De novo variants in the RNU4-2 snRNA cause a frequent neurodevelopmental syndrome.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 115 individuals with NDD. Most individuals (77.4%) have the same highly recurrent single base insertion (n.64_65insT). In 54 individuals where it could be determined, the de novo variants were all on the maternal allele. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to RNU4-1 and other U4 homologs. Using RNA-sequencing, we show how 5' splice site usage is systematically disrupted in individuals with RNU4-2 variants, consistent with the known role of this region during spliceosome activation. Finally, we estimate that variants in this 18 bp region explain 0.4% of individuals with NDD. This work underscores the importance of non-coding genes in rare disorders and will provide a diagnosis to thousands of individuals with NDD worldwide.

Characterization of meiotic recombination intermediates through gene knockouts in founder hybrid mice.

Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks, which requires choreographed binding of RPA, DMC1, and RAD51 to single-stranded DNA substrates. High-resolution in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the chromosome used as template for repair, revealing more mechanistic detail and enabling the structure of the recombination intermediates to be inferred. By applying CRISPR-Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this approach to explore the molecular detail of recombination when a key component is knocked out. As a proof of concept, we have generated hybrid biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in them. DMC1 is essential for meiotic recombination, and comparison of these maps with those from wild-type mice is informative about the structure and timing of critical recombination intermediates. We observe redistribution of RAD51 binding and complete abrogation of D-loop recombination intermediates at a molecular level in Dmc1 mutants. These data provide insight on the configuration of RPA in D-loop intermediates and suggest that stable strand exchange proceeds via multiple rounds of strand invasion with template switching in mouse. Our methodology provides a high-throughput approach for characterization of gene function in meiotic recombination at low animal cost.

Meiotic DNA breaks drive multifaceted mutagenesis in the human germ line.

Meiotic recombination commences with hundreds of programmed DNA breaks; however, the degree to which they are accurately repaired remains poorly understood. We report that meiotic break repair is eightfold more mutagenic for single-base substitutions than was previously understood, leading to de novo mutation in one in four sperm and one in 12 eggs. Its impact on indels and structural variants is even higher, with 100- to 1300-fold increases in rates per break. We uncovered new mutational signatures and footprints relative to break sites, which implicate unexpected biochemical processes and error-prone DNA repair mechanisms, including translesion synthesis and end joining in meiotic break repair. We provide evidence that these mechanisms drive mutagenesis in human germ lines and lead to disruption of hundreds of genes genome wide.

Altering the Binding Properties of PRDM9 Partially Restores Fertility across the Species Boundary.

Sterility or subfertility of male hybrid offspring is commonly observed. This phenomenon contributes to reproductive barriers between the parental populations, an early step in the process of speciation. One frequent cause of such infertility is a failure of proper chromosome pairing during male meiosis. In subspecies of the house mouse, the likelihood of successful chromosome synapsis is improved by the binding of the histone methyltransferase PRDM9 to both chromosome homologs at matching positions. Using genetic manipulation, we altered PRDM9 binding to occur more often at matched sites, and find that chromosome pairing defects can be rescued, not only in an intersubspecific cross, but also between distinct species. Using different engineered variants, we demonstrate a quantitative link between the degree of matched homolog binding, chromosome synapsis, and rescue of fertility in hybrids between Mus musculus and Mus spretus. The resulting partial restoration of fertility reveals additional mechanisms at play that act to lock-in the reproductive isolation between these two species.

Applications of Single-Cell DNA Sequencing.

Over the past decade, genomic analyses of single cells-the fundamental units of life-have become possible. Single-cell DNA sequencing has shed light on biological questions that were previously inaccessible across diverse fields of research, including somatic mutagenesis, organismal development, genome function, and microbiology. Single-cell DNA sequencing also promises significant future biomedical and clinical impact, spanning oncology, fertility, and beyond. While single-cell approaches that profile RNA and protein have greatly expanded our understanding of cellular diversity, many fundamental questions in biology and important biomedical applications require analysis of the DNA of single cells. Here, we review the applications and biological questions for which single-cell DNA sequencing is uniquely suited or required. We include a discussion of the fields that will be impacted by single-cell DNA sequencing as the technology continues to advance.

ZCWPW1 is recruited to recombination hotspots by PRDM9 and is essential for meiotic double strand break repair.

