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During meiotic recombination, homologue-templated repair of programmed DNA double-strand breaks (DSBs) produces relatively few crossovers and many difficult-to-detect non-crossovers. By intercrossing two diverged mouse subspecies over five generations and deep-sequencing 119 offspring, we detect thousands of crossover and non-crossover events genome-wide with unprecedented power and spatial resolution. We find that both crossovers and non-crossovers are strongly depleted at DSB hotspots where the DSB-positioning protein PRDM9 fails to bind to the unbroken homologous chromosome, revealing that PRDM9 also functions to promote homologue-templated repair. Our results show that complex non-crossovers are much rarer in mice than humans, consistent with complex events arising from accumulated non-programmed DNA damage. Unexpectedly, we also find that GC-biased gene conversion is restricted to non-crossover tracts containing only one mismatch. These results demonstrate that local genetic diversity profoundly alters meiotic repair pathway decisions via at least two distinct mechanisms, impacting genome evolution and Prdm9 -related hybrid infertility. During meiotic recombination, genetic information is transferred or exchanged between parental chromosome copies. Using a large hybrid mouse pedigree, the authors generated high-resolution maps of these transfer/exchange events and discovered new properties governing their processing and resolution.

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. Human cells have two copies of each chromosome: one from the mother, and one from the father. When cells divide to form sex cells, such as sperm or egg cells, the maternal and paternal chromosomes line up next to each other and swap some of their DNA. This process, known as genetic recombination, creates different versions of genes and ensures that we are all unique – or genetically diverse. Recombination is a complex process that is largely controlled by a protein called PRDM9. This protein binds DNA at particular spots on the chromosome and directs other proteins to carry out recombination nearby. However, not all of PRDM9’s binding sites are known, and not all regions that PRDM9 binds to undergo recombination. Until now, it was not understood why this is the case at fine scales. To investigate this further, Altemose et al. activated the human version of PRDM9 in human kidney cells grown in the laboratory. The results showed that PRDM9 often bound near the start sites of genes, although these regions rarely undergo recombination in humans. When PRDM9 bound near these sites, it sometimes turned the gene on, which suggests that it may also help to regulate the activity of genes. Moreover, a specific group of DNA-binding proteins, called KRAB-ZNF proteins, appear to suppress recombination wherever they bind, which explains why some PRDM9 binding sites do not recombine. Lastly, Altemose et al. discovered that the part of PRDM9 that binds to DNA can also bind to other copies of PRDM9 proteins. This self-binding ability might play a role in bringing together the maternal and paternal chromosomes at the correct spots during recombination. Together, these results shed new light on the recombination process, which is a driving force in the formation of new species and essential for fertility. A next step will be to study these results further in tissues of the reproductive organs. This will provide a better understanding of the forces that shape human evolution.

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.

Oxidative stress is a common etiological feature of neurological disorders, although the pathways that govern defence against reactive oxygen species (ROS) in neurodegeneration remain unclear. We have identified the role of oxidation resistance 1 (Oxr1) as a vital protein that controls the sensitivity of neuronal cells to oxidative stress; mice lacking Oxr1 display cerebellar neurodegeneration, and neurons are less susceptible to exogenous stress when the gene is over-expressed. A conserved short isoform of Oxr1 is also sufficient to confer this neuroprotective property both in vitro and in vivo. In addition, biochemical assays indicate that Oxr1 itself is susceptible to cysteine-mediated oxidation. Finally we show up-regulation of Oxr1 in both human and pre-symptomatic mouse models of amyotrophic lateral sclerosis, indicating that Oxr1 is potentially a novel neuroprotective factor in neurodegenerative disease.

Netherton syndrome is a severe autosomal recessive skin disorder characterized by congenital erythroderma, a specific hair-shaft abnormality, and atopic manifestations with high IgE levels. Recently, we identified SPINK5, which encodes the serine protease inhibitor Kazal-type 5 protein (LEKTI), as the defective gene in Netherton syndrome. Here we describe the intron-exon organization of the gene and characterize the SPINK5 mutations in patients from 21 families of different geographic origin, using denaturing high performance liquid chromatography and direct sequencing. We identified 18 mutations, of which 13 were novel and seven (39%) were recurrent. The majority of the mutations were clustered between exons 1–8 and exons 21–26. They comprised four nonsense mutations (22%), eight frameshift insertions or deletions (44%), and six splice-site defects (33%). All mutations predict the formation of premature termination codons. Northern blot analysis showed variable reduction of SPINK5 mutant transcript levels, suggesting variable efficiency of nonsense-mediated mRNA decay. Seven patients were homozygotes, eight were compound heterozygotes, and five were heterozygotes with only one identifiable SPINK5 mutation. Five mutations, one of which resulted in perinatal lethal disease in three families, were associated with certain ethnic groups. We also describe 45 intragenic polymorphisms in the patients studied. The clinical features of erythroderma, trichorrhexis invaginata, and atopic manifestations were present in the majority of affected individuals and ichthyosis linearis circumflexa was seen in 12 out of 24 patients. Interfamilial and intrafamilial variation in disease severity was observed, with no clear correlation between mutations and phenotype, suggesting that the degree of severity may be affected by other factors.

