Principles of chromosome organization for meiotic recombination.

In meiotic cells, chromosomes are organized as chromatin loop arrays anchored to a protein axis. This organization is essential to regulate meiotic recombination, from DNA double-strand break (DSB) formation to their repair. In mammals, it is unknown how chromatin loops are organized along the genome and how proteins participating in DSB formation are tethered to the chromosome axes. Here, we identify three categories of axis-associated genomic sites: PRDM9 binding sites, where DSBs form; binding sites of the insulator protein CTCF; and H3K4me3-enriched sites. We demonstrate that PRDM9 promotes the recruitment of MEI4 and IHO1, two proteins essential for DSB formation. In turn, IHO1 anchors DSB sites to the axis components HORMAD1 and SYCP3. We discovered that IHO1, HORMAD1, and SYCP3 are associated at the DSB ends during DSB repair. Our results highlight how interactions of proteins with specific genomic elements shape the meiotic chromosome organization for recombination.

Coupling crossover and synaptonemal complex in meiosis.

During meiosis, a molecular program induces DNA double-strand breaks (DSBs) and their repair by homologous recombination. DSBs can be repaired with or without crossovers. ZMM proteins promote the repair toward crossover. The sites of DSB repair are also sites where the axes of homologous chromosomes are juxtaposed and stabilized, and where a structure called the synaptonemal complex initiates, providing further regulation of both DSB formation and repair. How crossover formation and synapsis initiation are linked has remained unknown. The study by Pyatnitskaya and colleagues (pp. 53-69) in this issue of Genes & Development highlights the central role of the Saccharomyces cerevisiae ZMM protein Zip4 in this process.

Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation.

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil-based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners.

Mouse ANKRD31 Regulates Spatiotemporal Patterning of Meiotic Recombination Initiation and Ensures Recombination between X and Y Sex Chromosomes.

Orderly segregation of chromosomes during meiosis requires that crossovers form between homologous chromosomes by recombination. Programmed DNA double-strand breaks (DSBs) initiate meiotic recombination. We identify ANKRD31 as a key component of complexes of DSB-promoting proteins that assemble on meiotic chromosome axes. Genome-wide, ANKRD31 deficiency causes delayed recombination initiation. In addition, loss of ANKRD31 alters DSB distribution because of reduced selectivity for sites that normally attract DSBs. Strikingly, ANKRD31 deficiency also abolishes uniquely high rates of recombination that normally characterize pseudoautosomal regions (PARs) of X and Y chromosomes. Consequently, sex chromosomes do not form crossovers, leading to chromosome segregation failure in ANKRD31-deficient spermatocytes. These defects co-occur with a genome-wide delay in assembling DSB-promoting proteins on autosome axes and loss of a specialized PAR-axis domain that is highly enriched for DSB-promoting proteins in wild type. Thus, we propose a model for spatiotemporal patterning of recombination by ANKRD31-dependent control of axis-associated DSB-promoting proteins.

The impressionistic landscape of meiotic recombination.

Two high-resolution maps of meiotic recombination initiation sites across the genomes of budding yeast and mice illuminate broad similarities in the control of meiotic recombination in these diverse species but also highlight key differences. These studies offer new insights into the relationships between recombination, chromosome structure, and genome evolution.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

PRDM9 Methyltransferase Activity Is Essential for Meiotic DNA Double-Strand Break Formation at Its Binding Sites.

The programmed formation of hundreds of DNA double-strand breaks (DSBs) is essential for proper meiosis and fertility. In mice and humans, the location of these breaks is determined by the meiosis-specific protein PRDM9, through the DNA-binding specificity of its zinc-finger domain. PRDM9 also has methyltransferase activity. Here, we show that this activity is required for H3K4me3 and H3K36me3 deposition and for DSB formation at PRDM9-binding sites. By analyzing mice that express two PRDM9 variants with distinct DNA-binding specificities, we show that each variant generates its own set of H3K4me3 marks independently from the other variant. Altogether, we reveal several basic principles of PRDM9-dependent DSB site determination, in which an excess of sites are designated through PRDM9 binding and subsequent histone methylation, from which a subset is selected for DSB formation.

The TOPOVIBL meiotic DSB formation protein: new insights from its biochemical and structural characterization.

