RESUMO
During meiosis, nucleoprotein filaments of the strand exchange proteins RAD51 and DMC1 are crucial for repairing SPO11-generated DNA double-strand breaks (DSBs) by homologous recombination (HR). A balanced activity of positive and negative RAD51/DMC1 regulators ensures proper recombination. Fidgetin-like 1 (FIGNL1) was previously shown to negatively regulate RAD51 in human cells. However, FIGNL1's role during meiotic recombination in mammals remains unknown. Here, we decipher the meiotic functions of FIGNL1 and FIGNL1 Interacting Regulator of Recombination and Mitosis (FIRRM) using male germline-specific conditional knock-out (cKO) mouse models. Both FIGNL1 and FIRRM are required for completing meiotic prophase in mouse spermatocytes. Despite efficient recruitment of DMC1 on ssDNA at meiotic DSB hotspots, the formation of late recombination intermediates is defective in Firrm cKO and Fignl1 cKO spermatocytes. Moreover, the FIGNL1-FIRRM complex limits RAD51 and DMC1 accumulation on intact chromatin, independently from the formation of SPO11-catalyzed DSBs. Purified human FIGNL1ΔN alters the RAD51/DMC1 nucleoprotein filament structure and inhibits strand invasion in vitro. Thus, this complex might regulate RAD51 and DMC1 association at sites of meiotic DSBs to promote proficient strand invasion and processing of recombination intermediates.
Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Meiose , Camundongos Knockout , Rad51 Recombinase , Espermatócitos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Animais , Masculino , Meiose/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Humanos , Camundongos , Espermatócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Recombinação Homóloga , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Dano ao DNA , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Cromatina/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genéticaRESUMO
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.
RESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Histona-Lisina N-Metiltransferase , Meiose , Meiose/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Camundongos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Histonas/metabolismo , Histonas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Sítios de Ligação , Cromossomos/genética , Cromossomos/metabolismo , Cromatina/metabolismo , Cromatina/genética , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Recombinação Genética , MasculinoRESUMO
Molecular approaches are required to detect DNA double-strand break (DSB) events and to map and quantify them at high resolution. One of the most popular molecular methods in the field of meiotic recombination is the ChIP-SSDS (Chromatin immuno-precipitation and single-strand DNA sequencing). Here, we present two fully-automated Nextflow-based pipelines to analyze the sequencing data generated by this method. The first one identifies highly reproducible DSB sites, while the second provides a characterization of recovered DSB sites, including the description of the hotspot distribution and intensity along the genome and the overlap with specific regions such as gene features or known DSB hotspots. Finally, we discuss limitations/advantages and key points to consider when applying this method to specific genotypes or unconventional species.
Assuntos
Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , DNA de Cadeia Simples/genética , Genoma , Análise de Sequência de DNA , Meiose/genéticaRESUMO
Multiple pathways generate mutations at sites of meiotic recombination in humans.
Assuntos
Genoma Humano , Recombinação Homóloga , Meiose , Humanos , Meiose/genética , MutaçãoRESUMO
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.
Assuntos
Recombinação Homóloga , Proteínas de Saccharomyces cerevisiae , Animais , Camundongos , DNA , Meiose , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Meiosis requires the formation of programmed DNA double strand breaks (DSBs), essential for fertility and for generating genetic diversity. DSBs are induced by the catalytic activity of the TOPOVIL complex formed by SPO11 and TOPOVIBL. To ensure genomic integrity, DNA cleavage activity is tightly regulated, and several accessory factors (REC114, MEI4, IHO1, and MEI1) are needed for DSB formation in mice. How and when these proteins act is not understood. Here, we show that REC114 is a direct partner of TOPOVIBL, and identify their conserved interacting domains by structural analysis. We then analyse the role of this interaction by monitoring meiotic DSBs in female and male mice carrying point mutations in TOPOVIBL that decrease or disrupt its binding to REC114. In these mutants, DSB activity is strongly reduced genome-wide in oocytes, and only in sub-telomeric regions in spermatocytes. In addition, in mutant spermatocytes, DSB activity is delayed in autosomes. These results suggest that REC114 is a key member of the TOPOVIL catalytic complex, and that the REC114/TOPOVIBL interaction ensures the efficiency and timing of DSB activity.
