The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create an h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation. Overall design: ChIP-seq of a large number of modified and unmodified histones (H2A, H2A.Z, H2A.X, H3, H3K9me1, H3K9me2, H3K27me1, H1) in (WT) and triple H2A.W-null (h2a.w-2) plants to examine how the chromatin environment is perturbed upon loss of H2A.W. This was used in conjunction with ATAC-seq and bisulfite-seq of WT, h2a.w-2, double H1-null (h1) and quintuple H1 and H2A.W null (h1 h2a.w-2) to examine the relationship between H1 and H2A.W in regulating heterochromatin. Total (rRNA-depleted) RNA was also sequenced in WT and h2a.w-2 to identify any changes in gene or TE expression. Two replicates of ChIP-seq, ATAC-seq and bisulfite-see experiments were performed, while RNA-seq was done in triplicate. The h2a.w-2 line used in this study is a newly generated triple hta6 hta7 hta12 H2A.W null line. RNA-seq was also performed in h2a.w-1, the triple mutant generated in Yelagandula et al 2014, for comparison.