The TOPOVIL complex catalyzes the formation of DNA double strand breaks (DSB) that initiate meiotic homologous recombination, an essential step for chromosome segregation and genetic diversity during gamete production. TOPOVIL is composed of two subunits (SPO11 and TOPOVIBL) and is evolutionarily related to the archaeal TopoVI topoisomerase complex. SPO11 is the TopoVIA subunit orthologue and carries the DSB formation catalytic activity. TOPOVIBL shares homology with the TopoVIB ATPase subunit. TOPOVIBL is essential for meiotic DSB formation, but its molecular function remains elusive, partly due to the lack of biochemical studies. Here, we purified TOPOVIBLΔC25 and characterized its structure and mode of action in vitro. Our structural analysis revealed that TOPOVIBLΔC25 adopts a dynamic conformation in solution and our biochemical study showed that the protein remains monomeric upon incubation with ATP, which correlates with the absence of ATP binding. Moreover, TOPOVIBLΔC25 interacted with DNA, with a preference for some geometries, suggesting that TOPOVIBL senses specific DNA architectures. Altogether, our study identified specific TOPOVIBL features that might help to explain how TOPOVIL function evolved toward a DSB formation activity in meiosis.

Evolution and Diversity of the TopoVI and TopoVI-like Subunits With Extensive Divergence of the TOPOVIBL subunit.

Type II DNA topoisomerases regulate topology by double-stranded DNA cleavage and ligation. The TopoVI family of DNA topoisomerase, first identified and biochemically characterized in Archaea, represents, with TopoVIII and mini-A, the type IIB family. TopoVI has several intriguing features in terms of function and evolution. TopoVI has been identified in some eukaryotes, and a global view is lacking to understand its evolutionary pattern. In addition, in eukaryotes, the two TopoVI subunits (TopoVIA and TopoVIB) have been duplicated and have evolved to give rise to Spo11 and TopoVIBL, forming TopoVI-like (TopoVIL), a complex essential for generating DNA breaks that initiate homologous recombination during meiosis. TopoVIL is essential for sexual reproduction. How the TopoVI subunits have evolved to ensure this meiotic function is unclear. Here, we investigated the phylogenetic conservation of TopoVI and TopoVIL. We demonstrate that BIN4 and RHL1, potentially interacting with TopoVIB, have co-evolved with TopoVI. Based on model structures, this observation supports the hypothesis for a role of TopoVI in decatenation of replicated chromatids and predicts that in eukaryotes the TopoVI catalytic complex includes BIN4 and RHL1. For TopoVIL, the phylogenetic analysis of Spo11, which is highly conserved among Eukarya, highlighted a eukaryal-specific N-terminal domain that may be important for its regulation. Conversely, TopoVIBL was poorly conserved, giving rise to ATP hydrolysis-mutated or -truncated protein variants, or was undetected in some species. This remarkable plasticity of TopoVIBL provides important information for the activity and function of TopoVIL during meiosis.

Four-pronged negative feedback of DSB machinery in meiotic DNA-break control in mice.

In most taxa, halving of chromosome numbers during meiosis requires that homologous chromosomes (homologues) pair and form crossovers. Crossovers emerge from the recombination-mediated repair of programmed DNA double-strand breaks (DSBs). DSBs are generated by SPO11, whose activity requires auxiliary protein complexes, called pre-DSB recombinosomes. To elucidate the spatiotemporal control of the DSB machinery, we focused on an essential SPO11 auxiliary protein, IHO1, which serves as the main anchor for pre-DSB recombinosomes on chromosome cores, called axes. We discovered that DSBs restrict the DSB machinery by at least four distinct pathways in mice. Firstly, by activating the DNA damage response (DDR) kinase ATM, DSBs restrict pre-DSB recombinosome numbers without affecting IHO1. Secondly, in their vicinity, DSBs trigger IHO1 depletion mainly by another DDR kinase, ATR. Thirdly, DSBs enable homologue synapsis, which promotes the depletion of IHO1 and pre-DSB recombinosomes from synapsed axes. Finally, DSBs and three DDR kinases, ATM, ATR and PRKDC, enable stage-specific depletion of IHO1 from all axes. We hypothesize that these four negative feedback pathways protect genome integrity by ensuring that DSBs form without excess, are well-distributed, and are restricted to genomic locations and prophase stages where DSBs are functional for promoting homologue pairing and crossover formation.

Initiation of meiotic recombination: how and where? Conservation and specificities among eukaryotes.

