We used functional genomics to profile key recombination proteins in the Arabidopsis genome. Prior to this project, few genome-wide approaches had been taken to map recombination in plants. ERC support allowed us to innovate and generate these maps. A major output was sequencing short DNA oligos associated with SPO11-1, that initiate crossovers (Choi et al 2018 Genome Res). This revealed that DSBs associate with nucleosome free regions in gene promoters, and surprisingly, in specific classes transposons, including helitrons.
Hotspots were frequently DNA hypomethylated, leading us to repeat sequencing in the methylation mutants met1 and kyp suvh5 suvh6, which revealed increased meiotic DSBs in the repeat regions. Interestingly, met1 and kyp suvh5 suvh6 mutants show opposite effects on centromeric crossovers, implying divergent repair dependent on heterochromatic state (Underwood et al. 2018 Genome Res.).
As new long-read technologies emerged, new experimental possibilities arose, including assembly of repetitive genome regions. ERC support allowed us to innovate with these technologies and extend our work into previously unknown genome regions. Using these maps, and the recombination datasets generated in SynthHotSpot, we were able to show new relationships between the satellite repeats and meiotic recombination (Naish et al 2021 Science).
We generated genome-wide maps of key recombination factors using ChIP-seq, including the meiotic cohesin REC8. Surprisingly, this revealed REC8 high enrichment in heterochromatin. We repeated ChIP-seq in the kyp suvh5 suvh6 DNA methylation mutant, which demonstrated minimal changes to REC8 loading, showing independence from classical heterochromatin (Lambing et al 2020 Plant Cell).
We investigated the localisation the meiotic axis protein ASY1 via ChIP-seq and showed high correlation with REC8. Genetic analysis showed that recombination is highly dependent on ASY1 dosage, and that crossovers are restricted to sub-telomeric locations in asy1 and crossover interference is blocked (Lambing et al 2020 PNAS), developing a new model for how the chromosome axis controls crossovers.
We used sequencing to define crossovers at the local scale and mapped hotspots within disease resistance loci (Serra et al 2018 PLoS Genetics). We generated maps of crossovers genome wide in multiple genetic backgrounds, showing that while structural genetic variation suppresses recombination, intermediate levels of diversity associate with highest recombination (Rowan et al 2019 Genetics; Blackwell et al 2020 EMBO Journal). We also identified a novel recombination QTL in TAF4b that influences meiotic transcription and recombination (Lawrence et al 2019 Current Biology).
We used newly emerging tools to target proteins in order to create hotspots . First, we tested TALENs to target SPO11-1, but faced technical difficulties achieving complementing lines. We switched strategy to the dCas9 protein fused to SPO11-1 and MTOPVIB and complemented the respective mutants. Our gRNAs were active with Cas9. However, when the dCas9 fusions and gRNAs were combined in vivo this strategy was insufficient to create hotspots (Yelina et al. 2022 G3). Work performed in parallel has demonstrated the key role of the HEI10 E3 ligase (Ziolkowski et al 2017 Genes & Development; Serra et al 2018 PNAS) in controlling crossovers. Therefore, the technological platform established here will be used to tether HEI10 as the future strategy.
ERC support allowed us to make fundamental insights into recombination, epigenetic information and sequence polymorphism in plant genomes, which had a major effect on my career. It has allowed my group to pursue high risk – high reward projects and to reveal new insights into control of recombination. ERC support allowed us to build platform technologies and knowledge that will serve as a foundation for the next ten years of research. ERC support has enormously strengthened our international collaborations that are leading us in exciting directions.
