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

Abstract

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, including DNA methylation and H3K9 methylation. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. To analyze the in vivo function of H2A.W, we created a h2a.w null mutant via CRISPR-Cas9, h2a.w-2. We found that H2A.W is not essential for plant development, and that loss of H2A.W did not perturb histone methylation patterns. In contrast, we found a reduction of non-CG DNA methylation in pericentromeric heterochromatin and an increase in DNA methylation at euchromatic sites targeted by the RNA-directed DNA methylation pathway. Loss of DNA methylation in h2a.w-2 correlated with both increased H1 occupancy and decreased DNA accessibility at heterochromatin. Our results indicate that H2A.W helps stabilize the accessibility of heterochromatin and facilitates efficient DNA methylation by fine tuning the genomic distribution of H1.

Repression of CHROMOMETHYLASE 3 Prevents Epigenetic Collateral Damage in Arabidopsis

“…4K) (Mathieu et al, 2003;Soppe et al, 2002) and CMT3 prefers features associated with stable (Figs . 4E,F,S4G) (Bourguet et al, 2021;Osakabe et al, 2018;Yelagandula et al, 2014) over unstable nucleosomes such as H2A.Z with active marks (Figs. 4H,I,S4H).…”

“…Indeed, CHG hypermethylation across the four groups was positively correlated with levels of the stable histone variants H2A, H2A.X and most notably H2A.W that was recently shown to be required for CHG methylation (Figs . 4E,F,S4G) (Bourguet et al, 2021;Yelagandula et al, 2014). CMT3-induced CHG hypermethylation was also tightly associated with transcriptionally repressive H3K9me2 marks, which are required for interdependent feedback loops with CMT3 (Fig.…”

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis

“…clusters 2-4) (Figure 4-figure supplement 1I,J), we hypothesized that histone variants conferring differential nucleosome stabilities and chromatin accessibility may influence ectopic CMT3-induced hypermethylation (Osakabe et al, 2018). Indeed, CHG hypermethylation across the four groups was positively correlated with levels of the stable histone variants H2A, H2A.X and most notably H2A.W that was recently shown to be required for CHG methylation (Figure 4E,F, Figure 4-figure supplement 1K) (Bourguet et al, 2021;Yelagandula et al, 2014). CMT3-induced CHG hypermethylation was also tightly associated with transcriptionally repressive H3K9me2 marks, which are required for interdependent feedback loops with CMT3 (Figure 4G).…”

Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

“…Actually, a large genomic rearrangement in one of the T-DNA insertion mutant allele hindered proper functional analysis of H2A.W: the initially reported severe developmental and heterochromatin defects, rather than being caused by the loss of H2A.W function, were due to a duplication of the CMT3 locus. This was revealed by a newly constructed CRISPR-Cas9-induced null h2a.w triple mutant, which displayed no visible developmental phenotypes and had only minor effects on gene expression [ 51 ]. The CRISPR-Cas9 editing tool thus brings new perspectives, notably avoiding T-DNA-induced wide genomic rearrangements with unexpected effects on the epigenome.…”

“…A report describes the editing of histone H2A variants (H2AX and macroH2A, involved in DNA Damage Response) using CRISPR-Cas9 in human cells [ 52 ]. In plants, the above mentioned CRISPR-Cas9-induced new null h2a.w triple mutant (part 4.1) showed that H2A.W fine-tunes the accessibility of heterochromatin by preventing deposition of the linker Histone H1, thereby facilitating access to non-CG DNA methylation factors [ 51 ].…”