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.

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.

Natural variation and dosage of the HEI10 meiotic E3 ligase control Arabidopsis crossover recombination.

During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural Arabidopsis thaliana accessions, we identified two major recombination quantitative trait loci (rQTLs) that explain 56.9% of crossover variation in Col×Ler F2 populations. We mapped rQTL1 to semidominant polymorphisms in HEI10, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null hei10 mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional HEI10 copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest HEI10-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in Arabidopsis recombination. Our work reveals that HEI10 naturally limits Arabidopsis crossovers and has the potential to influence the response to selection.

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.

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.

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.

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.

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.

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes.

Epigenetic activation of meiotic recombination near Arabidopsis thaliana centromeres via loss of H3K9me2 and non-CG DNA methylation.

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.

Nucleosomes and DNA methylation shape meiotic DSB frequency in Arabidopsis thaliana transposons and gene regulatory regions.

Meiotic recombination initiates from DNA double-strand breaks (DSBs) generated by SPO11 topoisomerase-like complexes. Meiotic DSB frequency varies extensively along eukaryotic chromosomes, with hotspots controlled by chromatin and DNA sequence. To map meiotic DSBs throughout a plant genome, we purified and sequenced Arabidopsis thaliana SPO11-1-oligonucleotides. SPO11-1-oligos are elevated in gene promoters, terminators, and introns, which is driven by AT-sequence richness that excludes nucleosomes and allows SPO11-1 access. A positive relationship was observed between SPO11-1-oligos and crossovers genome-wide, although fine-scale correlations were weaker. This may reflect the influence of interhomolog polymorphism on crossover formation, downstream from DSB formation. Although H3K4me3 is enriched in proximity to SPO11-1-oligo hotspots at gene 5' ends, H3K4me3 levels do not correlate with DSBs. Repetitive transposons are thought to be recombination silenced during meiosis, to prevent nonallelic interactions and genome instability. Unexpectedly, we found high SPO11-1-oligo levels in nucleosome-depleted Helitron/Pogo/Tc1/Mariner DNA transposons, whereas retrotransposons were coldspots. High SPO11-1-oligo transposons are enriched within gene regulatory regions and in proximity to immunity genes, suggesting a role as recombination enhancers. As transposon mobility in plant genomes is restricted by DNA methylation, we used the met1 DNA methyltransferase mutant to investigate the role of heterochromatin in SPO11-1-oligo distributions. Epigenetic activation of meiotic DSBs in proximity to centromeres and transposons occurred in met1 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within A. thaliana genes and transposons, with significance for the diversity and evolution of plant genomes.

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.

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.

Massive crossover elevation via combination of HEI10 and recq4a recq4b during Arabidopsis meiosis.

During meiosis, homologous chromosomes undergo reciprocal crossovers, which generate genetic diversity and underpin classical crop improvement. Meiotic recombination initiates from DNA double-strand breaks (DSBs), which are processed into single-stranded DNA that can invade a homologous chromosome. The resulting joint molecules can ultimately be resolved as crossovers. In Arabidopsis, competing pathways balance the repair of ∼100-200 meiotic DSBs into ∼10 crossovers per meiosis, with the excess DSBs repaired as noncrossovers. To bias DSB repair toward crossovers, we simultaneously increased dosage of the procrossover E3 ligase gene HEI10 and introduced mutations in the anticrossovers helicase genes RECQ4A and RECQ4B As HEI10 and recq4a recq4b increase interfering and noninterfering crossover pathways, respectively, they combine additively to yield a massive meiotic recombination increase. Interestingly, we also show that increased HEI10 dosage increases crossover coincidence, which indicates an effect on interference. We also show that patterns of interhomolog polymorphism and heterochromatin drive recombination increases distally towards the subtelomeres in both HEI10 and recq4a recq4b backgrounds, while the centromeres remain crossover suppressed. These results provide a genetic framework for engineering meiotic recombination landscapes in plant genomes.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein-protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.

Understanding and Manipulating Meiotic Recombination in Plants.

Meiosis is a specialized cell division, essential in most reproducing organisms to halve the number of chromosomes, thereby enabling the restoration of ploidy levels during fertilization. A key step of meiosis is homologous recombination, which promotes homologous pairing and generates crossovers (COs) to connect homologous chromosomes until their separation at anaphase I. These CO sites, seen cytologically as chiasmata, represent a reciprocal exchange of genetic information between two homologous nonsister chromatids. This gene reshuffling during meiosis has a significant influence on evolution and also plays an essential role in plant breeding, because a successful breeding program depends on the ability to bring the desired combinations of alleles on chromosomes. However, the number and distribution of COs during meiosis is highly constrained. There is at least one CO per chromosome pair to ensure accurate segregation of homologs, but in most organisms, the CO number rarely exceeds three regardless of chromosome size. Moreover, their positions are not random on chromosomes but exhibit regional preference. Thus, genes in recombination-poor regions tend to be inherited together, hindering the generation of novel allelic combinations that could be exploited by breeding programs. Recently, much progress has been made in understanding meiotic recombination. In particular, many genes involved in the process in Arabidopsis (Arabidopsis thaliana) have been identified and analyzed. With the coming challenges of food security and climate change, and our enhanced knowledge of how COs are formed, the interest and needs in manipulating CO formation are greater than ever before. In this review, we focus on advances in understanding meiotic recombination and then summarize the attempts to manipulate CO formation. Last, we pay special attention to the meiotic recombination in polyploidy, which is a common genomic feature for many crop plants.

Arabidopsis PCH2 Mediates Meiotic Chromosome Remodeling and Maturation of Crossovers.

Meiotic chromosomes are organized into linear looped chromatin arrays by a protein axis localized along the loop-bases. Programmed remodelling of the axis occurs during prophase I of meiosis. Structured illumination microscopy (SIM) has revealed dynamic changes in the chromosome axis in Arabidopsis thaliana and Brassica oleracea. We show that the axis associated protein ASY1 is depleted during zygotene concomitant with synaptonemal complex (SC) formation. Study of an Atpch2 mutant demonstrates this requires the conserved AAA+ ATPase, PCH2, which localizes to the sites of axis remodelling. Loss of PCH2 leads to a failure to deplete ASY1 from the axes and compromizes SC polymerisation. Immunolocalization of recombination proteins in Atpch2 indicates that recombination initiation and CO designation during early prophase I occur normally. Evidence suggests that CO interference is initially functional in the mutant but there is a defect in CO maturation following designation. This leads to a reduction in COs and a failure to form COs between some homologous chromosome pairs leading to univalent chromosomes at metaphase I. Genetic analysis reveals that CO distribution is also affected in some chromosome regions. Together these data indicate that the axis remodelling defect in Atpch2 disrupts normal patterned formation of COs.

Inter-homolog crossing-over and synapsis in Arabidopsis meiosis are dependent on the chromosome axis protein AtASY3.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

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.

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.