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Genome Biology Jan 2024Centromeres load kinetochore complexes onto chromosomes, which mediate spindle attachment and allow segregation during cell division. Although centromeres perform a...
BACKGROUND
Centromeres load kinetochore complexes onto chromosomes, which mediate spindle attachment and allow segregation during cell division. Although centromeres perform a conserved cellular function, their underlying DNA sequences are highly divergent within and between species. Despite variability in DNA sequence, centromeres are also universally suppressed for meiotic crossover recombination, across eukaryotes. However, the genetic and epigenetic factors responsible for suppression of centromeric crossovers remain to be completely defined.
RESULTS
To explore the centromere-proximal meiotic recombination landscape, we map 14,397 crossovers against fully assembled Arabidopsis thaliana (A. thaliana) genomes. A. thaliana centromeres comprise megabase satellite repeat arrays that load nucleosomes containing the CENH3 histone variant. Each chromosome contains a structurally polymorphic region of ~3-4 megabases, which lack crossovers and include the satellite arrays. This polymorphic region is flanked by ~1-2 megabase low-recombination zones. These recombination-suppressed regions are enriched for Gypsy/Ty3 retrotransposons, and additionally contain expressed genes with high genetic diversity that initiate meiotic recombination, yet do not crossover. We map crossovers at high-resolution in proximity to CEN3, which resolves punctate centromere-proximal hotspots that overlap gene islands embedded in heterochromatin. Centromeres are densely DNA methylated and the recombination landscape is remodelled in DNA methylation mutants. We observe that the centromeric low-recombining zones decrease and increase crossovers in CG (met1) and non-CG (cmt3) mutants, respectively, whereas the core non-recombining zones remain suppressed.
CONCLUSION
Our work relates the genetic and epigenetic organization of A. thaliana centromeres and flanking pericentromeric heterochromatin to the zones of crossover suppression that surround the CENH3-occupied satellite repeat arrays.
Topics: Arabidopsis; DNA Methylation; Heterochromatin; Centromere; Meiosis
PubMed: 38254210
DOI: 10.1186/s13059-024-03163-4 -
The EMBO Journal Mar 2024The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the...
The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates the enrichment of axis proteins at nucleosome-rich islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a localized reduction of axis protein binding and meiotic DNA double-strand breaks (DSBs) in axis islands and leads to defects in chromosome synapsis. Synthetic effects with mutants of the Hop1 regulator Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation. Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of the CBR, suggesting that the mechanisms we uncover are broadly conserved.
Topics: Meiosis; Nucleosomes; Cryoelectron Microscopy; Phylogeny; Saccharomyces cerevisiae; DNA; Nuclear Proteins; Saccharomyces cerevisiae Proteins
PubMed: 38332377
DOI: 10.1038/s44318-024-00034-3 -
Nature Apr 2024RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which...
RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs). However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.
Topics: Humans; Cryoelectron Microscopy; DNA; DNA Breaks, Double-Stranded; DNA Repair; Nucleosomes; Protein Subunits; Rad51 Recombinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Mutation; Protein Domains; Histones; Protein Binding
PubMed: 38509361
DOI: 10.1038/s41586-024-07196-4 -
Scientific Reports Nov 2023Meiotic recombination through chromosomal crossovers ensures proper segregation of homologous chromosomes during meiosis, while also breaking down linkage disequilibrium...