Assuntos
Quebras de DNA de Cadeia Dupla , Meiose , Masculino , Feminino , Camundongos , Animais , Meiose/genética , Cromossomos , Espermatócitos , DNARESUMO
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.
Assuntos
Proteínas Arqueais , DNA Topoisomerases Tipo II , Filogenia , Sequência de Aminoácidos , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Proteínas Arqueais/química , Meiose/genética , Eucariotos/genética , Eucariotos/metabolismoRESUMO
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.
Assuntos
Troca Genética , Complexo Sinaptonêmico , Pareamento Cromossômico , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Meiose/genéticaRESUMO
One of the most fascinating aspects of meiosis is the extensive reorganization of the genome at the prophase of the first meiotic division (prophase I). The first steps of this reorganization are observed with the establishment of an axis structure, that connects sister chromatids, from which emanate arrays of chromatin loops. This axis structure, called the axial element, consists of various proteins, such as cohesins, HORMA-domain proteins, and axial element proteins. In many organisms, axial elements are required to set the stage for efficient sister chromatid cohesion and meiotic recombination, necessary for the recognition of the homologous chromosomes. Here, we review the different actors involved in axial element formation in Saccharomyces cerevisiae and in mouse. We describe the current knowledge of their localization pattern during prophase I, their functional interdependence, their role in sister chromatid cohesion, loop axis formation, homolog pairing before meiotic recombination, and recombination. We also address further challenges that need to be resolved, to fully understand the interplay between the chromosome structure and the different molecular steps that take place in early prophase I, which lead to the successful outcome of meiosis I.
RESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Meiose/genética , ATPases Associadas a Diversas Atividades Celulares/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/fisiologia , Pareamento Cromossômico , Retroalimentação Fisiológica , Gametogênese , Camundongos , Estágio Paquíteno , Cromossomos Sexuais , Transdução de SinaisRESUMO
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.
Assuntos
Evolução Molecular , Mamíferos/genética , Biossíntese de Proteínas , Transcriptoma/genética , Animais , Encéfalo/metabolismo , Galinhas/genética , Feminino , Genes Ligados ao Cromossomo X/genética , Humanos , Fígado/metabolismo , Macaca/genética , Masculino , Camundongos , Gambás/genética , Especificidade de Órgãos/genética , Ornitorrinco/genética , Biossíntese de Proteínas/genética , RNA-Seq , Ribossomos/metabolismo , Cromossomos Sexuais/genética , Especificidade da Espécie , Espermatogênese/genética , Testículo/metabolismo , Regulação para CimaRESUMO
Meiotic recombination starts with the formation of DNA double-strand breaks (DSBs) at specific genomic locations that correspond to PRDM9-binding sites. The molecular steps occurring from PRDM9 binding to DSB formation are unknown. Using proteomic approaches to find PRDM9 partners, we identified HELLS, a member of the SNF2-like family of chromatin remodelers. Upon functional analyses during mouse male meiosis, we demonstrated that HELLS is required for PRDM9 binding and DSB activity at PRDM9 sites. However, HELLS is not required for DSB activity at PRDM9-independent sites. HELLS is also essential for 5-hydroxymethylcytosine (5hmC) enrichment at PRDM9 sites. Analyses of 5hmC in mice deficient for SPO11, which catalyzes DSB formation, and in PRDM9 methyltransferase deficient mice reveal that 5hmC is triggered at DSB-prone sites upon PRDM9 binding and histone modification, but independent of DSB activity. These findings highlight the complex regulation of the chromatin and epigenetic environments at PRDM9-specified hotspots.
