RESUMEN
We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes.
Asunto(s)
Arabidopsis , Roturas del ADN de Doble Cadena , Meiosis , Arabidopsis/genética , Meiosis/genética , Reparación del ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromosomas de las Plantas/genética , Profase Meiótica I/genética , Intercambio Genético , Replicación del ADN/genéticaRESUMEN
During meiotic prophase I, chromosomes undergo large-scale dynamics to allow homologous chromosome pairing, prior to which chromosome ends attach to the inner nuclear envelope and form a chromosomal bouquet. Chromosome pairing is crucial for homologous recombination and accurate chromosome segregation during meiosis. However, the specific mechanism by which homologous chromosomes recognize each other is poorly understood. Here, we investigated the process of homologous chromosome pairing during early prophase I of meiosis in rice (Oryza sativa) using pooled oligo probes specific to an entire chromosome or chromosome arm. We revealed that chromosome pairing begins from both ends and extends toward the center from early zygotene through late zygotene. Genetic analysis of both trisomy and autotetraploidy also showed that pairing initiation is induced by both ends of a chromosome. However, healed ends that lack the original terminal regions on telocentric and acrocentric chromosomes cannot initiate homologous chromosome pairing, even though they may still enter the telomere clustering region at the bouquet stage. Furthermore, a chromosome that lacks the distal parts on both sides loses the ability to pair with other intact chromosomes. Thus, the native ends of chromosomes play a crucial role in initiating homologous chromosome pairing during meiosis and likely have a substantial impact on genome differentiation.
Asunto(s)
Emparejamiento Cromosómico , Cromosomas de las Plantas , Meiosis , Oryza , Oryza/genética , Emparejamiento Cromosómico/genética , Cromosomas de las Plantas/genética , Meiosis/genética , Telómero/genética , Hibridación Fluorescente in Situ , Profase Meiótica I/genéticaRESUMEN
Meiotic recombination between homologous chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs). Approximately 10% of these DSBs result in crossovers (COs), sites of physical DNA exchange between homologs that are critical to correct chromosome segregation. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers, the latter representing the defining marks of CO sites. The regulation of CO number and position is poorly understood, but undoubtedly requires the coordinated action of multiple repair pathways. In a previous report, we found gene-trap disruption of the DNA helicase, FANCJ (BRIP1/BACH1), elicited elevated numbers of MLH1 foci and chiasmata. In somatic cells, FANCJ interacts with numerous DNA repair proteins including MLH1, and we hypothesized that FANCJ functions with MLH1 to regulate the major CO pathway. To further elucidate the meiotic function of FANCJ, we produced three new Fancj mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, truncation of the N-terminal Helicase domain, and a C-terminal dual-tagged allele. We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, none of our Fancj mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 in meiosis. Instead, FANCJ co-localizes with BRCA1 and TOPBP1, forming discrete foci along the chromosome cores beginning in early meiotic prophase I and densely localized to unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Fancj mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data indicate a role for FANCJ in early DSB repair, but they rule out a role for FANCJ in MLH1-mediated CO events.
Asunto(s)
Meiosis , Profase Meiótica I , Animales , Masculino , Ratones , Alelos , ADN Helicasas/genética , Reparación del ADN/genética , Meiosis/genética , Profase Meiótica I/genéticaRESUMEN
During meiosis, the chromatin and transcriptome undergo prominent switches. Although recent studies have explored the genome reorganization during spermatogenesis, the chromatin remodeling in oogenesis and characteristics of homologous pairing remain largely elusive. We comprehensively compared chromatin structures and transcriptomes at successive substages of meiotic prophase in both female and male mice using low-input high-through chromosome conformation capture (Hi-C) and RNA sequencing (RNA-seq). Compartments and topologically associating domains (TADs) gradually disappeared and slowly recovered in both sexes. We found that homologs adopted different sex-conserved pairing strategies prior to and after the leptotene-to-zygotene transition, changing from long interspersed nuclear element (LINE)-enriched compartments B to short interspersed nuclear element (SINE)-enriched compartments A. We complemented marker genes and predicted the sex-specific meiotic sterile genes for each substage. This study provides valuable insights into the similarities and distinctions between sexes in chromosome architecture, homologous pairing, and transcriptome during meiotic prophase of both oogenesis and spermatogenesis.