Meiotic recombination through chromosomal crossovers ensures proper segregation of homologous chromosomes during meiosis, while also breaking down linkage disequilibrium and shuffling alleles at loci located on the same chromosome. Rates of recombination can vary between species, but also between and within individuals, sex and chromosomes within species. Indeed, the Atlantic salmon genome is known to have clear sex differences in recombination with female biased heterochiasmy and markedly different landscapes of crossovers between males and females. In male meiosis, crossovers occur strictly in the telomeric regions, whereas in female meiosis crossovers tend to occur closer to the centromeres. However, little is known about the genetic control of these patterns and how this differs at the individual level. Here, we investigate genetic variation in individual measures of recombination in > 5000 large full-sib families of a Norwegian Atlantic salmon breeding population with high-density SNP genotypes. We show that females had 1.6 × higher crossover counts (CC) than males, with autosomal linkage maps spanning a total of 2174 cM in females and 1483 cM in males. However, because of the extreme telomeric bias of male crossovers, female recombination is much more important for generation of new haplotypes with 8 × higher intra-chromosomal genetic shuffling than males. CC was heritable in females (h = 0.11) and males (h = 0.10), and shuffling was also heritable in both sex but with a lower heritability in females (h = 0.06) than in males (h = 0.11). Inter-sex genetic correlations for both traits were close to zero, suggesting that rates and distribution of crossovers are genetically distinct traits in males and females, and that there is a potential for independent genetic change in both sexes in the Atlantic Salmon. Together, these findings give novel insights into the genetic architecture of recombination in salmonids and contribute to a better understanding of how rates and distribution of recombination may evolve in eukaryotes more broadly.
Topics: Humans; Animals; Female; Male; Recombination, Genetic; Salmo salar; Chromosome Mapping; Genome; Chromosomes; Meiosis
PubMed: 37993527
DOI: 10.1038/s41598-023-47208-3 -
The EMBO Journal Jun 2024MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the...
MCM8 has emerged as a core gene in reproductive aging and is crucial for meiotic homologous recombination repair. It also safeguards genome stability by coordinating the replication stress response during mitosis, but its function in mitotic germ cells remains elusive. Here we found that disabling MCM8 in mice resulted in proliferation defects of primordial germ cells (PGCs) and ultimately impaired fertility. We further demonstrated that MCM8 interacted with two known helicases DDX5 and DHX9, and loss of MCM8 led to R-loop accumulation by reducing the retention of these helicases at R-loops, thus inducing genome instability. Cells expressing premature ovarian insufficiency-causative mutants of MCM8 with decreased interaction with DDX5 displayed increased R-loop levels. These results show MCM8 interacts with R-loop-resolving factors to prevent R-loop-induced DNA damage, which may contribute to the maintenance of genome integrity of PGCs and reproductive reserve establishment. Our findings thus reveal an essential role for MCM8 in PGC development and improve our understanding of reproductive aging caused by genome instability in mitotic germ cells.
PubMed: 38858601
DOI: 10.1038/s44318-024-00134-0 -
BioRxiv : the Preprint Server For... Oct 2023During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs),...
During meiotic prophase I, recombination between homologous parental chromosomes is initiated by the formation of hundreds of programmed double-strand breaks (DSBs), each of which must be repaired with absolute fidelity to ensure genome stability of the germline. One outcome of these DSB events is the formation of Crossovers (COs), the sites of physical DNA exchange between homologs that are critical to ensure the correct segregation of parental chromosomes. However, COs account for only a small (~10%) proportion of all DSB repair events; the remaining 90% are repaired as non-crossovers (NCOs), most by synthesis dependent strand annealing. Virtually all COs are formed by coordinated efforts of the MSH4/MSH5 and MLH1/MLH3 heterodimers. The number and positioning of COs is exquisitely controlled via mechanisms that remain poorly understood, but which undoubtedly require the coordinated action of multiple repair pathways downstream of the initiating DSB. In