Assuntos
5-Metilcitosina/análogos & derivados , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Histona-Lisina N-Metiltransferase/genética , 5-Metilcitosina/metabolismo , Animais , Sítios de Ligação , Endodesoxirribonucleases/metabolismo , Células HeLa , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteômica , Espermatócitos/citologia , Testículo/metabolismoRESUMO
Three independent studies show that a protein called ZCWPW1 is able to recognize the histone modifications that initiate the recombination of genetic information during meiosis.
Assuntos
Quebras de DNA de Cadeia Dupla , Histonas , Animais , DNA , Reparo do DNA , Epigênese Genética , Masculino , Meiose , Camundongos , LeituraRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesic drugs, such as acetaminophen (APAP), are frequently taken during pregnancy, even in combination. However, they can favour genital malformations in newborn boys and reproductive disorders in adults. Conversely, the consequences on postnatal ovarian development and female reproductive health after in utero exposure are unknown. Here, we found that in mice, in utero exposure to therapeutic doses of the APAP-ibuprofen combination during sex determination led to delayed meiosis entry and progression in female F1 embryonic germ cells. Consequently, follicular activation was reduced in postnatal ovaries through the AKT/FOXO3 pathway, leading in F2 animals to subfertility, accelerated ovarian aging with abnormal corpus luteum persistence, due to decreased apoptosis and increased AKT-mediated luteal cell survival. Our study suggests that administration of these drugs during the critical period of sex determination could lead in humans to adverse effects that might be passed to the offspring.
Assuntos
Acetaminofen/efeitos adversos , Envelhecimento/fisiologia , Ibuprofeno/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/patologia , Reprodução/fisiologia , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Feminino , Fertilidade , Proteína Forkhead Box O3/metabolismo , Células Germinativas/efeitos dos fármacos , Células Germinativas/patologia , Luteólise , Camundongos , Ovário/embriologia , Ovário/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Meiose/genética , Animais , Proteínas de Transporte/química , Segregação de Cromossomos/genética , Masculino , Camundongos , Regiões Pseudoautossômicas/genética , Espermatócitos/crescimento & desenvolvimento , Espermatócitos/metabolismo , Cromossomo X/genética , Cromossomo Y/genéticaRESUMO
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.
Assuntos
Meiose , Oócitos , Cromossomos Sexuais , Animais , Pareamento Cromossômico , Troca Genética , Feminino , Loci Gênicos , Cariótipo , Cariotipagem , Masculino , Camundongos , Cromossomo X , Cromossomo YRESUMO
Programmed formation of DNA double-strand breaks (DSBs) initiates the meiotic homologous recombination pathway. This pathway is essential for proper chromosome segregation at the first meiotic division and fertility. Meiotic DSBs are catalyzed by Spo11. Several other proteins are essential for meiotic DSB formation, including three evolutionarily conserved proteins first identified in Saccharomyces cerevisiae (Mer2, Mei4, and Rec114). These three S. cerevisiae proteins and their mouse orthologs (IHO1, MEI4, and REC114) co-localize on the axes of meiotic chromosomes, and mouse IHO1 and MEI4 are essential for meiotic DSB formation. Here, we show that mouse Rec114 is required for meiotic DSB formation. Moreover, MEI4 forms a complex with REC114 and IHO1 in mouse spermatocytes, consistent with cytological observations. We then demonstrated in vitro the formation of a stable complex between REC114 C-terminal domain and MEI4 N-terminal domain. We further determine the structure of the REC114 N-terminal domain that revealed similarity with Pleckstrin homology domains. These analyses provide direct insights into the architecture of these essential components of the meiotic DSB machinery.
RESUMO
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.
Assuntos
Mapeamento Cromossômico , Histona-Lisina N-Metiltransferase/genética , Meiose , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação a DNA , Evolução Molecular , Fertilidade , Heterozigoto , Histona-Lisina N-Metiltransferase/metabolismo , Recombinação Homóloga , Humanos , Infertilidade , Masculino , Camundongos , Conformação Proteica , Isolamento Reprodutivo , EspermatócitosRESUMO
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.