Meiotic recombination is essential for fertility in most sexually reproducing species. This process also creates new combinations of alleles and has important consequences for genome evolution. Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), which are repaired by homologous recombination. DSBs are catalyzed by the evolutionarily conserved SPO11 protein, assisted by several other factors. Some of them are absolutely required, whereas others are needed only for full levels of DSB formation and may participate in the regulation of DSB timing and frequency as well as the coordination between DSB formation and repair. The sites where DSBs occur are not randomly distributed in the genome, and remarkably distinct strategies have emerged to control their localization in different species. Here, I review the recent advances in the components required for DSB formation and localization in the various model organisms in which these studies have been performed.

PRDM9, a driver of the genetic map.

During meiosis, maternal and paternal chromosomes undergo exchanges by homologous recombination. This is essential for fertility and contributes to genome evolution. In many eukaryotes, sites of meiotic recombination, also called hotspots, are regions of accessible chromatin, but in many vertebrates, their location follows a distinct pattern and is specified by PR domain-containing protein 9 (PRDM9). The specification of meiotic recombination hotspots is achieved by the different activities of PRDM9: DNA binding, histone methyltransferase, and interaction with other proteins. Remarkably, PRDM9 activity leads to the erosion of its own binding sites and the rapid evolution of its DNA-binding domain. PRDM9 may also contribute to reproductive isolation, as it is involved in hybrid sterility potentially due to a reduction of its activity in specific heterozygous contexts.

Sex chromosome quadrivalents in oocytes of the African pygmy mouse Mus minutoides that harbors non-conventional sex chromosomes.

Eutherian mammals have an extremely conserved sex-determining system controlled by highly differentiated sex chromosomes. Females are XX and males XY, and any deviation generally leads to infertility, mainly due to meiosis disruption. The African pygmy mouse (Mus minutoides) presents an atypical sex determination system with three sex chromosomes: the classical X and Y chromosomes and a feminizing X chromosome variant, called X*. Thus, three types of females coexist (XX, XX*, and X*Y) that all show normal fertility. Moreover, the three chromosomes (X and Y on one side and X* on the other side) are fused to different autosomes, which results in the inclusion of the sex chromosomes in a quadrivalent in XX* and X*Y females at meiotic prophase. Here, we characterized the configurations adopted by these sex chromosome quadrivalents during meiotic prophase. The XX* quadrivalent displayed a closed structure in which all homologous chromosome arms were fully synapsed and with sufficient crossovers to ensure the reductional segregation of all chromosomes at the first meiotic division. Conversely, the X*Y quadrivalents adopted either a closed configuration with non-homologous synapsis of the X* and Y chromosomes or an open chain configuration in which X* and Y remained asynapsed and possibly transcriptionally silenced. Moreover, the number of crossovers was insufficient to ensure chromosome segregation in a significant fraction of nuclei. Together, these findings raise questions about the mechanisms allowing X*Y females to have a level of fertility as good as that of XX and XX* females, if not higher.

In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites.

In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed an extensive analysis of PRDM9 binding in mouse spermatocytes. Unexpectedly, we identified a noncanonical recruitment of PRDM9 to sites that lack recombination activity and the PRDM9 binding consensus motif. These sites include gene promoters, where PRDM9 is recruited in a DSB-dependent manner. Another subset reveals DSB-independent interactions between PRDM9 and genomic sites, such as the binding sites for the insulator protein CTCF. We propose that these DSB-independent sites result from interactions between hotspot-bound PRDM9 and genomic sequences located on the chromosome axis.

MCM8- and MCM9-deficient mice reveal gametogenesis defects and genome instability due to impaired homologous recombination.

We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.

Meiotic recombination in mammals: localization and regulation.

During meiosis, a programmed induction of DNA double-strand breaks (DSBs) leads to the exchange of genetic material between homologous chromosomes. These exchanges increase genome diversity and are essential for proper chromosome segregation at the first meiotic division. Recent findings have highlighted an unexpected molecular control of the distribution of meiotic DSBs in mammals by a rapidly evolving gene, PR domain-containing 9 (PRDM9), and genome-wide analyses have facilitated the characterization of meiotic DSB sites at unprecedented resolution. In addition, the identification of new players in DSB repair processes has allowed the delineation of recombination pathways that have two major outcomes, crossovers and non-crossovers, which have distinct mechanistic roles and consequences for genome evolution.

A new light on the meiotic DSB catalytic complex.

