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How different intrinsic sequence variations and regulatory modifications of histones combine in nucleosomes remain unclear. To test the importance of histone variants in the organization of chromatin we investigated how histone variants and histone modifications assemble in the Arabidopsis thaliana genome. We showed that a limited number of chromatin states divide euchromatin and heterochromatin into several subdomains. We found that histone variants are as significant as histone modifications in determining the composition of chromatin states. Particularly strong associations were observed between H2A variants and specific combinations of histone modifications. To study the role of H2A variants in organizing chromatin states we determined the role of the chromatin remodeler DECREASED IN DNA METHYLATION (DDM1) in the organization of chromatin states. We showed that the loss of DDM1 prevented the exchange of the histone variant H2A.Z to H2A.W in constitutive heterochromatin, resulting in significant effects on the definition and distribution of chromatin states in and outside of constitutive heterochromatin. We thus propose that dynamic exchanges of histone variants control the organization of histone modifications into chromatin states, acting as molecular landmarks.

Abstract Background Gene bodies of vertebrates and flowering plants are occupied by the histone variant H3.3 and DNA methylation. The origin and significance of these profiles remain largely unknown. DNA methylation and H3.3 enrichment profiles over gene bodies are correlated and both have a similar dependence on gene transcription levels. This suggests a mechanistic link between H3.3 and gene body methylation. Results We engineered an H3.3 knockdown in Arabidopsis thaliana and observed transcription reduction that predominantly affects genes responsive to environmental cues. When H3.3 levels are reduced, gene bodies show a loss of DNA methylation correlated with transcription levels. To study the origin of changes in DNA methylation profiles when H3.3 levels are reduced, we examined genome-wide distributions of several histone H3 marks, H2A.Z, and linker histone H1. We report that in the absence of H3.3, H1 distribution increases in gene bodies in a transcription-dependent manner. Conclusions We propose that H3.3 prevents recruitment of H1, inhibiting H1’s promotion of chromatin folding that restricts access to DNA methyltransferases responsible for gene body methylation. Thus, gene body methylation is likely shaped by H3.3 dynamics in conjunction with transcriptional activity.