During meiosis, homologous chromosomes pair and recombine, enabling balanced segregation and generating genetic diversity. In many vertebrates, double-strand breaks (DSBs) initiate recombination within hotspots where PRDM9 binds, and deposits H3K4me3 and H3K36me3. However, no protein(s) recognising this unique combination of histone marks have been identified. We identified Zcwpw1, containing H3K4me3 and H3K36me3 recognition domains, as having highly correlated expression with Prdm9. Here, we show that ZCWPW1 has co-evolved with PRDM9 and, in human cells, is strongly and specifically recruited to PRDM9 binding sites, with higher affinity than sites possessing H3K4me3 alone. Surprisingly, ZCWPW1 also recognises CpG dinucleotides. Male Zcwpw1 knockout mice show completely normal DSB positioning, but persistent DMC1 foci, severe DSB repair and synapsis defects, and downstream sterility. Our findings suggest ZCWPW1 recognition of PRDM9-bound sites at DSB hotspots is critical for synapsis, and hence fertility.

Sexual reproduction ­ that is, the combination of sex cells from two different individuals to produce an embryo ­ is one of the many mechanisms that have evolved to maintain genetic diversity. Most human cells contain 23 pairs of chromosomes, with each chromosome in a pair carrying either a paternal or maternal copy of the same gene. To form an embryo with the right number of chromosomes, each sex cell (the egg or sperm cell) must only contain one chromosome from each pair. Sex cells are produced from parent cells containing two sets of paternal and maternal chromosomes: these cells then divide twice to form four sex cells which contain only one chromosome from each pair. Before the parent cell divides, a process known as 'recombination' takes place, which allows chromosomes in a pair to exchange bits of genetic information. This reshuffling ensures that each chromosome in a sex cell is unique. A protein called PRDM9 helps control which sections of genetic information are recombined by modifying proteins attached to the chromosomes, marking them as locations for exchange. The DNA at each of these sites is then broken and repaired using the genetic sequence of the chromosome it is paired with as a template, thus causing the two chromosomes to swap genes. In 2019, a group of researchers found a set of genes in the testis of mice that are expressed at the same time as the gene for PRDM9. This suggested that another protein called ZCWPW1 is likely involved in recombination, but the precise role of this protein was unclear. To answer this question, Wells, Bitoun et al. ­ including many of the researchers involved in the 2019 study ­ examined human cells grown in the laboratory to determine where ZCWPW1 binds to in the chromosome. This revealed that ZCWPW1 can be found at the same sites as PRDM9, which is responsible for bringing it there. Furthermore, cells from male mice lacking the gene for ZCWPW1 cannot complete the exchange of genetic information between chromosomes, meaning that the mice are infertile. As such, ZCWPW1 seems to connect location selection by PRDM9 to the DNA repair mechanisms needed for gene exchange between chromosomes. Infertility is a significant issue for humans affecting as many as one in every six couples. Fertility is complex and many of the biological mechanisms involved are not fully understood. This work suggests that both PRDM9 and ZCWPW1 are key to the production of sex cells and may be worth investigating as factors that affect fertility in humans.

The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates.

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.

Molecular structures and mechanisms of DNA break processing in mouse meiosis.

Exonucleolytic resection, critical to repair double-strand breaks (DSBs) by recombination, is not well understood, particularly in mammalian meiosis. Here, we define structures of resected DSBs in mouse spermatocytes genome-wide at nucleotide resolution. Resection tracts averaged 1100 nt, but with substantial fine-scale heterogeneity at individual hot spots. Surprisingly, EXO1 is not the major 5' → 3' exonuclease, but the DSB-responsive kinase ATM proved a key regulator of both initiation and extension of resection. In wild type, apparent intermolecular recombination intermediates clustered near to but offset from DSB positions, consistent with joint molecules with incompletely invaded 3' ends. Finally, we provide evidence for PRDM9-dependent chromatin remodeling leading to increased accessibility at recombination sites. Our findings give insight into the mechanisms of DSB processing and repair in meiotic chromatin.

Factors influencing meiotic recombination revealed by whole-genome sequencing of single sperm.

