Mobile small RNAs regulate genome-wide DNA methylation

Abstract

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.

Keywords: RNA-directed DNA methylation; plant grafting; small RNA; transcriptional gene silencing; transposable element.

Fig. 1.

Genomic loci in Arabidopsis roots were classified according to their combination of sRNA and DNA methylation status. Grafts were made between shoots and roots of various Arabidopsis genotypes [combinations are indicated (Top), denoted shoot/root]. Target loci of sRNA and DNA methylation were identified by analyzing MethylC-seq and sRNA-seq data from roots of all graft combinations. Each locus classification was defined by a specific combination of sRNA and DNA methylation (mC) levels across the five graft combinations. “+” denotes a relatively high level of mC or sRNA, whereas “-” denotes a relatively low level. Classifications were designated A–C and are indicated (Left). See also SI Appendix, Fig. S1.

Fig. 2.

DNA methylation of mobile sRNA-associated loci (class A, direct; B, indirect) is reduced most strongly by drm1/2 mutation. The plots show the mean proportion of DNA methylation across class A and B loci in mutants of RNA-directed DNA methylation pathway components and wild-type (WT) plants. The legend (Right) indicates which color represents each mutant. Data are shown separately for non-CG (CHG and CHH) sequence contexts, which had the strongest association with mobile sRNAs. The proportion of methylation was calculated per base (between 0 and 1, unmethylated to fully methylated) for all loci within a class using published MethylC-seq data from RdDM mutants (31). Loci were then normalized to the same size, and the mean proportion of DNA methylation was calculated across them. The profiles of the mean proportion of methylation across the size-normalized loci are plotted between the dashed vertical lines (indicated by the solid black bars labeled “loci”). Mean proportions of methylation in flanking DNA, 4 kb upstream and downstream of the loci, are indicated to the left and right of the dashed vertical lines, respectively. Total numbers of loci assessed are given in parentheses.

Fig. 3.

Mobile sRNA directly targets DNA methylation at promoters containing transposable elements (TEs). (A) CHG and CHH (non-CG) context class A loci are enriched in TEs and in promoters containing TEs but are depleted in genes and coding sequences (CDSs). Very few CG context loci were identified. (B) Non-CG context class A loci preferentially target classes of type 1 retroelement, indicated by significant enrichment. They also, less strongly, target some type 2 DNA element classes. y-axis units in A and B normalize the feature/locus overlap by both sum of genome feature size and sum of methylation locus size, which permits comparison between columns. These units are the number of annotated features per total megabase (MB) of named feature per total MB of methylation in the locus class. Numbers of loci in each DNA methylation context are shown in parentheses. Significance levels: #, 0 < P < 10⁻⁵; +, 10⁻⁴ < P < 10⁻⁵; *, 10⁻³ < P < 10⁻⁴; !, 10⁻² < P < 10⁻³; blue, underrepresented;red, overrepresented (relative to background). (C) Class A loci are targeted by graft-transmissible 23- to 24-nt sRNAs (the known mobile class). Loci were normalized to the same size on the graph, indicated by the dark bar on the position axis, flanked by sRNA coverage 4 kb upstream and downstream of the locus. Numbers of loci assessed are given in parentheses.

Fig. 4.

Loci where DNA methylation is indirectly targeted by mobile sRNAs (class B) exhibit similar characteristics to loci that are directly targeted (class A). (A) Non-CG (CHG and CHH) context B loci are significantly enriched in TEs and in promoters containing TEs but are depleted in promoters that do not contain TEs, genes, and CDSs. Very few CG context loci were identified. (B) Non-CG context B loci target most classes of TEs but show most significant association with classes of type 1 retroelement. They also, less strongly, target some type 2 DNA element classes. y-axis units normalize the feature/locus overlap by both sum of feature size and sum of methylation locus size, which permits comparison between columns. These units are the number of annotated features per total MB of named feature per total MB of methylation in the locus class. Significance levels: #, 0 < P < 10⁻⁵; +, 10⁻⁴ < P < 10⁻⁵; *, 10⁻³ < P < 10⁻⁴; !, 10⁻² < P < 10⁻³; blue, underrepresented; red, overrepresented (relative to background). Numbers of loci assessed are given in parentheses.

Fig. 5.

C24-derived sRNAs can target DNA methylation de novo at unmethylated regions of the Col-0 genome. Three such de novo loci are shown in AnnoJ genome browser (www.annoj.org) screenshots. The screenshots display sRNA reads and methylated cytosine residues (mCs) for graft combinations C24/C24, Col/Col, and C24/Col. Results from two independent biological replicates, suffixed 1 and 2, are shown for each graft combination. Note the groups of sRNA reads present only in C24/C24 and C24/Col, which correlate with the presence of mC. Red and green sRNAs indicate that they map to the Watson (W) and Crick (C) strands, respectively. Gold, blue, and pink mC positions indicate methylation in the CG, CHG, and CHH contexts, respectively.