Investigating the interplay between chromosome axes and homologous recombination in arabidopsis meiosis

During meiosis and mitosis, the chromatin is organised stochastically in loop arrays. The formation and stabilisation of chromatin loop arrays is dependent on both cohesin and condensin but independent on the presence of a chromosome axis. The meiotic chromosome axes differ significantly from the mitotic axes. Components of the meiotic chromosome axes are thought to localise on the chromatin during S-phase and G2 in budding yeast and Arabidopsis, respectively, and promote the formation of meiotic DNA double-strand breaks (DSBs). In addition, several evidences suggest that the chromosome axes are required for the repair of SPO11-dependent DSBs off the sister. The status of the meiotic axes may also have an important role in the designation of future crossover sites and crossover interference. However, the composition of the chromosome axes, the regulation of its morphology and its function during meiosis are poorly understood in plants. The aims of this study consisted at further investigating the function of the axis-associated protein AtASY1 during DSB formation and DSB repair. The interaction between AtASY1 and the structural axis component AtASY3 was tested. In addition, the interplay between chromosome axes and the localisation of the AtSPO11-accessory protein AtPRD3 was analysed. This study showed that the formation of meiotic DSBs is spatially and temporally regulated by the formation of a nascent axis in Arabidopsis. The formation of the axes was independent on the formation of DSBs. However, the repair of DSBs off the sister and the formation of inter-homologue crossovers led to the remodelling of the axes in a pathway dependent on AtPCH2, an AAA ATPase family member. In addition, the phosphorylation level of AtASY1 T295 was increased in response to DSB formation. These highlight the coordination between the formation of DSBs/progression of DSB repair by homologous recombination and the remodelling of the chromosome axes

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

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.Research was supported by grants from the European Research Council Consolidator award SynthHotSpot and Proof-of-Concept award HEIREC and BBSRC ERA-CAPs Grant BB/M004937/1

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.We thank Korbinian Schneeberger and Beth Rowan for advice implementing TIGER and Ler polymorphism data, Donna Bond for pJawohl-Act2, Quentin Gouil for the bisulfite sequencing protocol, Simon Andrews and Felix Krueger for advice using SeqMonk, Gregory Copenhaver and Avi Levy for fluorescent lines, Raphael Mercier for zip4-2 fancm-1, Chris Franklin for the ASY1 antibody, and the Gurdon Institute Imaging Facility for access to microscopes. Research was supported by a Broodbank Fellowship (to N.E.Y.), a Royal Society University Research Fellowship (to I.R.H.), grant GAT2962 from the Gatsby Charitable Foundation (to I.R.H.), and Biotechnology and Biological Sciences Research Council grant BB/L006847/1 (to I.R.H.).This is the final version of the article. It first appeared from Cold Spring Habour Laboratory Press via http://dx.doi.org/10.1101/gad.270876.11

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.This work was supported by a BBSRC DTP studentship (EJL), European Research Area Network for Coodinating Action in Plant Sciences/BBSRC “DeCOP” (BB/M004937/1) (CAL), a BBSRC David Phillips Fellowship (BB/L025043/1) (HG, XF), the European Research Council (CoG ‘SynthHotspot’) (AJT, CAL, IRH) and (StG ‘SexMeth’) (XF) and a Sainsbury Charitable Foundation Studentship (ARB)

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 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.Marie Curie International Training Network COMREC European Research Council (ERC) National Research Foundation of Korea Suh Kyungbae Foundatio

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