Asunto(s)
Meiosis , Espermatogénesis , Masculino , Femenino , Ratones , Animales , Meiosis/genética , Espermatogénesis/genética , Profase , Profase Meiótica I/genética , Cromatina/genética , Oogénesis/genética , Emparejamiento Cromosómico/genéticaRESUMEN
Nonhomologous chromosome interactions take place in both somatic and meiotic cells. Prior to this study, we had discovered special contacts through the SYCP3 (synaptonemal complex protein 3) filament between the short arms of nonhomologous acrocentrics at the pachytene stage in the Alay mole vole, and these contacts demonstrate several patterns from proximity to the complete fusion stage. Here, we investigated the nonhomologous chromosome contacts in meiotic prophase I. It turned out that such contacts do not introduce changes into the classic distribution of DNA double-strand breaks. It is noteworthy that not all meiotic contacts were localized in the H3k9me3-positive heterochromatic environment. Both in the mid zygotene and in the early-mid diplotene, three types of contacts (proximity, touching, and anchoring/tethering) were observed, whereas fusion seems to be characteristic only for pachytene. The number of contacts in the mid pachytene is significantly higher than that in the zygotene, and the distance between centromeres in nonhomologous contacts is also the smallest in mid pachytene for all types of contacts. Thus, this work provides a new insight into the behavior of meiotic contacts during prophase I and points to avenues of further research.
Asunto(s)
Meiosis , Profase Meiótica I , Animales , Profase Meiótica I/genética , Roedores/genética , Arvicolinae/genética , Centrómero/genéticaRESUMEN
Meiotic homologous recombination during fetal development dictates proper chromosome segregation in adult mammalian oocytes. Successful homologous synapsis and recombination during Meiotic Prophase I (MPI) depends on telomere-led chromosome movement along the nuclear envelope. In mice, all chromosomes are acrocentric, while other mammalian species carry a mixture of acrocentric and metacentric chromosomes. Such differences in telomeric structures may explain the exceptionally low aneuploidy rates in mice. Here, we tested whether the presence of metacentric chromosomes carrying Robertsonian translocations (RbT) affects the rate of homologous recombination or aneuploidy. We found a delay in MPI progression in RbT-carrier vs. wild-type (WT) fetal ovaries. Furthermore, resolution of distal telomere clusters, associated with synapsis initiation, was delayed and centromeric telomere clusters persisted until later MPI substages in RbT-carrier oocytes compared to WT oocytes. When chromosomes fully synapsed, higher percentages of RbT-carrier oocytes harbored at least one chromosome pair lacking MLH1 foci, which indicate crossover sites, compared to WT oocytes. Aneuploidy rates in ovulated eggs were also higher in RbT-carrier females than in WT females. In conclusion, the presence of metacentric chromosomes among acrocentric chromosomes in mouse oocytes delays MPI progression and reduces the efficiency of homologous crossover, resulting in a higher frequency of aneuploidy.
Asunto(s)
Meiosis , Oocitos , Aneuploidia , Animales , Cromosomas , Femenino , Mamíferos , Meiosis/genética , Profase Meiótica I/genética , Ratones , Telómero/genética , Translocación GenéticaRESUMEN
The bivalent chromosomes that are generated during prophase of meiosis I comprise a pair of homologous chromosomes. Homolog pairing during prophase I must include mechanisms that avoid or eliminate entanglements between non-homologous chromosomes. In Drosophila spermatocytes, non-homologous associations are disrupted by chromosome territory formation, while linkages between homologous chromosomes are maintained by special conjunction proteins. These proteins function as alternative for crossovers that link homologs during canonical meiosis but are absent during the achiasmate Drosophila male meiosis. How and where within bivalents the alternative homolog conjunction proteins function is still poorly understood. To clarify the rules that govern territory formation and alternative homolog conjunction, we have analyzed spermatocytes with chromosomal aberrations. We examined territory formation after acute chromosome cleavage by Cas9, targeted to the dodeca satellite adjacent to the centromere of chromosome 3 specifically in spermatocytes. Moreover, we studied territory organization, as well as the eventual orientation of chromosomes during meiosis I, in spermatocytes with stable structural aberrations, including heterozygous reciprocal autosomal translocations. Our observations indicate that alternative homolog conjunction is applied in a spatially confined manner. Comparable to crossovers, only a single conjunction spot per chromosome arm appears to be applied usually. These conjunction spots resist separation by the dispersing forces that drive apart homologous pericentromeric heterochromatin and embedded centromeres within territories, as well as the distinct chromosomal entities into peripheral, maximally separated territories within the spermatocyte nucleus.
