacts with Rad51 and Dmc1 recombinases and stimulates their activity up to 35-fold. Our data reveal an interplay among Hop2, Mnd1 and Rad51 and Dmc1 in the formation of the first recombination intermediates during meiosis.

The Hop2 protein has a direct role in promoting interhomolog interactions during mouse meiosis.

The S. cerevisiae Hop2 protein and its fission yeast homolog Meu13 are required for proper homologous chromosome pairing and recombination during meiosis. The mechanism of this requirement is, however, not understood. The previous studies in Saccharomyces suggested that Hop2 is a guardian of meiotic chromosome synapsis with the ability to prevent or resolve deleterious associations between nonhomologous chromosomes. We have generated a Hop2 knockout mouse that shows profound meiotic defects with a distinct and novel phenotype. Hop2(-/-) spermatocytes arrest at the stage of pachytene-like chromosome condensation. Axial elements are fully developed, but synapsis of any kind is very limited. Immunofluorescence analysis of meiotic chromosome spreads indicates that while meiotic double-stranded breaks are formed and processed in the Hop2 knockout, they fail to be repaired. In aggregate, the Hop2 phenotype is consistent with a direct role for the mouse Hop2 protein in promoting homologous chromosome synapsis.