Genetic recombination occurs during meiosis, the key developmental program of gametogenesis. Recombination in mammals has been recently linked to the activity of a histone H3 methyl-transferase, PRDM91–6, the product of the only known speciation gene in mammals7. PRDM9 is thought to determine the preferred recombination sites – recombination hotspots – through sequence-specific binding of its highly polymorphic multi-Zn-finger domain8. Nevertheless, Prdm9 knockout mice are proficient at initiating recombination 9. Here we map and analyze the genome-wide distribution of recombination initiation sites in Prdm9 knockout mice and in two mouse strains with different Prdm9 alleles and their F1 hybrid. We show that PRDM9 determines the positions of practically all hotspots in the mouse genome, with the remarkable exception of the pseudoautosomal region – the only area of the genome that undergoes recombination in 100% of cells10. Surprisingly, hotspots are still observed in Prdm9 knockout mice and as in wild-type, these hotspots are found at H3K4 trimethylation marks. However, in the absence of PRDM9, the majority of 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.

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 co-localization 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.

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Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in1-5). To enable a comprehensive analysis of hotspot-associated DNA and chromatin characteristics we developed a direct molecular approach for mapping meiotic DNA double stranded breaks that initiate recombination. Here, we present the genome-wide distribution of recombination initiation sites in the mouse genome, constituting the first physical map of recombination hotspots in a multi-cellular organism. Hotspot centres are mapped with approximately 200-nucleotide precision that enables 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 centre 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. Importantly, the mapping technique developed here does not depend on availability of genetic markers and hence can be easily adapted for 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.

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.

CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function.[Keywords: RNA-binding protein; noncoding RNA; CTCF; insulator function] Supplemental material is available at http://www.genesdev.org.

Sex chromosomes in males of most eutherian species share only a diminutive homologous segment, the pseudoautosomal region (PAR), wherein double-strand break (DSB) formation, pairing, and crossing over must occur for correct meiotic segregation 1,2. How cells ensure PAR recombination is unknown. Here we delineate an unexpected dynamic ultrastructure of the PAR and identify controlling cis-and transacting factors that make this the hottest area of DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyper-accumulate in the PAR, its chromosome axes elongate, and the sister chromatids separate. These phenomena are linked to heterochromatic mo-2 minisatellite arrays and require MEI4 and ANKRD31 proteins but not axis components REC8 or HORMAD1. We propose that the repetitive PAR sequence confers unique chromatin and higher order structures crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, remarkably, oocytes can be reprogrammed to display spermatocyte-like PAR DSB levels simply by delaying or preventing synapsis. Thus, sexually dimorphic behavior of the PAR rests in part on kinetic differences between the sexes for a race between maturation of PAR structure, DSB formation, and completion of pairing and synapsis. Our findings establish a mechanistic paradigm of sex chromosome recombination during meiosis. During meiotic recombination, DSBs must occur within the tiny (~700 kb 3,4) mouse PAR 2-6. Since on average one DSB forms per ten megabases, the PAR would risk frequent recombination failure if it behaved like a typical autosomal segment 2. Consequently, the PAR 2 has disproportionately frequent DSBs and recombination 2,6-8 (Supplementary Discussion). Mechanisms promoting such frequent DSBs are unknown in any species. DSBs arise concomitantly with linear axial structures that anchor chromatin loops wherein DSBs occur 9,10. Axes begin to form during replication and become assembly sites for proteins that promote SPO11 DSBs 11-13. PAR chromatin in spermatocytes forms relatively short loops on a long axis 2. However, only a low-resolution view of PAR structure was available and the controlling cis-and transacting factors were unknown. Moreover, it was unclear how spermatocytes but not oocytes make the PAR so hyperrecombinogenic. A distinctive PAR ultrastructure X and Y usually pair late, with PARs paired in less than 20% of spermatocytes at late zygonema when most autosomes are paired 2,14. At this stage, unsynapsed PAR axes (SYCP2/3) appeared thickened relative to other unsynapsed axes and had bright HORMAD1/2 staining (Fig. 1a and Extended Data Fig. 1a,b) 15. Moreover, the PAR was highly enriched for REC114, MEI4, MEI1, and IHO1-essential for genome-wide DSB formation 16-19-plus ANKRD31, a REC114 partner essential for PAR DSBs 20,21. All five proteins (RMMAI) colocalized in several bright "blobs" for most of prophase I (Fig. 1a and Extended Data Fig. 1c). Two blobs were on X and Y PARs and others highlighted specific autosome en...

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 (ChIPseq). 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.