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

Abstract

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 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 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 mutants, coincident with reduced nucleosome occupancy, gain of transcription, and H3K4me3. Together, our work reveals a complex relationship between chromatin and meiotic DSBs within genes and transposons, with significance for the diversity and evolution of plant genomes.

A pseudomolecule‐scale genome assembly of the liverwort Marchantia polymorpha

“…DNA methylation is known to be important in modulating recombination rates in the model plant A. thaliana , which is realized in a strong anticorrelation between recombination rates and DNA methylation (Yelina et al , ; Zhao et al , ; Choi et al , ; Dluzewska et al , ; Tock and Henderson, ; Underwood et al , ). In line with that, we found that recombination rate and the level of DNA methylation were significantly anticorrelated, both in M. polymorpha and P. patens .…”

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

“…An important question raised by our study is why pericentromeric crossover frequency increases in the H3K9me2/non-CG pathway mutants reported here, but not in met1 and ddm1 where reduced pericentromeric crossover frequency is observed (Colomé-Tatché et al 2012;Melamed-Bessudo and Levy 2012;Mirouze et al 2012;Yelina et al 2012Yelina et al , 2015, despite both kyp suvh5 suvh6 and met1 showing increased SPO11-1-oligos in proximity to centromeres (Choi et al 2018). MET1 and DDM1 play major roles in the maintenance of DNA methylation in the CG context.…”

“…We propose that, while both CG and non-CG DNA methylation inhibit centromeric meiotic DSBs (Choi et al 2018), only non-CG methylation and/or H3K9me2 inhibit crossovers. In agreement with this idea, euchromatic crossover hotspots in A. thaliana can be silenced by RNA-directed DNA methylation associated with gain of H3K9me2 and both CG and non-CG methylation .…”

“…SPO11 enzymes are related to topoisomerase-VI transesterases and become covalently bound to ∼20-to 50-base target site oligonucleotides during DSB formation (Keeney and Kleckner 1995;Pan et al 2011;Lange et al 2016). We have purified and sequenced A. thaliana SPO11-1-oligos in order to map patterns of meiotic DSBs genome-wide, using a complementing SPO11-1-Myc spo11-1 line (Choi et al 2018). In order to profile meiotic DSBs in H3K9me2/non-CG DNA methylation mutants, we generated a SPO11-1-Myc spo11-1 kyp suvh5 suvh6 line and used this to purify and sequence SPO11-1-oligos (Supplemental Table S16).…”

“…A detailed protocol and analysis methodology are provided in an accompanying manuscript (Choi et al 2018). A complementing SPO11-1-Myc spo11-1 line was crossed with kyp suvh5 suvh6 triple mutants and SPO11-1-Myc spo11-1 kyp suvh5 suvh6 plants identified for analysis.…”