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Sex chromosomes in males of most eutherian mammals share only a small homologous segment, the pseudoautosomal region (PAR), in which the formation of double-strand breaks (DSBs), pairing and crossing over must occur for correct meiotic segregation 1 , 2 . How cells ensure that recombination occurs in the PAR is unknown. Here we present a dynamic ultrastructure of the PAR and identify controlling cis - and trans -acting factors that make the PAR the hottest segment for DSB formation in the male mouse genome. Before break formation, multiple DSB-promoting factors hyperaccumulate in the PAR, its chromosome axes elongate and the sister chromatids separate. These processes are linked to heterochromatic mo-2 minisatellite arrays, and require MEI4 and ANKRD31 proteins but not the axis components REC8 or HORMAD1. We propose that the repetitive DNA sequence of the PAR confers unique chromatin and higher-order structures that are crucial for recombination. Chromosome synapsis triggers collapse of the elongated PAR structure and, notably, oocytes can be reprogrammed to exhibit spermatocyte-like levels of DSBs in the PAR simply by delaying or preventing synapsis. Thus, the sexually dimorphic behaviour of the PAR is in part a result of kinetic differences between the sexes in a race between the maturation of the PAR structure, formation of DSBs and completion of pairing and synapsis. Our findings establish a mechanistic paradigm for the recombination of sex chromosomes during meiosis. In mice, the pseudoautosomal region of the sex chromosomes undergoes a dynamic structural rearrangement to promote a high rate of DNA double-strand breaks and to ensure X–Y recombination.

Meiotic recombination involves the generation of double-strand breaks, that needs to be carefully controlled to avoid fetal aneuploidy. In worms and yeast, crossover numbers are constant regardless of the amount of double-strand breaks. Jasin and colleagues now show that such crossover homeostasis mechanisms exist at two stages in mammalian meiosis. Humans suffer from high rates of fetal aneuploidy, often arising from the absence of meiotic crossover recombination between homologous chromosomes 1 . Meiotic recombination is initiated by double-strand breaks (DSBs) generated by the SPO11 transesterase 2 . In yeast and worms, at least one buffering mechanism, crossover homeostasis, maintains crossover numbers despite variation in DSB numbers 3 , 4 , 5 , 6 , 7 , 8 . We show here that mammals exhibit 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—exhibited 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 towards 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.

Meiosis requires that each chromosome find its homologous partner and undergo 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 in the PAR are genetically distinct from both 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.

Lack of crossing-over in meiosis can trigger an apoptotic response at metaphase I by the spindle assembly checkpoint (SAC). In contrast to females, segregation of sex chromosomes in males poses a particular challenge as recombination and chiasma formation is restricted to the pseudoautosomal region, the small region of homology between X and Y chromosomes. Existing data indicate that low levels of crossover failure in male meiosis can be tolerated without compromising fertility, while high levels of X-Y dissociation (in ≥70 % of cells) result in widespread apoptosis and subsequent infertility, demonstrated earlier, e.g., in Spo11β-only mice. Here, we explore the threshold of X-Y recombination failure frequency that is compatible with fertility. We show that in Spo11β-onlyᵐᵇ mice with a mixed genetic background, in contrast to Spo11β-only mice with a C57BL/6 background, X-Y pairing fails in ~50 % of cells but this still allows for sperm production without any overt impact on fertility. We also review data on apoptosis and fertility from other achiasmate mouse models and propose that the incidence of homolog dissociation that can be tolerated in vivo without compromising male fertility lies between 50 and 70 %.

Exploring non-genetic evolution of cell states during cancer treatments has become attainable by recent advances in lineage-tracing methods. However, transcriptional changes that drive cells into resistant fates may be subtle, necessitating high resolution analysis. Here, we present ReSisTrace that uses shared transcriptomic features of sister cells to predict the states priming treatment resistance. Applying ReSisTrace in ovarian cancer cells perturbed with olaparib, carboplatin or natural killer (NK) cells reveals pre-resistant phenotypes defined by proteostatic and mRNA surveillance features, reflecting traits enriched in the upcoming subclonal selection. Furthermore, we show that DNA repair deficiency renders cells susceptible to both DNA damaging agents and NK killing in a context-dependent manner. Finally, we leverage the obtained pre-resistance profiles to predict and validate small molecules driving cells to sensitive states prior to treatment. In summary, ReSisTrace resolves pre-existing transcriptional features of treatment vulnerability, facilitating both molecular patient stratification and discovery of synergistic pre-sensitizing therapies. Transcriptional cell states can drive treatment resistance in cancer. Here, the authors develop ReSisTrace to predict cell states that are primed to resist ovarian cancer treatment and validate their findings using small molecule inhibitors.

