Mobile small RNAs regulate genome-wide DNA methylation.

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.

We thank Matthew D. Schultz and Yupeng He for assistance with analyses; Huaming Chen for assistance with web browser development; and Roberto Solano, Robert J. Schmitz, Taiji Kawakatsu, Chongyuan Luo, Ryan Lister, Ronan C. O’Malley, and Lindsay Robinson for helpful discussions. M.G.L. was funded by an EU Marie Curie FP7 International Outgoing Fellowship (252475). Work in J.R.E.’s laboratory is funded by the Gordon and Betty Moore Foundation (3034) and the National Science Foundation (MCB-1024999). J.R.E. is an investigator of the Howard Hughes Medical Institute. C.W.M. was funded by a Clare College (Cambridge, United Kingdom) Junior Research Fellowship. Work in the D.C.B. laboratory was supported by the Gatsby Charitable Foundation, EU FP7 Collaborative Project Grant AENEAS, and ERC Advanced Investigator Grant ERC-2013-AdG 340642. D.C.B. Q:29 is the Royal Society Edward Penley Abraham Research Professor.

This is the author accepted manuscript. The final version is available from the National Academy of Sciences via http://dx.doi.org/10.1073/pnas.1515072113