Recombination is critical to meiosis and evolution, yet many aspects of the physical exchange of DNA via crossovers remain poorly understood. We report an approach for single-cell whole-genome DNA sequencing by which we sequenced 217 individual hybrid mouse sperm, providing a kilobase-resolution genome-wide map of crossovers. Combining this map with molecular assays measuring stages of recombination, we identified factors that affect crossover probability, including PRDM9 binding on the non-initiating template homolog and telomere proximity. These factors also influence the time for sites of recombination-initiating DNA double-strand breaks to find and engage their homologs, with rapidly engaging sites more likely to form crossovers. We show that chromatin environment on the template homolog affects positioning of crossover breakpoints. Our results also offer insights into recombination in the pseudoautosomal region.

Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice.

The DNA-binding protein PRDM9 directs positioning of the double-strand breaks (DSBs) that initiate meiotic recombination in mice and humans. Prdm9 is the only mammalian speciation gene yet identified and is responsible for sterility phenotypes in male hybrids of certain mouse subspecies. To investigate PRDM9 binding and its role in fertility and meiotic recombination, we humanized the DNA-binding domain of PRDM9 in C57BL/6 mice. This change repositions DSB hotspots and completely restores fertility in male hybrids. Here we show that alteration of one Prdm9 allele impacts the behaviour of DSBs controlled by the other allele at chromosome-wide scales. These effects correlate strongly with the degree to which each PRDM9 variant binds both homologues at the DSB sites it controls. Furthermore, higher genome-wide levels of such 'symmetric' PRDM9 binding associate with increasing fertility measures, and comparisons of individual hotspots suggest binding symmetry plays a downstream role in the recombination process. These findings reveal that subspecies-specific degradation of PRDM9 binding sites by meiotic drive, which steadily increases asymmetric PRDM9 binding, has impacts beyond simply changing hotspot positions, and strongly support a direct involvement in hybrid infertility. Because such meiotic drive occurs across mammals, PRDM9 may play a wider, yet transient, role in the early stages of speciation.

Recombination in the human Pseudoautosomal region PAR1.

The pseudoautosomal region (PAR) is a short region of homology between the mammalian X and Y chromosomes, which has undergone rapid evolution. A crossover in the PAR is essential for the proper disjunction of X and Y chromosomes in male meiosis, and PAR deletion results in male sterility. This leads the human PAR with the obligatory crossover, PAR1, to having an exceptionally high male crossover rate, which is 17-fold higher than the genome-wide average. However, the mechanism by which this obligatory crossover occurs remains unknown, as does the fine-scale positioning of crossovers across this region. Recent research in mice has suggested that crossovers in PAR may be mediated independently of the protein PRDM9, which localises virtually all crossovers in the autosomes. To investigate recombination in this region, we construct the most fine-scale genetic map containing directly observed crossovers to date using African-American pedigrees. We leverage recombination rates inferred from the breakdown of linkage disequilibrium in human populations and investigate the signatures of DNA evolution due to recombination. Further, we identify direct PRDM9 binding sites using ChIP-seq in human cells. Using these independent lines of evidence, we show that, in contrast with mouse, PRDM9 does localise peaks of recombination in the human PAR1. We find that recombination is a far more rapid and intense driver of sequence evolution in PAR1 than it is on the autosomes. We also show that PAR1 hotspot activities differ significantly among human populations. Finally, we find evidence that PAR1 hotspot positions have changed between human and chimpanzee, with no evidence of sharing among the hottest hotspots. We anticipate that the genetic maps built and validated in this work will aid research on this vital and fascinating region of the genome.

The landscape of recombination in African Americans.

Recombination, together with mutation, gives rise to genetic variation in populations. Here we leverage the recent mixture of people of African and European ancestry in the Americas to build a genetic map measuring the probability of crossing over at each position in the genome, based on about 2.1 million crossovers in 30,000 unrelated African Americans. At intervals of more than three megabases it is nearly identical to a map built in Europeans. At finer scales it differs significantly, and we identify about 2,500 recombination hotspots that are active in people of West African ancestry but nearly inactive in Europeans. The probability of a crossover at these hotspots is almost fully controlled by the alleles an individual carries at PRDM9 (P value < 10(-245)). We identify a 17-base-pair DNA sequence motif that is enriched in these hotspots, and is an excellent match to the predicted binding target of PRDM9 alleles common in West Africans and rare in Europeans. Sites of this motif are predicted to be risk loci for disease-causing genomic rearrangements in individuals carrying these alleles. More generally, this map provides a resource for research in human genetic variation and evolution.