Long interspersed nuclear elements (LINE-1s/L1s) are a group of retrotransposons that can copy themselves within a genome. In humans, it is the most successful transposon in nucleotide content. L1 expression is generally mild in normal human tissues, but the activity has been shown to increase significantly in many cancers. Few studies have examined L1 expression at single-cell resolution, thus it is undetermined whether L1 reactivation occurs solely in malignant cells within tumors. One of the cancer types with frequent L1 activity is high-grade serous ovarian carcinoma (HGSOC). Here, we identified locus-specific L1 expression with 3′ single-cell RNA sequencing in pre- and post-chemotherapy HGSOC sample pairs from 11 patients, and in fallopian tube samples from five healthy women. Although L1 expression quantification with the chosen technique was challenging due to the repetitive nature of the element, we found evidence of L1 expression primarily in cancer cells, but also in other cell types, e.g. cancer-associated fibroblasts. The expression levels were similar in samples taken before and after neoadjuvant chemotherapy, indicating that L1 transcriptional activity was unaffected by clinical platinum-taxane treatment. Furthermore, L1 activity was negatively associated with the expression of MYC target genes, a finding that supports earlier literature of MYC being an L1 suppressor.

The production of gametes (sperm and eggs in mammals) involves two sequential cell divisions, meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate to different daughter cells, and meiosis II resembles mitotic divisions in that sister chromatids separate. While in principle the process is identical in males and females, the time frame and susceptibility to chromosomal defects, including achiasmy and cohesion weakening, and the response to mis-segregating chromosomes are not. In this review, we compare and contrast meiotic spindle assembly checkpoint function and aneuploidy in the two sexes.

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 1 , 2 , 3 , 4 , 5 . The relative influences of meiotic crossover distribution and population history on LD remain unclear, however 5 . In particular, it is uncertain to what extent crossovers are clustered into 'hot spots, 6 , 7 , 8 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 9 . 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.

Key Points Meiotic recombination events are distributed nonrandomly across the genome. Single-molecule methods for the recovery of recombinant DNA molecules directly from sperm DNA have allowed recombination analysis to be carried out at high resolution in mammals. Recent studies indicate that most meiotic recombination events (in both yeast and mammals) occur at highly localized hot spots (1–2 kb in width), whereas the bulk of the DNA is 'cold'. Recombination hot spots in mice and humans are sites of initiation and resolution of both crossovers and non-crossover gene conversions. Studies in model organisms, primarily budding yeast, provide direct insight into the molecular mechanisms behind hot-spot activity in mammals. The primary determinant of the recombination distribution in yeast is the distribution of the double-strand breaks (DSBs) that initiate recombination. Recombination hot spots are DSB hot spots. The frequency of DSBs in yeast is determined by the large-scale structural features of a chromosome as well as by local chromatin structure. In principle, regional variation in crossover frequencies might also reflect differences in the likelihood that a given DSB will give rise to a crossover as opposed to a non-crossover product. The fact that recombination hot spots are highly localized and separated by recombinationally 'cold' DNA is important in structuring the genome into linkage disequilibrium (LD)/haplotype blocks. Until recently, recombination studies in humans and mice had identified only a few anecdotal examples of crossover hot spots. Recently, the pace of discovery has accelerated. In every genomic segment that has been examined at sufficiently high resolution, recombination events have a punctate recombination distribution: they are clustered within small (1–2-kb) regions that are surrounded by large stretches of recombinationally suppressed DNA. Here, we review progress in understanding the distribution of mammalian recombination events, tie mammalian results together with informative studies in budding yeast and discuss the consequences of these findings for genome diversity and evolution.

Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism. In most copy events, only the L1 sequence is inserted, however, they can also mobilize the flanking non-repetitive region by a process known as 3′ transduction. L1 insertions can contribute to genome plasticity and cause potentially tumorigenic genomic instability. However, detecting the activity of a particular source L1 and identifying new insertions stemming from it is a challenging task with current methodological approaches. We developed a long-distance inverse PCR (LDI-PCR) based approach to monitor the mobility of active L1 elements based on their 3′ transduction activity. LDI-PCR requires no prior knowledge of the insertion target region. By applying LDI-PCR in conjunction with Nanopore sequencing (Oxford Nanopore Technologies) on one L1 reported to be particularly active in human cancer genomes, we detected 14 out of 15 3′ transductions previously identified by whole genome sequencing in two different colorectal tumour samples. In addition we discovered 25 novel highly subclonal insertions. Furthermore, the long sequencing reads produced by LDI-PCR/Nanopore sequencing enabled the identification of both the 5′ and 3′ junctions and revealed detailed insertion sequence information.