The histone variant H2A.W restricts heterochromatic crossovers in <i>Arabidopsis</i>

Meiotic crossovers rearrange allele combinations and create offspring diversity. Crossovers occur nonrandomly along chromosomes, predominantly in distal euchromatin and less in pericentromeric heterochromatin marked with histone H3 lysine 9 dimethylation (H3K9me2) and the H2A variant H2A.W in <i>Arabidopsis thaliana</i>. Loss of H3K9me2 increases heterochromatic crossovers, but how H2A.W affects crossover formation in pericentromeric regions is unknown. Here, we report that H2A.W is required to restrict heterochromatic crossovers in <i>Arabidopsis</i>. Using meiosis-specific microRNA-induced gene silencing (meiMIGS) and fluorescence-tagged recombination reporters, we show that meiotic knockdown of <i>H2A.W.6</i>, <i>H2A.W.7</i>, and <i>H2A.W.12</i> (<i>meiMIGS-H2A.W.6/7/12</i>) increases pericentromeric crossovers. High-resolution genomic maps of crossovers show that <i>meiMIGS-H2A.W.6/7/12</i> enhances heterochromatic crossovers, similar to <i>meiMIGS</i> plants silencing the H3K9me2 pathway. Consistently, genome-wide crossover maps show that the mutants <i>h2a.w.6</i>, <i>h2a.w.7</i>, <i>h2a.w.6 h2a.w.7</i>, and <i>h2a.w.6 h2a.w.7 h2a.w.12,</i> but not <i>h2a.w.12,</i> exhibit a similar increase in heterochromatic crossovers to <i>meiMIGS-H2A.W.6/7/12</i>, demonstrating that H2A.W.6 and H2A.W.7 limit heterochromatic crossovers. Profiling of genome-wide nucleosome density using micrococcal nuclease sequencing reveals that <i>h2a.w</i> mutants with increased heterochromatic crossovers have increased heterochromatin accessibility, with lower H3K9me2 levels during meiosis. Our findings shed light on the role of H2A.W variants as heterochromatin compaction factors that suppress meiotic crossovers within the pericentromeric regions.