DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.

The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.