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

Energy supply of countryside based on geothermal deposit

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes

Recombination Rate Heterogeneity within Arabidopsis Disease Resistance Genes.

Meiotic crossover frequency varies extensively along chromosomes and is typically concentrated in hotspots. As recombination increases genetic diversity, hotspots are predicted to occur at immunity genes, where variation may be beneficial. A major component of plant immunity is recognition of pathogen Avirulence (Avr) effectors by resistance (R) genes that encode NBS-LRR domain proteins. Therefore, we sought to test whether NBS-LRR genes would overlap with meiotic crossover hotspots using experimental genetics in Arabidopsis thaliana. NBS-LRR genes tend to physically cluster in plant genomes; for example, in Arabidopsis most are located in large clusters on the south arms of chromosomes 1 and 5. We experimentally mapped 1,439 crossovers within these clusters and observed NBS-LRR gene associated hotspots, which were also detected as historical hotspots via analysis of linkage disequilibrium. However, we also observed NBS-LRR gene coldspots, which in some cases correlate with structural heterozygosity. To study recombination at the fine-scale we used high-throughput sequencing to analyze ~1,000 crossovers within the RESISTANCE TO ALBUGO CANDIDA1 (RAC1) R gene hotspot. This revealed elevated intragenic crossovers, overlapping nucleosome-occupied exons that encode the TIR, NBS and LRR domains. The highest RAC1 recombination frequency was promoter-proximal and overlapped CTT-repeat DNA sequence motifs, which have previously been associated with plant crossover hotspots. Additionally, we show a significant influence of natural genetic variation on NBS-LRR cluster recombination rates, using crosses between Arabidopsis ecotypes. In conclusion, we show that a subset of NBS-LRR genes are strong hotspots, whereas others are coldspots. This reveals a complex recombination landscape in Arabidopsis NBS-LRR genes, which we propose results from varying coevolutionary pressures exerted by host-pathogen relationships, and is influenced by structural heterozygosity.Research in the Henderson laboratory was supported by a Royal Society University Research Fellowship, Gatsby Charitable Foundation grant 2962, BBSRC grant BB/N007557/1 and National Natural Science Foundation of China grant 61403318. KC was funded by an EMBO long term postdoctoral fellowship ALTF 807-2009. PAZ was supported by a Polish Mobility Plus Fellowship 605/MOB/2011/0. GPC is funded by a National Science Foundation Grant (MCB-1121563). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLOS at http://dx.doi.org/10.1371/journal.pgen.1006179

Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes

Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters

PRDM9 directs human meiotic crossover hotspots to intergenic sequence motifs, whereas budding yeast hotspots overlap low nucleosome density regions in gene promoters. To investigate hotspots in plants, which lack PRDM9, we used coalescent analysis of Arabidopsis genetic variation. Crossovers increase towards gene promoters and terminators, and hotspots are associated with active chromatin modifications, including H2A.Z, histone H3K4me3, low nucleosome density and low DNA methylation. Hotspot-enriched A-rich and CTT-repeat DNA motifs occur upstream and downstream of transcriptional start respectively. Crossovers are asymmetric around promoters and highest over CTT-motifs and H2A.Z-nucleosomes. Pollen-typing, segregation and cytogenetic analysis show decreased crossovers in the arp6 H2A.Z deposition mutant, at multiple scales. During meiosis H2A.Z and DMC1/RAD51 recombinases form overlapping chromosomal foci. As arp6 reduces DMC1/RAD51 foci, H2A.Z may promote formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hotspots within eukaryotes and PRDM9 is a derived state within vertebrates

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

Quantification and Sequencing of Crossover Recombinant Molecules from Arabidopsis Pollen DNA

During meiosis, homologous chromosomes undergo recombination, which can result in formation of reciprocal crossover molecules. Crossover frequency is highly variable across the genome, typically occurring in narrow hotspots, which has a significant effect on patterns of genetic diversity. Here we describe methods to measure crossover frequency in plants at the hotspot scale (bp–kb), using allele-specific PCR amplification from genomic DNA extracted from the pollen of F1 heterozygous plants. We describe (1) titration methods that allow amplification, quantification and sequencing of single crossover molecules, (2) quantitative PCR methods to more rapidly measure crossover frequency, and (3) application of high-throughput sequencing for study of crossover distributions within hotspots. We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions.11Nscopu

Coexpression of MEIOTIC-TOPOISOMERASE VIB-dCas9 with guide RNAs specific to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis.

During meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms, crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double-strand breaks that are catalyzed by SPO11 complexes, which consist of 2 catalytic (SPO11-1 and SPO11-2) and 2 noncatalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to coexpress an MTOPVIB-dCas9 fusion protein with guide RNAs specific to the 3a crossover hotspot. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.BBSRC, Broodbank Trust, Gatsby Charitable Foundatio

Coexpression of MEIOTIC-TOPOISOMERASE VIB-dCas9 with guide RNAs specific to a recombination hotspot is insufficient to increase crossover frequency in Arabidopsis.

Funder: Broodbank FellowshipFunder: Gatsby Grant to Exceptional ResearchersDuring meiosis, homologous chromosomes pair and recombine, which can result in reciprocal crossovers that increase genetic diversity. Crossovers are unevenly distributed along eukaryote chromosomes and show repression in heterochromatin and the centromeres. Within the chromosome arms, crossovers are often concentrated in hotspots, which are typically in the kilobase range. The uneven distribution of crossovers along chromosomes, together with their low number per meiosis, creates a limitation during crop breeding, where recombination can be beneficial. Therefore, targeting crossovers to specific genome locations has the potential to accelerate crop improvement. In plants, meiotic crossovers are initiated by DNA double-strand breaks that are catalyzed by SPO11 complexes, which consist of 2 catalytic (SPO11-1 and SPO11-2) and 2 noncatalytic subunits (MTOPVIB). We used the model plant Arabidopsis thaliana to coexpress an MTOPVIB-dCas9 fusion protein with guide RNAs specific to the 3a crossover hotspot. We observed that this was insufficient to significantly change meiotic crossover frequency or pattern within 3a. We discuss the implications of our findings for targeting meiotic recombination within plant genomes.BBSRC, Broodbank Trust, Gatsby Charitable Foundatio