Asunto(s)
Drosophila , Espermatocitos , Animales , Centrómero/genética , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Masculino , Meiosis/genética , Profase Meiótica I/genética , Profase , Espermatocitos/metabolismoRESUMEN
To generate gametes, sexually reproducing organisms need to achieve a reduction in ploidy, via meiosis. Several mechanisms are set in place to ensure proper reductional chromosome segregation at the first meiotic division (MI), including chromosome remodeling during late prophase I. Chromosome remodeling after crossover formation involves changes in chromosome condensation and restructuring, resulting in a compact bivalent, with sister kinetochores oriented to opposite poles, whose structure is crucial for localized loss of cohesion and accurate chromosome segregation. Here, we review the general processes involved in late prophase I chromosome remodeling, their regulation, and the strategies devised by different organisms to produce bivalents with configurations that promote accurate segregation.
Asunto(s)
Segregación Cromosómica , Profase Meiótica I , Segregación Cromosómica/genética , Cinetocoros , Meiosis/genética , Profase Meiótica I/genéticaRESUMEN
Regulation of transcriptional activity during meiosis depends on the interrelated processes of recombination and synapsis. In eutherian mammal spermatocytes, transcription levels change during prophase-I, being low at the onset of meiosis but highly increased from pachytene up to the end of diplotene. However, X and Y chromosomes, which usually present unsynapsed regions throughout prophase-I in male meiosis, undergo a specific pattern of transcriptional inactivation. The interdependence of synapsis and transcription has mainly been studied in mammals, basically in mouse, but our knowledge in other unrelated phylogenetically species is more limited. To gain new insights on this issue, here we analyzed the relationship between synapsis and transcription in spermatocytes of the grasshopper Eyprepocnemis plorans. Autosomal chromosomes of this species achieve complete synapsis; however, the single X sex chromosome remains always unsynapsed and behaves as a univalent. We studied transcription in meiosis by immunolabeling with RNA polymerase II phosphorylated at serine 2 and found that whereas autosomes are active from leptotene up to diakinesis, the X chromosome is inactive throughout meiosis. This inactivation is accompanied by the accumulation of, at least, two repressive epigenetic modifications: H3 methylated at lysine 9 and H2AX phosphorylated at serine 139. Furthermore, we identified that X chromosome inactivation occurs in premeiotic spermatogonia. Overall, our results indicate: (i) transcription regulation in E. plorans spermatogenesis differs from the canonical pattern found in mammals and (ii) X chromosome inactivation is likely preceded by a process of heterochromatinization before the initiation of meiosis.
Asunto(s)
Saltamontes/genética , Espermatogénesis/genética , Inactivación del Cromosoma X/genética , Cromosoma X/genética , Animales , Emparejamiento Cromosómico/genética , Epigénesis Genética/genética , Femenino , Silenciador del Gen/fisiología , Histonas/genética , Lisina/genética , Masculino , Meiosis/genética , Profase Meiótica I/genética , ARN Polimerasa II/genética , Espermatocitos/fisiología , Cromosoma Y/genéticaRESUMEN
Accurate repair of DNA double-strand breaks (DSBs) in developing germ cells is critical to promote proper chromosome segregation and to maintain genome integrity. To directly detect homolog-independent (intersister/intrachromatid) meiotic DSB repair, we exploited the genetics and germline physiology of C. elegans to (1) induce a single DSB in nuclei across discrete stages of meiotic prophase I; (2) detect repair of that DSB as a homolog-independent crossover or noncrossover; and (3) sequence the resultant product to assess mechanisms of recombination. For complete details on the use and execution of this protocol, please refer to Toraason et al. (2021).
