Control of meiotic crossover interference by a proteolytic chaperone network.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

Incorrect recombination partner associations contribute to meiotic instability of neo-allopolyploid Arabidopsis suecica.

Combining two or more related homoeologous genomes in a single nucleus, newly formed allopolyploids must rapidly adapt meiosis to restore balanced chromosome segregation, production of euploid gametes and fertility. The poor fertility of such neo-allopolyploids thus strongly selects for the limitation or avoidance of genetic crossover formation between homoeologous chromosomes. In this study, we have reproduced the interspecific hybridization between Arabidopsis thaliana and Arabidopsis arenosa leading to the allotetraploid Arabidopsis suecica and have characterized the first allopolyploid meioses. First-generation neo-allopolyploid siblings vary considerably in fertility, meiotic behavior and levels of homoeologous recombination. We show that centromere dynamics at early meiosis is altered in synthetic neo-allopolyploids compared with evolved A. suecica, with a significant increase in homoeologous centromere interactions at zygotene. At metaphase I, the presence of multivalents involving homoeologous chromosomes confirms that homoeologous recombination occurs in the first-generation synthetic allopolyploid plants and this is associated with a significant reduction in homologous recombination, compared to evolved A. suecica. Together, these data strongly suggest that the fidelity of recombination partner choice, likely during the DNA invasion step, is strongly impaired during the first meiosis of neo-allopolyploids and requires subsequent adaptation.

Meiotic chromosome organization and its role in recombination and cancer.

Chromosomes adopt specific conformations to regulate various cellular processes. A well-documented chromosome configuration is the highly compacted chromosome structure during metaphase. More regional chromatin conformations have also been reported, including topologically associated domains encompassing mega-bases of DNA and local chromatin loops formed by kilo-bases of DNA. In this review, we discuss the changes in chromatin conformation taking place between somatic and meiotic cells, with a special focus on the establishment of a proteinaceous structure, called the chromosome axis, at the beginning of meiosis. The chromosome axis is essential to support key meiotic processes such as chromosome pairing, homologous recombination, and balanced chromosome segregation to transition from a diploid to a haploid stage. We review the role of the chromosome axis in meiotic chromatin organization and provide a detailed description of its protein composition. We also review the conserved and distinct roles between species of axis proteins in meiotic recombination, which is a major factor contributing to the creation of genetic diversity and genome evolution. Finally, we discuss situations where the chromosome axis is deregulated and evaluate the effects on genome integrity and the consequences from protein deregulation in meiocytes exposed to heat stress, and aberrant expression of genes encoding axis proteins in mammalian somatic cells associated with certain types of cancers.

Differentiated function and localisation of SPO11-1 and PRD3 on the chromosome axis during meiotic DSB formation in Arabidopsis thaliana.

During meiosis, DNA double-strand breaks (DSBs) occur throughout the genome, a subset of which are repaired to form reciprocal crossovers between chromosomes. Crossovers are essential to ensure balanced chromosome segregation and to create new combinations of genetic variation. Meiotic DSBs are formed by a topoisomerase-VI-like complex, containing catalytic (e.g. SPO11) proteins and auxiliary (e.g. PRD3) proteins. Meiotic DSBs are formed in chromatin loops tethered to a linear chromosome axis, but the interrelationship between DSB-promoting factors and the axis is not fully understood. Here, we study the localisation of SPO11-1 and PRD3 during meiosis, and investigate their respective functions in relation to the chromosome axis. Using immunocytogenetics, we observed that the localisation of SPO11-1 overlaps relatively weakly with the chromosome axis and RAD51, a marker of meiotic DSBs, and that SPO11-1 recruitment to chromatin is genetically independent of the axis. In contrast, PRD3 localisation correlates more strongly with RAD51 and the chromosome axis. This indicates that PRD3 likely forms a functional link between SPO11-1 and the chromosome axis to promote meiotic DSB formation. We also uncovered a new function of SPO11-1 in the nucleation of the synaptonemal complex protein ZYP1. We demonstrate that chromosome co-alignment associated with ZYP1 deposition can occur in the absence of DSBs, and is dependent on SPO11-1, but not PRD3. Lastly, we show that the progression of meiosis is influenced by the presence of aberrant chromosomal connections, but not by the absence of DSBs or synapsis. Altogether, our study provides mechanistic insights into the control of meiotic DSB formation and reveals diverse functional interactions between SPO11-1, PRD3 and the chromosome axis.

Arabidopsis HEAT SHOCK FACTOR BINDING PROTEIN is required to limit meiotic crossovers and HEI10 transcription.

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

CTAB DNA Extraction and Genotyping-by-Sequencing to Map Meiotic Crossovers in Plants.