a previous report we found evidence suggesting that the DNA helicase and Fanconi Anemia repair protein, FANCJ (BRIP1/BACH1), functions to regulate meiotic recombination in mouse. A gene-trap disruption of showed an elevated number of MLH1 foci and COs. FANCJ is known to interact with numerous DNA repair proteins in somatic cell repair contexts, including MLH1, BLM, BRCA1, and TOPBP1, and we hypothesized that FANCJ regulates CO formation through a direct interaction with MLH1 to suppress the major CO pathway. To further elucidate the function of FANCJ in meiosis, we produced three new mutant mouse lines via CRISPR/Cas9 gene editing: a full-gene deletion, a mutant line lacking the MLH1 interaction site and the N-terminal region of the Helicase domain, and a C-terminal 6xHIS-HA dual-tagged allele of . We also generated an antibody against the C-terminus of the mouse FANCJ protein. Surprisingly, while Fanconi-like phenotypes are observed within the somatic cell lineages of the full deletion line, none of the mutants show any change in either MLH1 focus counts during pachynema or total CO number at diakinesis of prophase I of meiosis. We find evidence that FANCJ and MLH1 do not interact in meiosis; further, FANCJ does not co-localize with MSH4, MLH1, or MLH3 during late prophase I. Instead, FANCJ forms discrete foci along the chromosome cores beginning in early meiotic prophase I, occasionally co-localizing with MSH4, and then becomes densely localized on unsynapsed chromosome axes in late zygonema and to the XY chromosomes in early pachynema. Strikingly, this localization strongly overlaps with BRCA1 and TOPBP1. mutants also exhibit a subtle persistence of DSBs in pachynema. Collectively, these data suggest a role for FANCJ in early DSB repair events, and possibly in the formation of NCOs, but they rule out a role for FANCJ in MLH1-mediated CO events. Thus, the role of FANCJ in meiotic cells involves different pathways and different interactors to those described in somatic cell lineages.
PubMed: 37873301
DOI: 10.1101/2023.10.06.561296 -
Bio-protocol Jul 2023During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to...
During the first meiotic prophase in mouse, repair of SPO11-induced DNA double-strand breaks (DSBs), facilitating homologous chromosome synapsis, is essential to successfully complete the first meiotic cell division. Recombinases RAD51 and DMC1 play an important role in homology search, but their mechanistic contribution to this process is not fully understood. Super-resolution, single-molecule imaging of RAD51 and DMC1 provides detailed information on recombinase accumulation on DSBs during meiotic prophase. Here, we present a detailed protocol of recombination foci analysis of three-color direct stochastic optical reconstruction microscopy (dSTORM) imaging of SYCP3, RAD51, and DMC1, fluorescently labeled by antibody staining in mouse spermatocytes. This protocol consists of sample preparation, data acquisition, pre-processing, and data analysis. The sample preparation procedure includes an updated version of the nuclear spreading of mouse testicular cells, followed by immunocytochemistry and the preparation steps for dSTORM imaging. Data acquisition consists of three-color dSTORM imaging, which is extensively described. The pre-processing that converts fluorescent signals to localization data also includes channel alignment and image reconstruction, after which regions of interest (ROIs) are identified based on RAD51 and/or DMC1 localization patterns. The data analysis steps then require processing of the fluorescent signal localization within these ROIs into discrete nanofoci, which can be further analyzed. This multistep approach enables the systematic investigation of spatial distributions of proteins associated with individual DSB sites and can be easily adapted for analyses of other foci-forming proteins. All computational scripts and software are freely accessible, making them available to a broad audience. Key features Preparation of spread nuclei, resulting in a flattened preparation with easy antibody-accessible chromatin-associated proteins on dSTORM-compatible coverslips. dSTORM analysis of immunofluorescent repair foci in meiotic prophase nuclei. Detailed descriptions of data acquisition, (pre-)processing, and nanofoci feature analysis applicable to all proteins that assemble in immunodetection as discrete foci. Graphical overview.
PubMed: 37497444
DOI: 10.21769/BioProtoc.4780 -
BioRxiv : the Preprint Server For... Apr 2024DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In meiosis, this 5'-to-3' resection...