Asunto(s)
Cromátides , Intercambio Genético/genética , Reparación del ADN/genética , Profase Meiótica I/genética , Biología Molecular/métodos , Animales , Caenorhabditis elegans/genética , Cromátides/química , Cromátides/genética , Cromátides/metabolismo , Segregación Cromosómica/genética , Roturas del ADN de Doble CadenaRESUMEN
The programmed formation of DNA double-strand breaks (DSBs) in meiotic prophase I initiates the homologous recombination process that yields crossovers between homologous chromosomes, a prerequisite to accurately segregating chromosomes during meiosis I (MI). In the budding yeast Saccharomyces cerevisiae, proteins required for meiotic DSB formation (DSB proteins) accumulate to higher levels specifically on short chromosomes to ensure that these chromosomes make DSBs. We previously demonstrated that as-yet undefined cis-acting elements preferentially recruit DSB proteins and promote higher levels of DSBs and recombination and that these intrinsic features are subject to selection pressure to maintain the hyperrecombinogenic properties of short chromosomes. Thus, this targeted boosting of DSB protein binding may be an evolutionarily recurrent strategy to mitigate the risk of meiotic mis-segregation caused by karyotypic constraints. However, the underlining mechanisms are still elusive. Here, we discuss possible scenarios in which components of the meiotic chromosome axis (Red1 and Hop1) bind to intrinsic features independent of the meiosis-specific cohesin subunit Rec8 and DNA replication, promoting preferential binding of DSB proteins to short chromosomes. We also propose a model where chromosome position in the nucleus, influenced by centromeres, promotes the short-chromosome boost of DSB proteins.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Cromosomas Fúngicos/genética , Proteínas de Unión al ADN/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Segregación Cromosómica/genética , Roturas del ADN de Doble Cadena , Recombinación Homóloga , Profase Meiótica I/genética , Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Parthenogenetic species of Caucasian rock lizards of the genus Darevksia are important evidence for reticulate evolution and speciation by hybridization in vertebrates. Female-only lineages formed through interspecific hybridization have been discovered in many groups. Nevertheless, critical mechanisms of oogenesis and specifics of meiosis that provide long-term stability of parthenogenetic species are still unknown. Here we report cytogenetic characteristics of somatic karyotypes and meiotic prophase I nuclei in the diploid parthenogenetic species Darevskia unisexualis from the new population "Keti" in Armenia which contains an odd number of chromosomes 2n = 37, instead of the usual 2n = 38. We revealed 36 acrocentric chromosomes and a single metacentric autosomal chromosome, resulting from Robertsonian translocation. Comparative genomic hybridization revealed that chromosome fusion occurred between two chromosomes inherited from the maternal species, similar to another parthenogenetic species D. rostombekowi. To trace the chromosome behaviour in meiosis, we performed an immunocytochemical study of primary oocytes' spread nuclei and studied chromosome synapsis during meiotic prophase I in D. unisexualis based on analysis of synaptonemal complexes (SCs). We found meiotic SC-trivalent composed of one metacentric and two acrocentric chromosomes. We confirmed that the SC was assembled between homeologous chromosomes inherited from two parental species. Immunostaining of the pachytene and diplotene nuclei revealed a mismatch repair protein MLH1 loaded to all autosomal SC bivalents. Possible mechanisms of meiotic recombination between homeologous chromosomes are discussed.
Asunto(s)
Lagartos/genética , Profase Meiótica I/genética , Homólogo 1 de la Proteína MutL/genética , Animales , Células Cultivadas , Reparación de la Incompatibilidad de ADN , Reordenamiento Génico , Lagartos/fisiología , Partenogénesis , Complejo SinaptonémicoRESUMEN
Meiosis is a specialized cell division for producing haploid gametes in sexually reproducing organisms. In this study, we have independently identified a novel meiosis protein male meiosis recombination regulator (MAMERR)/4930432K21Rik and showed that it is indispensable for meiosis prophase I progression in male mice. Using super-resolution structured illumination microscopy, we found that MAMERR functions at the same double-strand breaks as the replication protein A and meiosis-specific with OB domains/spermatogenesis associated 22 complex. We generated a Mamerr-deficient mouse model by deleting exons 3-6 and found that most of Mamerr-/- spermatocytes were arrested at pachynema and failed to progress to diplonema, although they exhibited almost intact synapsis and progression to the pachytene stage along with XY body formation. Further mechanistic studies revealed that the recruitment of DMC1/RAD51 and heat shock factor 2-binding protein in Mamerr-/- spermatocytes was only mildly impaired with a partial reduction in double-strand break repair, whereas a substantial reduction in ubiquitination on the autosomal axes and on the XY body appeared as a major phenotype in Mamerr-/- spermatocytes. We propose that MAMERR may participate in meiotic prophase I progression by regulating the ubiquitination of key meiotic proteins on autosomes and XY chromosomes, and in the absence of MAMERR, the repressed ubiquitination of key meiotic proteins leads to pachytene arrest and cell death.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cromosomas/genética , Meiosis/genética , Profase Meiótica I/genética , Animales , Emparejamiento Cromosómico/genética , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Masculino , Ratones , Recombinación Genética/genética , Espermatocitos/citología , Espermatogénesis/genéticaRESUMEN
In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.
