There is considerable interest in understanding patterns of linkage disequilibrium (LD) in the human genome, to aid investigations of human evolution and facilitate association studies in complex disease. The relative influences of meiotic crossover distribution and population history on LD remain unclear, however. In particular, it is uncertain to what extent crossovers are clustered into 'hot spots, that might influence LD patterns. As a first step to investigating the relationship between LD and recombination, we have analyzed a 216-kb segment of the class II region of the major histocompatibility complex (MHC) already characterized for familial crossovers. High-resolution LD analysis shows the existence of extended domains of strong association interrupted by patchwork areas of LD breakdown. Sperm typing shows that these areas correspond precisely to meiotic crossover hot spots. All six hot spots defined share a remarkably similar symmetrical morphology but vary considerably in intensity, and are not obviously associated with any primary DNA sequence determinants of hot-spot activity. These hot spots occur in clusters and together account for almost all crossovers in this region of the MHC. These data show that, within the MHC at least, crossovers are far from randomly distributed at the molecular level and that recombination hot spots can profoundly affect LD patterns.

Meiosis requires that each chromosome finds its homologous partner and undergoes at least one crossover. X-Y chromosome segregation hinges on efficient crossing-over in a very small region of homology, the pseudoautosomal region (PAR). We find that mouse PAR DNA occupies unusually long chromosome axes, potentially as shorter chromatin loops, predicted to promote double-strand break (DSB) formation. Most PARs show delayed appearance of RAD51/DMC1 foci, which mark DSB ends, and all PARs undergo delayed DSB-mediated homologous pairing. Analysis of Spo11β isoform-specific transgenic mice revealed that late RAD51/DMC1 foci on the PAR are genetically distinct from early PAR foci and global foci, and that late PAR foci promote efficient X-Y pairing, recombination and male fertility. Our findings uncover specific mechanisms that surmount the unique challenges of X-Y recombination.

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Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes1. Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase2. In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers3–8. We show here that mammals display progressive homeostatic control of recombination. In wild-type mouse spermatocytes, focus numbers for early recombination proteins (RAD51, DMC1) were highly variable from cell to cell, whereas foci of the crossover marker MLH1 showed little variability. Furthermore, mice with greater or fewer copies of the Spo11 gene — with correspondingly greater or fewer numbers of early recombination foci — displayed relatively invariant crossover numbers. Homeostatic control is enforced during at least two stages, after the formation of early recombination intermediates and later while these intermediates mature toward crossovers. Thus, variability within the mammalian meiotic program is robustly managed by homeostatic mechanisms to control crossover formation, probably to suppress aneuploidy. Meiotic recombination exemplifies how order can be progressively implemented in a self-organizing system despite natural cell-to-cell disparities in the underlying biochemical processes.

Different organisms display widely different numbers of the programmed double-strand breaks (DSBs) that initiate meiotic recombination (e.g., hundreds per meiocyte in mice and humans vs. dozens in nematodes), but little is known about what drives these species-specific DSB set points or the regulatory pathways that control them. Here we examine male mice with a lowered dosage of SPO11, the meiotic DSB catalyst, to gain insight into the effect of reduced DSB numbers on mammalian chromosome dynamics. An approximately twofold DSB reduction was associated with the reduced ability of homologs to synapse along their lengths, provoking prophase arrest and, ultimately, sterility. In many spermatocytes, chromosome subsets displayed a mix of synaptic failure and synapsis with both homologous and nonhomologous partners (''chromosome tangles''). The X chromosome was nearly always involved in tangles, and small autosomes were involved more often than large ones. We conclude that homolog pairing requirements dictate DSB set points during meiosis. Importantly, our results reveal that karyotype is a key factor: Smaller autosomes and heteromorphic sex chromosomes become weak links when DSBs are reduced below a critical threshold. Unexpectedly, unsynapsed chromosome segments trapped in tangles displayed an elevated density of DSB markers later in meiotic prophase. The unsynapsed portion of the X chromosome in wild-type males also showed evidence that DSB numbers increased as prophase progressed. These findings point to the existence of a feedback mechanism that links DSB number and distribution with interhomolog interactions.

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...

Homologous recombination deficiency (HRD) correlates with platinum sensitivity in patients with ovarian cancer, which clinically is the most useful predictor of sensitivity to PARPi. To date, there are no reliable diagnostic tools to anticipate response to platinum-based chemotherapy, thus we aimed to develop an functional HRD detection test that could predict both platinum-sensitivity and patient eligibility to targeted drug treatments. We obtained a functional HR score by quantifying homologous recombination (HR) repair after ionizing radiation-induced DNA damage in primary ovarian cancer samples ( = 32). Samples clustered in 3 categories: HR-deficient, HR-low, and HR-proficient. We analyzed the HR score association with platinum sensitivity and treatment response, platinum-free interval (PFI) and overall survival (OS), and compared it with other clinical parameters. In parallel, we performed DNA-sequencing of HR genes to assess if functional HRD can be predicted by currently offered genetic screening. Low HR scores predicted primary platinum sensitivity with high statistical significance ( = 0.0103), associated with longer PFI (HR-deficient vs. HR-proficient: 531 vs. 53 days), and significantly correlated with improved OS (HR score <35 vs. ≥35, hazard ratio = 0.08, = 0.0116). At the genomic level, we identified a few unclear mutations in HR genes and the mutational signature associated with HRD, but, overall, genetic screening failed to predict functional HRD. We developed an assay that detects tumor functional HRD and an HR score able to predict platinum sensitivity, which holds the clinically relevant potential to become the routine companion diagnostic in the management of patients with ovarian cancer..

Recombination, demographic history, drift and selection influence the extent of linkage disequilibrium (LD) in the human genome, but their relative contributions remain unclear. To investigate the effect of meiotic recombination versus population history on LD, three populations with different demographic histories (UK north Europeans, Saami and Zimbabweans) were genotyped for high-frequency single-nucleotide polymorphisms (SNPs) across a 75 kb DNA segment of the MHC class II region. This region spans three well-characterized recombination hotspots and a 60 kb long LD block. Despite a high level of underlying haplotype diversity and considerable divergence in haplotype composition between populations, all three populations showed very similar patterns of LD. Surprisingly, the entire 60 kb LD block was present even in Africans, although it was relatively difficult to detect owing to a systematic deficiency of high frequency SNPs. In contrast, DNA within recombination hotspots did not show this low nucleotide diversity in Africans. Thus, while population history has some influence on LD, our findings suggest that recombination hotspots play a major global role in shaping LD patterns as well as helping to maintain localized SNP diversity in this region of the MHC.