Domains rearranged methyltransferase3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis

Abstract

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase domains rearranged methylase 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

Keywords: DNA methylation; RNA polymerase; epigenetic regulation; gene silencing; non-coding RNA.

Fig. 1.

DRM3 has moderate effects on global DNA methylation at RNA-directed DNA methylation targets. (A) Overlap of differentially methylated regions (DMRs) identified in drm3, drm2, and nrpe1 mutants. CHH methylation levels over (B) drm2 hypo-DMRs, (C) drm3 hypo-DMRs, and (D) drm2 hypo-DMRs excluding drm3 hypo-DMRs in WT, drm2, nrpe1, and drm3. The CHH methylation in drm3 is significantly reduced compared with WT (P < 2.2e−16; Wilcoxon signed rank test). CHH methylation levels in drm2 and nrpe1 are both significantly reduced compared with drm3 (P < 2.2e−16; Wilcoxon signed rank test).

Fig. 2.

DRM3 acts downstream of the production of siRNAs. (A) Northern blots show accumulation levels of small RNAs at 5S rDNA, AtSN1, and siRNA1003 loci. The siRNA02 and miR165a serve as two internal controls. (B) Average distribution of 24-nt siRNA reads over drm2 CHH hypo-DMRs. The siRNA abundance is significantly reduced compared with WT in all mutants (P < 2.2e−16; Wilcoxon signed rank test). Levels of 24-nt siRNAs in WT and mutants over previously defined siRNA clusters affected in the “downstream” (C) and “upstream” (D) RdDM mutants. The 24-nt siRNA levels are only significantly reduced in drm3 (P = 4.611e−10; Wilcoxon signed rank test), drm2 (P < 2.2e−16; Wilcoxon signed rank test), and nrpe1 (P < 2.2e−16; Wilcoxon signed rank test) compared with those of WT in the downstream clusters (C).

Fig. 3.

DRM3 is associated with Pol V in vivo. (A) Complementation test of drm3 mutant with pDRM3::HA-DRM3 at the MEA-ISR locus by restriction digestion with methylation-sensitive MspI and Southern blot. Me, methylated DNA; unMe, unmethylated DNA. (B) Coimmunoprecipitation assays confirming DRM3–NRPE1 interaction. Input lanes confirm the expression of the epitope-tagged proteins in the parental lines and F1. F1 represents the first generation from a cross between the two parental lines.

Fig. 4.

DRM2 and DMR3 differentially affect Pol V occupancy. (A) Distribution of log2 ratios of Pol V enrichment in mutant vs. WT over previously defined Pol V sites. The drm2 shows a significantly greater loss of Pol V compared with drm3 (P < 2.2e−16; Wilcoxon signed rank test), and nrpe1 shows a greater reduction than either drm2 or drm3 (P < 2.2e−16; Wilcoxon signed rank test). (B) Scatter plots of the relationship between Pol V ChIP-seq fold-change in nrpe1, drm2, and drm3 mutants relative to WT and the significance of that change (P value; Fisher’s exact test) at defined Pol V binding sites (n = 4,317). P value cutoff (P < 1e−5) used to define Pol V sites with either a neutral change or a significant gain or loss of Pol V for each mutant is represented by a horizontal dotted line. (C) Venn diagrams representing the overlaps of Pol V sites (total = 4,317) that lose or gain Pol V in drm2 and drm3 mutants.

Fig. 5.

Pol V association with chromatin in drm mutants is correlated to DNA methylation levels. (A) Methylation levels for different cytosine contexts in drm2 at Pol V sites classified as experiencing a “loss,” “gain,” or “no change” in Pol V occupancy in drm2. (B) Same representation as A, except methylation levels in a drm3 mutant are shown, and the Pol V sites are classified as experiencing a loss, gain, or no change in Pol V occupancy in drm3. The methylation levels of total methylated C (mC) and methylated CG (mCG) are significantly reduced (P < 2.2e−16; Wilcoxon rank sum test) at loss sites compared with gain or no change sites for both drm2 and drm3. (C) Distribution of R² values of the fit of predicted Pol V signal to the actual observed Pol V ChIP-seq signal using models built upon methylation, cytosine content, and sRNA data derived from each respective genotype. Models were trained using 3/4 of the defined Pol V peaks and tested against the remaining 1/4 of sites. Training and testing were repeated 25 times for each genotype. The WT R² value is significantly less than both the drm2 and drm3 values (P = 5.96e−08; Wilcoxon signed rank test). (D) Distribution of R² values of predicted versus actual Pol V ChIP-seq signal for models trained on drm2 data as in C using the full set of parameters (“Full”) or subtracting one parameter (−mC, removing methylation data for a given context; −C, removing cytosine abundance data for a given context; −sRNA, removing 24-nt sRNA data). Both the −mCG and −sRNA models perform significantly worse than the next worse model (−CHH, P = 5.96e−08; Wilcoxon signed rank test). (E) Distribution of log2 ratios between predicted Pol V ChIP-seq signal in drm2 versus predicted signal in WT at Pol V sites classified as “Loss,” “Gain,” or “no change” in Pol V enrichment in drm2. The model was trained using drm3 data (without mCHH or sRNAs) at Pol V sites, excluding drm2 “Loss” or “Gain” sites. (F) Analogous to E, except that sites tested were classified based on Pol V behavior in drm3 and the model was generated using drm2 data, excluding the drm3 “Loss” or “Gain” sites. The loss category is significantly lower than the no change category (P < 2.2e−16; Wilcoxon rank sum test) for both drm2 and drm3.

Fig. 6.

DRM3 is partially required for Pol V-dependent noncoding RNA transcript accumulations. Quantitative RT-PCR analysis of the abundance of noncoding RNA transcripts from Pol V-enriched loci that either gained or did not lose Pol V occupancy in nrpe1 and drm3 mutants. Transcript levels were analyzed in WT, drm3, and nrpe1 plants and normalized to the levels of ACTIN7. Error bars represent the SD of more than five replicates.