Reciprocal DNA crossovers between chromosomes form new allelic combinations and contribute to the formation of novel genetic diversity. Crossovers are formed during meiosis of germ cells and these recombination events have influenced plant genome evolution and are used during breeding to create improved plant varieties. Meiotic crossovers are not uniformly formed across the genome but instead occur in regions with low nucleosome density. The recombination landscape differs between the model plant organism Arabidopsis thaliana and crops such as rice and maize. Genotyping-by-sequencing is a technique that can detect crossover location and provide information on the recombination landscape genome-wide. This technique can be used to compare crossover position between ecotypes, species, and mutant lines to gain information on factors controlling meiotic recombination. In this protocol, we describe the steps to purify DNA from plant tissue, prepare 96 DNA libraries in parallel and perform quality control before next-generation sequencing.

Super-resolution Chromatin Visualization Using a Combined Method of Fluorescence In Situ Hybridization and Structured Illumination Microscopy in Solanum lycopersicum.

Chromatin organization influences gene and transposon expression, and regulates various cellular processes. Higher order chromatin structure has been widely studied using genomic approaches and microscopy image analyses. Chromosome conformation capture and sequencing the junction of DNA fragments enables the study of both chromatin interaction and chromosome folding. However, certain cell types are embedded in other cell types which complicate the process of studying them using high-throughput genomic approaches. To overcome this limitation, high-resolution microscopy techniques are now available to investigate chromatin organization in single cells. In this chapter, we provide a detailed protocol to prepare chromosome spreading from tomato nuclei, to label genomic loci by fluorescence in situ hybridization, and to visualize these locations at high resolution with Structured Illumination microscopy.

The genetic and epigenetic landscape of the Arabidopsis centromeres.

Centromeres attach chromosomes to spindle microtubules during cell division and, despite this conserved role, show paradoxically rapid evolution and are typified by complex repeats. We used long-read sequencing to generate the Col-CEN Arabidopsis thaliana genome assembly that resolves all five centromeres. The centromeres consist of megabase-scale tandemly repeated satellite arrays, which support CENTROMERE SPECIFIC HISTONE H3 (CENH3) occupancy and are densely DNA methylated, with satellite variants private to each chromosome. CENH3 preferentially occupies satellites that show the least amount of divergence and occur in higher-order repeats. The centromeres are invaded by ATHILA retrotransposons, which disrupt genetic and epigenetic organization. Centromeric crossover recombination is suppressed, yet low levels of meiotic DNA double-strand breaks occur that are regulated by DNA methylation. We propose that Arabidopsis centromeres are evolving through cycles of satellite homogenization and retrotransposon-driven diversification.

Loss of chromatin remodeler DDM1 causes segregation distortion in Arabidopsis thaliana.

MAIN CONCLUSION: In ddm1 mutants, the DNA methylation is primarily affected in the heterochromatic region of the chromosomes, which is associated with the segregation distortion of SNPs in the F2 progenies. Segregation distortion (SD) is common in most genetic mapping experiments and a valuable resource to determine how gene loci induce deviation. Meiotic DNA crossing over and SD are under the control of several types of epigenetic modifications. DNA methylation is an important regulatory epigenetic modification that is inherited across generations. In the present study, we investigated the relationship between SD and DNA methylation. The ecotypes Col-0/C24 and chromatin remodeler mutants ddm1-10/Col and ddm1-15/C24 were reciprocally crossed to obtain F2 generations. A total of 300 plants for each reciprocally crossed plant in the F2 generations were subjected to next-generation sequencing to detect the single-nucleotide polymorphisms (SNPs) as DNA markers. All SNPs were analyzed using the Chi-square test method to determine their segregation ratio in F2 generations. Through the segregation ratio, whole-genome SNPs were classified into 16 classes. In class 10, the SNPs in the reciprocal crosses of wild type showed the expected Mendelian ratio of 1:2:1, while those in the reciprocal crosses of ddm1 mutants showed distortion. In contrast, all SNPs in class 16 displayed a normal 1:2:1 ratio, and class 1 showed SD, regardless of wild type or mutants, as assessed using CAPS (cleaved amplified polymorphic sequences) marker analysis to confirm the next-generation sequencing. In ddm1 mutants, the DNA methylation is highly reduced throughout the whole genome and more significantly in the heterochromatic regions of chromosomes. Our results showed that the ddm1 mutants exhibit low levels of DNA methylation, which facilitates the SD of SNPs primarily located in the heterochromatic region of chromosomes by reducing the heterozygous ratio. The present study will provide a strong base for future research focusing on the impact of DNA methylation on trait segregation and plant evolution.

Rewiring Meiosis for Crop Improvement.

