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, 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 a 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.

Crosstalk between H2A variant-specific modifications impacts vital cell functions

“…We hypothesized that the expression patterns of H2A.W.6 and H2A.W.7 differ, leading to exposure of these two variants to contrasting activities that deposit and maintain KSPK phosphorylation. Contrary to our hypothesis, we observed that phosphorylation of KSPK in H2A.W.7 was still absent even when expressed under the control of the H2A.W.6 promoter in the triple mutant h2a.w-2 completely deprived of H2A.W [43] (Fig 4D). Furthermore, we observed phosphorylation of KSPK of the C-terminal tail of H2A.W.6 when fused to the core of H2A.W.7 irrespective of the promoter used (Fig 4D).…”

“…Contrary to our hypothesis, we observed that phosphorylation of KSPK in H2A.W.7 was still absent even when expressed under the control of the H2A.W.6 promoter in the triple mutant h2a . w-2 completely deprived of H2A.W [ 43 ] ( Fig 4D ). Furthermore, we observed phosphorylation of KSPK of the C-terminal tail of H2A.W.6 when fused to the core of H2A.W.7 irrespective of the promoter used ( Fig 4D ).…”

“…w . 12 ) [ 20 , 43 ]. Transgenic lines expressing WT and hypomorphic SQ motif mutants of H2A.W.7 in the hta7 mutant background as published [ 18 ].…”

“…For the final GreenGate reaction pGGZ003_ccdB, the entry clones were combined with empty destination vector. The final constructs were transformed into the triple hta6 hta7 hta12 mutant which is a complete knock-out line deprived of H2A.W [ 43 ].…”

“…We used the following mutant lines hta6 (SALK_024544.32; h2a.w.6), hta7 (GK_149G05; h2a.w.7), and hta12 (SAIL_667_G10; h2a.w.12) [20,43]. Transgenic lines expressing WT and hypomorphic SQ motif mutants of H2A.W.7 in the hta7 mutant background as published [18].…”