Asunto(s)
Cromosomas Fúngicos/genética , Recombinación Homóloga , Meiosis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Centrómero/genética , Segregación Cromosómica , Cromosomas Fúngicos/metabolismo , Roturas del ADN de Doble Cadena , Momento de Replicación del ADN , Meiosis/genética , Profase Meiótica I/genética , Recombinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telómero/genética , Factores de TiempoRESUMEN
Robertsonian translocations are common chromosomal alterations. Chromosome variability affects human health and natural evolution. Despite the significance of such mutations, no mechanisms explaining the emergence of such translocations have yet been demonstrated. Several models have explored possible changes in interphase nuclei. Evidence for non-homologous chromosomes end joining in meiosis is scarce, and is often limited to uncovering mechanisms in damaged cells only. This study presents a primarily qualitative analysis of contacts of non-homologous chromosomes by short arms, during meiotic prophase I in the mole vole, Ellobius alaicus, a species with a variable karyotype, due to Robertsonian translocations. Immunocytochemical staining of spermatocytes demonstrated the presence of four contact types for non-homologous chromosomes in meiotic prophase I: (1) proximity, (2) touching, (3) anchoring/tethering, and (4) fusion. Our results suggest distinct mechanisms for chromosomal interactions in meiosis. Thus, we propose to change the translocation mechanism model from 'contact first' to 'contact first in meiosis'.
Asunto(s)
Arvicolinae/genética , Cromosomas/genética , Meiosis/genética , Translocación Genética/genética , Animales , Núcleo Celular/genética , Reparación del ADN por Unión de Extremidades/genética , Humanos , Interfase/genética , Profase Meiótica I/genética , Mutación/genéticaRESUMEN
The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.
Asunto(s)
Regulación de la Expresión Génica , Meiosis/genética , Profase Meiótica I/genética , Metafase/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Alelos , Animales , Masculino , Mesotelina , Ratones , Ratones Transgénicos , Oocitos/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factores SexualesRESUMEN
To maintain the integrity of the genome, meiotic DNA double strand breaks (DSBs) need to form by the meiosis-specific nuclease Spo11 and be repaired by homologous recombination. One class of products formed by recombination are crossovers, which are required for proper chromosome segregation in the first meiotic division. The synaptonemal complex (SC) is a protein structure that connects homologous chromosomes during meiotic prophase I. The proper assembly of the SC is important for recombination, crossover formation, and the subsequent chromosome segregation. Here we identify the components of Cullin RING E3 ubiquitin ligase 4 (CRL4) that play a role in SC assembly in Caenorhabditis elegans. Mutants of the CRL4 complex (cul-4, ddb-1, and gad-1) show defects in SC assembly manifested in the formation of polycomplexes (PCs), impaired progression of meiotic recombination, and reduction in crossover numbers. PCs that are formed in cul-4 mutants lack the mobile properties of wild type SC, but are likely not a direct target of ubiquitination. In C. elegans, SC assembly does not require recombination and there is no evidence that PC formation is regulated by recombination as well. However, in one cul-4 mutant PC formation is dependent upon early meiotic recombination, indicating that proper assembly of the SC can be diminished by recombination in some scenarios. Lastly, our studies suggest that CUL-4 deregulation leads to transposition of the Tc3 transposable element, and defects in formation of SPO-11-mediated DSBs. Our studies highlight previously unknown functions of CRL4 in C. elegans meiosis and show that CUL-4 likely plays multiple roles in meiosis that are essential for maintaining genome integrity.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Endodesoxirribonucleasas/genética , Recombinación Homóloga/genética , Ligasas/genética , Complejo Sinaptonémico/genética , Animales , Caenorhabditis elegans/genética , Emparejamiento Cromosómico/genética , Segregación Cromosómica/genética , Intercambio Genético , Roturas del ADN de Doble Cadena , Células Germinativas/crecimiento & desarrollo , Meiosis/genética , Profase Meiótica I/genéticaRESUMEN
