Embryo-specific epigenetic mechanisms reconstitute the CHH methylation landscape during Arabidopsis embryogenesis

Abstract

The modification of flowering plant DNA by CHH methylation acts primarily to silence transposable elements, of which many active copies are present in Arabidopsis thaliana. During embryogenesis, the CHH methylation landscape is dramatically reprogrammed, resulting in exceedingly high levels of this modification upon mature embryo formation. The mechanisms constituting the remodeling process, and its function in embryos, are unclear. Here, we isolate embryos from Arabidopsis plants harboring mutations for key components of the pathways that confer CHH methylation, namely RNA-directed DNA methylation (RdDM) and the Chromomethylase 2 (CMT2) pathways. We reveal that embryos are more methylated than leaves at shared CMT2 and RdDM targeting loci, accounting for most embryonic CHH hypermethylation. While the majority of embryo CHH methylated loci overlap with those in somatic tissues, a subset of conventional pericentric CMT2-methylated loci are instead targeted by RdDM in embryos. These loci, termed embRdDM exhibit intermediate H3K9me2 levels, associated with increased chromatin accessibility. Strikingly, more than 50% of the embRdDM loci in pollen vegetative (nurse) cells and ddm1 mutant somatic tissues are also targeted by RdDM, and these tissues were also reported to exhibit increased chromatin accessibility in pericentric heterochromatin. Furthermore, the root columella stem cell niche also displays CHH hypermethylation and an enriched presence of small RNAs at embRdDM loci. Finally, we observe a significant overlap of CHH hypermethylated loci with endosperm DEMETER targeting sites, suggesting that non-cell autonomous communication within the seed may contribute to the epigenetic landscape of the embryo. However, similar overlap with vegetative cell DEMETER targets indicates that the chromatin landscape that allows DEMETER access is mirrored in developing embryos, permitting CHH methylation catalysis at the same loci. Our findings demonstrate that both conserved and embryo-specific epigenetic mechanisms reshape CHH methylation profiles in the dynamic chromatin environment of embryogenesis.

Distinct regulatory pathways contribute to dynamic CHH methylation patterns in transposable elements throughout Arabidopsis embryogenesis

“…In addition, when we mapped the position of these non-sRNA overlapping CHH methylated regions, we found that they were tightly centromeric, and as such likely to be conferred by CMT2 ( Supplementary Figure S1 ). To further investigate the positive correlation of CHH methylation spreading with sRNA clusters, we visualized individual overlapping loci, and observed that expanding sRNA expression was coincident with CHH methylation spreading and accumulation ( Figures 2D–F ), providing support for previous observations that increased embryo CHH methylation is conferred by the RdDM pathway ( Hsieh et al., 2023 ).…”

“…RdDM targets are methylated by de novo methyltransferases, DRM1 and 2. Recent papers described the delineation of embryo CHH loci into classes based on their mechanism of catalysis using drm1/2 and cmt2 mutants ( Kawakatsu et al., 2017 ; Hsieh et al., 2023 ). Briefly, if an embryonic CHH DMR is targeted by CMT2 both in the vegetative (leaves) and reproductive embryo stages, that DMR is denoted as a conserved CMT2 (cCMT2) DMR.…”

“…If a DMR is targeted by DRM1/2 both in leaves and embryos, that DMR is denoted as a conserved RdDM (cRdDM) DMR. If a DMR is targeted by CMT2 or unmethylated in leaves but becomes exclusively targeted by DRM1/2 in embryos, that DMR is denoted an embryonic RdDM (eRdDM) DMR ( Hsieh et al., 2023 )…”

“…These regions were referred to as “CMT2-to-RdDM loci” by the authors. Some eRdDM DMRs exhibited similar traits to CMT2-to-RdDM loci, as they were initially methylated by CMT2 in somatic tissues and subsequently switch to the RdDM pathway after fertilization during embryonic development ( Hsieh et al., 2023 ). This prompted us to investigate the extent of overlap between eRdDM DMRs and CMT2-to-RdDM loci by He et al.…”

“…To determine the dependency of TE methylation on CMT2 and RdDM pathways, we utilized previously identified lists of differentially methylated regions (DMRs) ( Stroud et al., 2013 ), and recently derived CHH methylation pathway DMR classifications ( Hsieh et al., 2023 ): DMRs that are regulated by the CMT2 or RdDM pathways in both leaf and embryos are classified as cCMT2 DMRs or cRdDM DMRs, respectively. DMRs that are regulated by CMT2 or unmethylated in the leaf and become newly targeted by the RdDM pathway in embryo are classified as eRdDM DMRs.…”