Meiosis is a specialized cell division that contributes to halve the genome content and reshuffle allelic combinations between generations in sexually reproducing eukaryotes. During meiosis, a large number of programmed DNA double-strand breaks (DSBs) are formed throughout the genome. Repair of meiotic DSBs facilitates the pairing of homologs and forms crossovers which are the reciprocal exchange of genetic information between chromosomes. Meiotic recombination also influences centromere organization and is essential for proper chromosome segregation. Accordingly, meiotic recombination drives genome evolution and is a powerful tool for breeders to create new varieties important to food security. Modifying meiotic recombination has the potential to accelerate plant breeding but it can also have detrimental effects on plant performance by breaking beneficial genetic linkages. Therefore, it is essential to gain a better understanding of these processes in order to develop novel strategies to facilitate plant breeding. Recent progress in targeted recombination technologies, chromosome engineering, and an increasing knowledge in the control of meiotic chromosome segregation has significantly increased our ability to manipulate meiosis. In this review, we summarize the latest findings and technologies on meiosis in plants. We also highlight recent attempts and future directions to manipulate crossover events and control the meiotic division process in a breeding perspective.

Natural variation identifies SNI1, the SMC5/6 component, as a modifier of meiotic crossover in Arabidopsis.

The frequency and distribution of meiotic crossovers are tightly controlled; however, variation in this process can be observed both within and between species. Using crosses of two natural Arabidopsis thaliana accessions, Col and Ler, we mapped a crossover modifier locus to semidominant polymorphisms in SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), which encodes a component of the SMC5/6 complex. The sni1 mutant exhibits a modified pattern of recombination across the genome with crossovers elevated in chromosome distal regions but reduced in pericentromeres. Mutations in SNI1 result in reduced crossover interference and can partially restore the fertility of a Class I crossover pathway mutant, which suggests that the protein affects noninterfering crossover repair. Therefore, we tested genetic interactions between SNI1 and both RECQ4 and FANCM DNA helicases, which showed that additional Class II crossovers observed in the sni1 mutant are FANCM independent. Furthermore, genetic analysis of other SMC5/6 mutants confirms the observations of crossover redistribution made for SNI1 The study reveals the importance of the SMC5/6 complex in ensuring the proper progress of meiotic recombination in plants.

HIGH CROSSOVER RATE1 encodes PROTEIN PHOSPHATASE X1 and restricts meiotic crossovers in Arabidopsis.

Meiotic crossovers are tightly restricted in most eukaryotes, despite an excess of initiating DNA double-strand breaks. The majority of plant crossovers are dependent on class I interfering repair, with a minority formed via the class II pathway. Class II repair is limited by anti-recombination pathways; however, similar pathways repressing class I crossovers have not been identified. Here, we performed a forward genetic screen in Arabidopsis using fluorescent crossover reporters to identify mutants with increased or decreased recombination frequency. We identified HIGH CROSSOVER RATE1 (HCR1) as repressing crossovers and encoding PROTEIN PHOSPHATASE X1. Genome-wide analysis showed that hcr1 crossovers are increased in the distal chromosome arms. MLH1 foci significantly increase in hcr1 and crossover interference decreases, demonstrating an effect on class I repair. Consistently, yeast two-hybrid and in planta assays show interaction between HCR1 and class I proteins, including HEI10, PTD, MSH5 and MLH1. We propose that HCR1 plays a major role in opposition to pro-recombination kinases to restrict crossovers in Arabidopsis.

Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.

ASY1 acts as a dosage-dependent antagonist of telomere-led recombination and mediates crossover interference in Arabidopsis.

During meiosis, interhomolog recombination produces crossovers and noncrossovers to create genetic diversity. Meiotic recombination frequency varies at multiple scales, with high subtelomeric recombination and suppressed centromeric recombination typical in many eukaryotes. During recombination, sister chromatids are tethered as loops to a polymerized chromosome axis, which, in plants, includes the ASY1 HORMA domain protein and REC8-cohesin complexes. Using chromatin immunoprecipitation, we show an ascending telomere-to-centromere gradient of ASY1 enrichment, which correlates strongly with REC8-cohesin ChIP-seq data. We mapped crossovers genome-wide in the absence of ASY1 and observe that telomere-led recombination becomes dominant. Surprisingly, asy1/+ heterozygotes also remodel crossovers toward subtelomeric regions at the expense of the pericentromeres. Telomeric recombination increases in asy1/+ occur in distal regions where ASY1 and REC8 ChIP enrichment are lowest in wild type. In wild type, the majority of crossovers show interference, meaning that they are more widely spaced along the chromosomes than expected by chance. To measure interference, we analyzed double crossover distances, MLH1 foci, and fluorescent pollen tetrads. Interestingly, while crossover interference is normal in asy1/+, it is undetectable in asy1 mutants, indicating that ASY1 is required to mediate crossover interference. Together, this is consistent with ASY1 antagonizing telomere-led recombination and promoting spaced crossover formation along the chromosomes via interference. These findings provide insight into the role of the meiotic axis in patterning recombination frequency within plant genomes.

