DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons

Abstract

The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution.

Figure 1

Identification of NRPE1 enriched sites by ChIP-seq and characterization of epigenetic marks at those sites. (a) Chromosomal view of NRPE1-FLAG ChIP-seq reads relative to an untagged wild type (WT) control with a schematic representation of each chromosome shown below. The chromosome numbers represent the approximation of the centromere location with the boxes indicating pericentromeric heterochromatin. (b) Heatmaps showing the % methylation levels in all three sequence contexts as well as 24 nucleotide small RNA abundance (reads per bp per million 21 nt mapping reads) for all NRPE1 sites (+/− 2,000 bp from midpoint) in WT (top) and nrpe1 mutants (bottom) as well as a scatter plot showing the relative NRPE1 enrichment at each site. (c) Distribution of the % methylation of the central 100 base pairs of NRPE1 sites in wild type and nrpe1 mutants. (d) Metaplot (+/− 2,000 base pairs from NRPE1 binding midpoints shown by triangle) showing the relative change of epigenetic marks in shown in (b) for nrpe1 mutants. (e) Metaplot showing the changes of RNA-seq reads in nrpe1 mutants relative to WT.

Figure 2

The DDR complex is required for stable Pol V association with chromatin. (a) Heat map of NRPE1 enrichment at sites defined in ChIP-seq experiments in wild type and mutants. The genotype of each library is indicated at the far right side. (b) Boxplot (whiskers extend to +/− 1.5 inter-quartile range (IQR)) of NRPE1 enrichment at sites shown in (a) for various genotypes. (c) Quantitative RT-PCR analysis of transcripts originating from NRPE1 enrichment sites. IGN22 is a previously published Pol V target, P2 to P9 are newly identified Pol V binding sites and NP is a non-NRPE1 enrichment region. (d) Quantitative RT-PCR analysis of transcripts originating from NRPE1 enrichment sites in nrpe1, drd1 and dms3 mutants. Error bars represent the standard deviation of three biological replicates.

Figure 3

NRPE1 is enriched at gene promoters. (a) Relative enrichment of the observed overlap between NRPE1 sites and gene features compared to the average overlap of 10,000 genome-shuffled experiments. (b) Metaplots for WT and nrpe1 genomes of DNA methylation for each cytosine context at NRPE1-associated promoters and non-NRPE1-associated promoters. (c–d) Metaplots (c) and heatmaps (d) of ChIP-seq reads at NRPE1-associated promoters versus non-NRPE1-associated promoters. For each panel black triangles denote the transcriptional start site (TSS) with plots extending + or − 2,000 bp upstream and downstream.

Figure 4

NRPE1 is enriched at the intersection of promoters and transposons. (a) NRPE1-associated promoters are enriched for promoters overlapping with transposons (P<2.2e-16, Fisher’s Exact Test). (b) Metaplot showing NRPE1 enrichment at transposon-proximal (within 1 kb) and -distal (>1 kb) protein coding genes for ± 2,000 bp upstream and downstream and over the gene body (shown in % coverage of gene 5′ to 3′). (c) NRPE1-associated transposons (those overlapping with an NRPE1 site) are enriched for gene-proximal transposons (P<2.2e-16, Fisher’s Exact Test). (d) Metplots of NRPE1 enrichment at NRPE1-associated transposons organized by size class.

Figure 5

Loss of NRPE1 causes changes in protein coding gene expression. (a) Boxplots (whiskers extend +/−1.5 IQR) of log2 ratios of normalized RNA-seq read counts for nrpe1 mutants over those for wild type plants for protein coding genes with an NRPE1 site in their promoter. Each boxplot represents a subclass of those genes based on the distance between the TSS and the NRPE1 site. * indicates P<0.05 (Mann-Whitney Test). (b) Metaplot showing the normalized RNA-seq reads for different RdDM mutants at and around the TSS of protein coding genes with an NRPE1 site within 50 bp upstream of the TSS.

Figure 6

NRPE1 is enriched at transposons that are relatively new in the A. thaliana genome. (a) NRPE1-associated transposons are enriched for transposons unique to A. thaliana (P<2.2e-16, Fisher’s Exact Test). (b) Relative transposon abundance (transposons per promoter/total number transposons of that type) at NRPE1-associated (+) and non-NRPE1-associated (−) promoters. (c) Metaplot showing the ChIP-seq read ratios over unique and ancient transposons. (d) Log2 ratio of RNA-seq reads in nrpe1 mutants compared to WT. (e) NRPE1 is significantly enriched at “unique” transposons found at promoters as compared to either all promoter-associated transposons or “ancient” promoter-associated transposons (P<2.2e-16, Mann-Whitney Test).