DNA double-strand breaks (DSBs) are nucleolytically processed to generate single-stranded DNA tails for homologous recombination. In meiosis, this 5'-to-3' resection involves initial nicking by the Mre11-Rad50-Xrs2 complex (MRX) plus Sae2, then exonucleolytic digestion by Exo1. Chromatin remodeling adjacent to meiotic DSBs is thought to be necessary for resection, but the relevant remodeling activity was unknown. Here we show that the SWI/SNF-like ATPase Fun30 plays a major, non-redundant role in resecting meiotic DSBs. A null mutation shortened resection tract lengths almost as severely as an (nuclease-dead) mutation, and resection was further shortened in the double mutant. Fun30 associates with chromatin in response to meiotic DSBs, and the constitutive positioning of nucleosomes governs resection endpoint locations in the absence of Fun30. We infer that Fun30 directly promotes both the MRX- and Exo1-dependent steps in resection, possibly by removing nucleosomes from broken chromatids. Moreover, we found that the extremely short resection in the double mutant is accompanied by compromised interhomolog recombination bias, leading to defects in recombination and chromosome segregation. Thus, this study also provides insight about the minimal resection lengths needed for robust recombination.
PubMed: 38659928
DOI: 10.1101/2024.04.17.589955 -
The Journal of Cell Biology Feb 2024During meiosis, cohesin and meiosis-specific proteins organize chromatin into an axis-loop architecture, coordinating homologous synapsis, recombination, and ordered...
During meiosis, cohesin and meiosis-specific proteins organize chromatin into an axis-loop architecture, coordinating homologous synapsis, recombination, and ordered chromosome segregation. However, how the meiotic chromosome axis is assembled and differentiated with meiotic progression remains elusive. Here, we explore the dynamic recruitment of two long arms of the bivalent proteins, LAB-1 and LAB-2, in Caenorhabditis elegans. LAB proteins directly interact with the axis core HORMA complexes and weak interactions contribute to their recruitment. LAB proteins phase separate in vitro, and this capacity is promoted by HORMA complexes. During early prophase, synapsis oppositely regulates the axis enrichment of LAB proteins. After the pachytene exit, LAB proteins switch from a reciprocal localization pattern to a colocalization pattern, and the normal dynamic pattern of LAB proteins is altered in meiotic mutants. We propose that LAB recruitment senses axis differentiation, and phase separation of meiotic structures helps subdomain establishment and accurate segregation of the chromosomes.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Cycle Proteins; Chromosome Pairing; Chromosome Segregation; Chromosomes; Meiosis; Chromosomal Proteins, Non-Histone
PubMed: 38010234
DOI: 10.1083/jcb.202212035 -
BioRxiv : the Preprint Server For... Jan 2024Male germ cells, which are responsible for producing millions of genetically diverse sperm through meiosis in the testis, rely on lactate as their central energy...
Male germ cells, which are responsible for producing millions of genetically diverse sperm through meiosis in the testis, rely on lactate as their central energy metabolite. Recent study has revealed that lactate induces epigenetic modification in cells through histone lactylation, a post-translational modification involving the addition of lactyl groups to lysine residues on histones. Here we report dynamic histone lactylation at histone H4-lysine 5 (K5), -K8, and -K12 during meiosis prophase I in mouse spermatogenesis. By profiling genome-wide occupancy of histone H4-K8 lactylation (H4K8la), which peaks at zygotene, our data show that H4K8la mark is observed at the promoters of genes exhibiting active expression with Gene Ontology (GO) functions enriched for meiosis. Notably, our data also demonstrate that H4K8la is closely associated with recombination hotspots, where machinery involved in the processing DNA double-stranded breaks (DSBs), such as SPO11, DMC1, RAD51, and RPA2, is engaged. In addition, H4K8la was also detected at the meiosis-specific cohesion sites (marked by RAD21L and REC8) flanking the recombination hotspots. Collectively, our findings suggest that histone lactylation serves as a novel mechanism through which lactate regulates germ cell meiosis.
PubMed: 38328171
DOI: 10.1101/2024.01.25.576681