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. PRDM9 is a DNA-binding protein that controls the position of double-strand breaks in meiosis, and the gene that encodes it is responsible for hybrid infertility between closely related mouse species; this hybrid infertility is eliminated by introducing the zinc-finger domain sequence from the human version of the PRDM9 gene, a change which alters both the position of double-strand breaks and the symmetry of PRDM9 binding and suggests that PRDM9 may have a more general but transient role in the early stages of speciation. Mechanism of action of Prdm9 'speciation gene' PRDM9 is a DNA-binding protein that controls the position of double-strand breaks in meiosis, and the Prdm9 gene that encodes it is the only known example of a mammalian speciation gene, being responsible for hybrid infertility between closely related mouse species. These authors show that hybrid infertility in mice can be eliminated by introducing the zinc-finger domain sequence from the human version of the gene, thereby altering the position of double-strand breaks. PRDM9 may therefore have a general but transient role in the early stages of speciation.

One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models.

We have established that the gene AF4, which had long been recognized as disrupted in childhood leukemia, also plays a role in the CNS. Af4 is mutated in the robotic mouse that is characterized by ataxia and Purkinje cell loss. To determine the molecular basis of this mutation, we carried out a yeast two-hybrid screen and show that Af4 binds the E3 ubiquitin ligases Drosophila seven in absentia (sina) homologues (Siah)-1a and Siah-2 in the brain. Siah-1a and Af4 are expressed in Purkinje cells and colocalize in the nucleus of human embryonic kidney 293T and P19 cells. In vitro binding assays and coimmunoprecipitation reveal a significant reduction in affinity between Siah-1a and robotic mutant Af4 compared with wild-type, which correlates with the almost complete abolition of mutant Af4 degradation by Siah-1a. These data strongly suggest that an accumulation of mutant Af4 occurs in the robotic mouse due to a reduction in its normal turnover by the proteasome. A significant increase in the transcriptional activity of mutant Af4 relative to wild-type was obtained in mammalian cells, suggesting that the activity of Af4 is controlled through Siah-mediated degradation. Another member of the Af4 family, Fmr2, which is involved in mental handicap in humans, binds Siah proteins in a similar manner. These results provide evidence that a common regulatory mechanism exists that controls levels of the Af4/Fmr2 protein family. The robotic mouse thus provides a unique opportunity to understand how these proteins play a role in disorders as diverse as leukemia, mental retardation, and neuro-degenerative disease.

The devastating nature and lack of effective treatments associated with neurodegenerative diseases have stimulated a world-wide search for the elucidation of their molecular basis to which mouse models have made a major contribution. In combination with transgenic and knockout technologies, large-scale mouse mutagenesis is a powerful approach for the identification of new genes and associated signalling pathways controlling neuronal cell death and survival. Here we review the characterization of the robotic mouse, a novel model of autosomal dominant cerebellar ataxia isolated from an ENU-mutagenesis programme, which develops adult-onset region-specific Purkinje cell loss and cataracts, and displays defects in early T-cell maturation and general growth retardation. The mutated protein, Af4, is a member of the AF4/LAF4/FMR2 (ALF) family of putative transcription factors previously implicated in childhood leukaemia and FRAXE mental retardation. The mutation, which lies in a highly conserved region among the ALF family members, significantly reduces the binding affinity of Af4 to the E3 ubiquitin-ligase Siah-1a, isolated with Siah-2 as interacting proteins in the brain. This leads to a markedly slower turnover of mutant Af4 by the ubiquitin-proteasome pathway and consequently to its abnormal accumulation in the robotic mouse. Importantly, the conservation of the Siah-binding domain of Af4 in all other family members reveals that Siah-mediated proteasomal degradation is a common regulatory mechanism that controls the levels, and thereby the function, of the ALF family. The robotic mouse represents a unique model in which to study the newly revealed role of Af4 in the maintenance of vital functions of Purkinje cells in the cerebellum and further the understanding of its implication in lymphopoeisis.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a putative serine protease inhibitor encoded by serine protease inhibitor Kazal-type 5 (SPINK5). It is strongly expressed in differentiated keratinocytes in normal skin but expression is markedly reduced or absent in Netherton syndrome (NS), a severe ichthyosis caused by SPINK5 mutations. At present, however, both the precise intracellular localization and biological roles of LEKTI are not known. To understand the functional role of LEKTI, we examined the localization of LEKTI together with kallikrein (KLK)7 and KLK5, possible targets of LEKTI, in the human epidermis, by confocal laser scanning microscopy and immunoelectron microscopy. In normal skin, LEKTI, KLK7, and KLK5 were all found in the lamellar granule (LG) system, but were separately localized. LEKTI was expressed earlier than KLK7 and KLK5. In NS skin, LEKTI was absent and an abnormal split in the superficial stratum granulosum was seen in three of four cases. Collectively, these results suggest that in normal skin the LG system transports and secretes LEKTI earlier than KLK7 and KLK5 preventing premature loss of stratum corneum integrity/cohesion. Our data provide new insights into the biological functions of LG and the pathogenesis of NS.