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs). More than 15 years ago, Spo11 was identified as the protein responsible for meiotic DSB formation, notably because of its striking similarities with the A subunit of topoisomerase VI (TopoVI). TopoVI are enzymes that modify DNA topology by generating transient DSBs and are active as heterotetramers, composed of two A and two B subunits. A2 dimers catalyse the DNA cleavage reaction, whereas the B subunits regulate A2 conformation, DNA capture, cleavage and re-ligation. The recent identification in plants and mammals of a B-like TopoVI subunit that interacts with SPO11 and is required for meiotic DSB formation makes us to reconsider our understanding of the meiotic DSB catalytic complex. We provide here an overview of the knowledge on TopoVI structure and mode of action and we compare them with their meiotic counterparts. This allows us to discuss the nature, structure and functions of the meiotic TopoVI-like complex during meiotic DSB formation.

The PRDM9 KRAB domain is required for meiosis and involved in protein interactions.

PR domain-containing protein 9 (PRDM9) is a major regulator of the localization of meiotic recombination hotspots in the human and mouse genomes. This role involves its DNA-binding domain, which is composed of a tandem array of zinc fingers, and PRDM9-dependent trimethylation of histone H3 at lysine 4. PRDM9 is a member of the PRDM family of transcription regulators, but unlike other family members, it contains a Krüppel-associated box (KRAB)-related domain that is predicted to be a potential protein interaction domain. Here, we show that truncation of the KRAB domain of mouse PRDM9 leads to loss of PRDM9 function and altered meiotic prophase and gametogenesis. In addition, we identified proteins that interact with the KRAB domain of PRDM9 in yeast two-hybrid assay screens, particularly CXXC1, a member of the COMPASS complex. We also show that CXXC1 interacts with IHO1, an essential component of the meiotic double-strand break (DSB) machinery. As CXXC1 is orthologous to Saccharomyces cerevisiae Spp1 that links DSB sites to the DSB machinery on the chromosome axis, we propose that these molecular interactions involved in the regulation of meiotic DSB formation are conserved in mouse meiosis.

Functional conservation of Mei4 for meiotic DNA double-strand break formation from yeasts to mice.

Meiotic recombination is initiated by the programmed induction of DNA double-strand breaks (DSBs) catalyzed by the evolutionarily conserved Spo11 protein. Studies in yeast have shown that DSB formation requires several other proteins, the role and conservation of which remain unknown. Here we show that two of these Saccharomyces cerevisiae proteins, Mei4 and Rec114, are evolutionarily conserved in most eukaryotes. Mei4(-/-) mice are deficient in meiotic DSB formation, thus showing the functional conservation of Mei4 in mice. Cytological analyses reveal that, in mice, MEI4 is localized in discrete foci on the axes of meiotic chromosomes that do not overlap with DMC1 and RPA foci. We thus propose that MEI4 acts as a structural component of the DSB machinery that ensures meiotic DSB formation on chromosome axes. We show that mouse MEI4 and REC114 proteins interact directly, and we identify conserved motifs as required for this interaction. Finally, the unexpected, concomitant absence of Mei4 and Rec114, as well as of Mnd1, Hop2, and Dmc1, in some eukaryotic species (particularly Neurospora crassa, Drosophila melanogaster, and Caenorhabditis elegans) suggests the existence of Mei4-Rec114-dependent and Mei4-Rec114-independent mechanisms for DSB formation, and a functional relationship between the chromosome axis and DSB formation.

MEI4 ­ a central player in the regulation of meiotic DNA double-strand break formation in the mouse.

The formation of programmed DNA double-strand breaks (DSBs) at the beginning of meiotic prophase marks the initiation of meiotic recombination. Meiotic DSB formation is catalyzed by SPO11 and their repair takes place on meiotic chromosome axes. The evolutionarily conserved MEI4 protein is required for meiotic DSB formation and is localized on chromosome axes. Here, we show that HORMAD1, one of the meiotic chromosome axis components, is required for MEI4 localization. Importantly, the quantitative correlation between the level of axis-associated MEI4 and DSB formation suggests that axis-associated MEI4 could be a limiting factor for DSB formation. We also show that MEI1, REC8 and RAD21L are important for proper MEI4 localization. These findings on MEI4 dynamics during meiotic prophase suggest that the association of MEI4 to chromosome axes is required for DSB formation, and that the loss of this association upon DSB repair could contribute to turning off meiotic DSB formation.