Spatial and functional relationships among Pol V-associated loci, Pol IV-dependent siRNAs, and cytosine methylation in the Arabidopsis epigenome

Abstract

Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.

Figure 1.

A model for RNA-directed DNA methylation in Arabidopsis thaliana. RNA Pol IV transcripts are used as templates by the Pol IV-interacting protein RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). DICER-LIKE 3 (DCL3) cleaves resulting dsRNAs into 24-nt siRNA products, one strand of which is loaded into an ARGONAUTE 4 (AGO4) RISC complex. Independent of siRNA biogenesis, the DDR complex enables transcription by RNA Pol V, whose nascent transcripts serve as scaffolds for the binding of AGO–RISC complexes. AGO4 also interacts with the C-terminal domain of the Pol V largest subunit and RDM1. In turn, RDM1 interacts with the de novo DNA methyltransferase DRM2.

Figure 2.

Pol V ChIP-seq and evidence for Pol V transcription at associated loci. (A) Browser views for a subset of genomic regions associated with Pol V, identified by ChIP-seq. Individual reads corresponding to the two DNA strands are represented by green or red horizontal bars. Annotated genes or transposable elements (TEs) are shown. (B) RT–PCR detection of Pol V-dependent transcripts at the Pol V peak loci shown in A.

Figure 3.

Correlations among Pol V peaks, cytosine methylation, and 24-nt siRNAs. (A) Example of Pol V ChIP-seq, DNA methylation, and small RNA data tracks displayed in the AnnoJ browser for the region including the SINE element AT2TE82000. (B) RT–PCR analysis of AT2TE82000 expression in wild type or an nrpe1 mutant or nrpe1 mutant recused by an NRPE1 transgene. (C) Genomic Pol V peaks were subdivided into five bins, each corresponding to 20% of the peak. The numbers of methylcytosines (in different contexts) or 24-nt siRNAs within each bin are plotted. (D) Pie chart depicting the relationships among 24-nt siRNAs, methylcytosines in CHH motifs, or total methylcytosines within Pol V peaks. (E) The number of Pol V peaks overlapping transposons or genes among the blue or red subclasses of Pol V peaks shown in D (for CHH methylation) is shown.

Figure 4.

Motifs enriched at Pol V peak regions. (A) Complementary sequences that were identified using BioProspector as being overrepresented at Pol V-associated loci. (B) Number of consensus repeats (vertical axis) within the 1157 Pol V peaks (horizontal axis). Peaks overlapping genes encoding proline- or glycine-rich proteins are colored black or rust, respectively. (C) AnnoJ browser tracks of the region surrounding AT1G26250, a gene encoding a proline-rich protein with 115 copies of the CNCCNCCNCC motif. (D) Histone H3 Lys 9 and Lys 14 acetylation at the AT1G26250 and ACTIN2 genes in Col-0, nrpe1, or nrpe1 complemented with an NRPE1-Flag transgene.

Figure 5.

Genome-wide distribution of Pol V and a Pol V-associated motif. The number of Pol V (black line) and Pol V-associated motifs (red line) within a 500-kb fixed window is plotted for each chromosome. Data were normalized relative to the highest value position for each chromosome and smoothed using spline interpolation. Light-blue shading indicates the approximate position of each centromere. Black circles mark the approximate position of heterochromatic knobs on chromosomes 4 and 5.

Figure 6.

Pol IV- and Pol V-dependent small RNAs and DNA methylation. (A) Chromosomal distribution of uniquely mapping small RNAs within a 500-kb sliding window, shown for chromosomes 1 and 5. Wild-type Col-0 and nrpd1, nrpe1, and nrp(d/e)2 mutants are compared. Peak heights are normalized relative to the highest level detected for each chromosome. (B) Relative amounts of methylcytosines in CG, CHG, or CHH contexts (where H represents A, C, or T) in wild-type Col-0 and nrpd1, nrpe1, or nrp(d/e)2 mutants. (C) Venn diagrams comparing the number and genomic positions of methylated cytosines in CG, CHG, or CHH contexts in wild-type Col-0 and nrpd1, nrpe1, or nrp(d/e)2 mutants. (D) Chromosomal distributions of methylated CH, CHG, or CHH sites, shown for chromosomes 1 and 5. Wild-type Col-0 and nrpd1, nrpe1, and nrp(d/e)2 mutants are compared using a 500-kb fixed window size. Peak heights are normalized relative to the highest level detected for each chromosome.