Interacting Genomic Landscapes of REC8-Cohesin, Chromatin, and Meiotic Recombination in Arabidopsis.

Meiosis recombines genetic variation and influences eukaryote genome evolution. During meiosis, DNA double-strand breaks (DSBs) enter interhomolog repair to yield crossovers and noncrossovers. DSB repair occurs as replicated sister chromatids are connected to a polymerized axis. Cohesin rings containing the REC8 kleisin subunit bind sister chromatids and anchor chromosomes to the axis. Here, we report the genomic landscape of REC8 using chromatin immunoprecipitation sequencing (ChIP-seq) in Arabidopsis (Arabidopsis thaliana). REC8 associates with regions of high nucleosome occupancy in multiple chromatin states, including histone methylation at H3K4 (expressed genes), H3K27 (silent genes), and H3K9 (silent transposons). REC8 enrichment is associated with suppression of meiotic DSBs and crossovers at the chromosome and fine scales. As REC8 enrichment is greatest in transposon-dense heterochromatin, we repeated ChIP-seq in kyp suvh5 suvh6 H3K9me2 mutants. Surprisingly, REC8 enrichment is maintained in kyp suvh5 suvh6 heterochromatin and no defects in centromeric cohesion were observed. REC8 occupancy within genes anti-correlates with transcription and is reduced in COPIA transposons that reactivate expression in kyp suvh5 suvh6 Abnormal axis structures form in rec8 that recruit DSB-associated protein foci and undergo synapsis, which is followed by chromosome fragmentation. Therefore, REC8 occupancy correlates with multiple chromatin states and is required to organize meiotic chromosome architecture and interhomolog recombination.

Chromatin Immunoprecipitation of Meiotically Expressed Proteins from Arabidopsis thaliana Flowers.

During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.

Natural Variation in TBP-ASSOCIATED FACTOR 4b Controls Meiotic Crossover and Germline Transcription in Arabidopsis.

Meiotic crossover frequency varies within genomes, which influences genetic diversity and adaptation. In turn, genetic variation within populations can act to modify crossover frequency in cis and trans. To identify genetic variation that controls meiotic crossover frequency, we screened Arabidopsis accessions using fluorescent recombination reporters. We mapped a genetic modifier of crossover frequency in Col × Bur populations of Arabidopsis to a premature stop codon within TBP-ASSOCIATED FACTOR 4b (TAF4b), which encodes a subunit of the RNA polymerase II general transcription factor TFIID. The Arabidopsis taf4b mutation is a rare variant found in the British Isles, originating in South-West Ireland. Using genetics, genomics, and immunocytology, we demonstrate a genome-wide decrease in taf4b crossovers, with strongest reduction in the sub-telomeric regions. Using RNA sequencing (RNA-seq) from purified meiocytes, we show that TAF4b expression is meiocyte enriched, whereas its paralog TAF4 is broadly expressed. Consistent with the role of TFIID in promoting gene expression, RNA-seq of wild-type and taf4b meiocytes identified widespread transcriptional changes, including in genes that regulate the meiotic cell cycle and recombination. Therefore, TAF4b duplication is associated with acquisition of meiocyte-specific expression and promotion of germline transcription, which act directly or indirectly to elevate crossovers. This identifies a novel mode of meiotic recombination control via a general transcription factor.

Tackling Plant Meiosis: From Model Research to Crop Improvement.

Genetic engineering and traditional plant breeding, which harnesses the natural genetic variation that arises during meiosis, will have key roles to improve crop varieties and thus deliver Food Security in the future. Meiosis, a specialized cell division producing haploid gametes to maintain somatic diploidy following their fusion, assures genetic variation by regulated genetic exchange through homologous recombination. However, meiotic recombination events are restricted in their total number and their distribution along chromosomes limiting allelic variations in breeding programs. Thus, modifying the number and distribution of meiotic recombination events has great potential to improve and accelerate plant breeding. In recent years much progress has been made in understanding meiotic progression and recombination in plants. Many genes and factors involved in these processes have been identified primarily in Arabidopsis thaliana but also more recently in crops such as Brassica, rice, barley, maize, or wheat. These advances put researchers in the position to translate acquired knowledge to various crops likely improving and accelerating breeding programs. However, although fundamental aspects of meiotic progression and recombination are conserved between species, differences in genome size and organization (due to repetitive DNA content and ploidy level) exist, particularly among plants, that likely account for differences in meiotic progression and recombination patterns found between species. Thus, tools and approaches are needed to better understand differences and similarities in meiotic progression and recombination among plants, to study fundamental aspects of meiosis in a variety of plants including crops and non-model species, and to transfer knowledge into crop species. In this article, we provide an overview of tools and approaches available to study plant meiosis, highlight new techniques, give examples of areas of future research and review distinct aspects of meiosis in non-model species.