Meiosis is a specialized type of cell division that creates haploid germ cells and ensures their genetic diversity through homologous recombination. We show that the H3K4me3 reader ZCWPW1 is specifically required for meiosis prophase I progression in male but not in female germ cells in mice. Loss of Zcwpw1 in male mice caused a complete failure of synapsis, resulting in meiotic arrest at the zygotene to pachytene stage, accompanied by incomplete DNA double-strand break repair and lack of crossover formation, leading to male infertility. In oocytes, deletion of Zcwpw1 only somewhat slowed down meiosis prophase I progression; Zcwpw1-/- oocytes were able to complete meiosis, and Zcwpw1-/- female mice had normal fertility until mid-adulthood. We conclude that the H3K4me3 reader ZCWPW1 is indispensable for meiosis synapsis in males but is dispensable for females. Our results suggest that ZCWPW1 may represent a previously unknown, sex-dependent epigenetic regulator of germ cell meiosis in mammals.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Reparación del ADN por Unión de Extremidades/genética , Código de Histonas/genética , Profase Meiótica I/genética , Oocitos/citología , Espermatozoides/citología , Animales , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Femenino , Histonas/genética , Infertilidad Masculina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores SexualesRESUMEN
Spermatogenesis is tightly regulated by ubiquitination and proteasomal degradation, especially during spermiogenesis, in which histones are replaced by protamine. However, the functions of proteasomal activity in meiosis I and II remain elusive. Here, we show that PSMA8-associated proteasomes are essential for the degradation of meiotic proteins and the progression of meiosis I during spermatogenesis. PSMA8 is expressed in spermatocytes from the pachytene stage, and assembles a type of testis-specific core proteasome. Deletion of PSMA8 decreases the abundance of proteasome in testes. Meiotic proteins that are normally degraded at late prophase I, such as RAD51 and RPA1, remain stable in PSMA8-deleted spermatocytes. Moreover, PSMA8-null spermatocytes exhibit delayed M-phase entry and are finally arrested at this stage, resulting in male infertility. However, PSMA8 is neither expressed nor required for female meiotic progression. Thus, meiosis I progression in spermatogenesis, particularly entry into and exit from M-phase, requires the proteasomal activity of PSMA8-associated proteasomes.
Asunto(s)
Profase Meiótica I/genética , Complejo de la Endopetidasa Proteasomal/genética , Espermatogénesis/genética , Testículo/enzimología , Animales , División Celular/genética , Femenino , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Fase Paquiteno/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Espermatocitos/enzimología , Espermatocitos/metabolismoRESUMEN
During meiotic prophase I, double-strand breaks (DSBs) initiate homologous recombination leading to non-crossovers (NCOs) and crossovers (COs). In mouse, 10% of DSBs are designated to become COs, primarily through a pathway dependent on the MLH1-MLH3 heterodimer (MutLγ). Mlh3 contains an endonuclease domain that is critical for resolving COs in yeast. We generated a mouse (Mlh3DN/DN) harboring a mutation within this conserved domain that is predicted to generate a protein that is catalytically inert. Mlh3DN/DN males, like fully null Mlh3-/- males, have no spermatozoa and are infertile, yet spermatocytes have grossly normal DSBs and synapsis events in early prophase I. Unlike Mlh3-/- males, mutation of the endonuclease domain within MLH3 permits normal loading and frequency of MutLγ in pachynema. However, key DSB repair factors (RAD51) and mediators of CO pathway choice (BLM helicase) persist into pachynema in Mlh3DN/DN males, indicating a temporal delay in repair events and revealing a mechanism by which alternative DSB repair pathways may be selected. While Mlh3DN/DN spermatocytes retain only 22% of wildtype chiasmata counts, this frequency is greater than observed in Mlh3-/- males (10%), suggesting that the allele may permit partial endonuclease activity, or that other pathways can generate COs from these MutLγ-defined repair intermediates in Mlh3DN/DN males. Double mutant mice homozygous for the Mlh3DN/DN and Mus81-/- mutations show losses in chiasmata close to those observed in Mlh3-/- males, indicating that the MUS81-EME1-regulated crossover pathway can only partially account for the increased residual chiasmata in Mlh3DN/DN spermatocytes. Our data demonstrate that mouse spermatocytes bearing the MLH1-MLH3DN/DN complex display the proper loading of factors essential for CO resolution (MutSγ, CDK2, HEI10, MutLγ). Despite these functions, mice bearing the Mlh3DN/DN allele show defects in the repair of meiotic recombination intermediates and a loss of most chiasmata.