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. 2016 Dec 1;30(23):2565-2570.
doi: 10.1101/gad.289553.116. Epub 2016 Dec 16.

Evidence for ARGONAUTE4-DNA Interactions in RNA-directed DNA Methylation in Plants

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Free PMC article

Evidence for ARGONAUTE4-DNA Interactions in RNA-directed DNA Methylation in Plants

Sylvie Lahmy et al. Genes Dev. .
Free PMC article

Abstract

RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4-DNA interaction could be responsible for the unique specificities of RdDM.

Keywords: Argonaute; DNA methylation; RdDM.

Figures

Figure 1.
Figure 1.
Pol V and SPT5L AGO hook motifs are functionally redundant and essential for RdDM. (A, left panel) Analysis of DNA methylation by Chop PCR at SB2-2 and soloLTR loci in Col-0, nrpe1, Pol V+(1,2), and Pol V(1,2) lines. Genomic DNA digested with HaeIII, DdeI, and AluI methylation-sensitive enzymes was used as a template for PCR. Undigested DNA (input) was used as a control. (Right panel) Analysis of DNA methylation by Chop PCR at RdDM targets in nrpe1, spt5l, Col-0, SPT+(1,2), and truncated SPT(1,2) lines. Genomic DNA digested with AluI and HaeIII methylation-sensitive enzymes was used as a template for PCR. The At2g19920 locus was used as a control. (B, top panel) Analysis of DNA methylation by Chop PCR at RdDM targets in Col-0, spt5l, nrpe1, Pol V/SPT, SPT(1), and Pol V(2) lines. Genomic DNA was digested with HaeIII and AluI methylation-sensitive enzymes. The At2g19920 locus was used as a control. (Middle panel) Transcript levels at two RdDM targets (IGLINE and AT3g48131) in Col-0, spt5l, nrpe1, Pol V/SPT, SPT(1), and Pol V(2) lines. ACTIN2 was used as a loading control, and −RT reactions show the absence of genomic DNA contamination. (Bottom panel) Detection of Pol V/NRPE1 and SPT variants by Western blot. Coomassie blue staining was used as a loading control. (C) Whole-genome bisulfite analysis of DNA methylation in Col-0, nrpe1, spt5l, Pol V(2), Pol V+(2), SPT(1), SPT+(1), Pol V/SPT, and Pol V+/SPT lines. Box plots represent whole-genome CHH methylation ratios at regions previously identified as targeted by Pol V (Zhong et al. 2012). A dependent two-group Wilcoxon signed rank test was run on methylation at the Pol V sites for Pol V/SPT versus Pol V+/SPT and revealed that there is a significant difference in methylation levels. P < 2.2 × 10−16.
Figure 2.
Figure 2.
AGO hook motifs are essential determinants of AGO4 recruitment to RdDM loci. (A) ChIP analysis of Pol V (top panel) and AGO4 (bottom panel) binding in nrpe1, Pol V(2), and SPT(1) complemented lines and a Pol V/SPT cross line. The tested targets are indicated at the right. Actin2 and Actin7 were used as negative controls. After formaldehyde cross-link, Pol V complexes were immunoprecipitated using either the anti-NRPE5 or anti-AGO4 antibodies. Values are means ± SD from two independent amplifications. (B, top panel) Quantitative RT–PCR (qRT–PCR) analysis of Pol V lncRNA levels. IGN5, P9, IGN22, IGN23, and IGN27 transcript accumulation was tested in nrpe1, Pol V(2), and Pol V/SPT lines. (Rel. Tran. Lev.) Relative transcript level normalized to Actin and Pol V(2) using the ΔΔCt method. (Bottom panel) Analysis of DNA methylation by Chop PCR at IGN23, IGN25, and IGN27 loci. Genomic DNA was digested with HaeIII or MscI methylation-sensitive enzymes and used as a template for PCR. The At2g19920 locus has no HaeIII site and was used as a control (cont). DNA methylation was assessed in Col-0, nrpe1, Pol V(2), and Pol V/SPT lines.
Figure 3.
Figure 3.
Pol V lncRNAs or the act of their transcription primarily mediate the association of Pol V to chromatin. (A) Amino acid sequences of the catalytic sites (metal A) of different polymerase subunits from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli. (B) qRT–PCR analysis of Pol V lncRNA levels in Col-0 and nrpe1-11 and nrpe1-3 mutants. (Rel. Tran. Lev.) Relative transcript level. Values represent the means ± SD of three independent experiments. (C) ChIP analysis of Pol V binding in nrpe1-11 and nrpe1-3 mutants at different RdDM targets, as indicated at the right. Actin2 and Actin7 were used as controls. Values are means ± SD of two independent amplifications. After formaldehyde cross-link, Pol V complexes were immunoprecipitated using either the anti-NRPE5 or anti-NRPE1 antibody.
Figure 4.
Figure 4.
Detection of AGO4–DNA interactions at RdDM loci by LChIP. (A) Scheme of LChIP. (B) LChIP analysis of Pol V binding in a Pol V+-complemented line. Nonirradiated (NI) or irradiated [I (UV)] nuclei were used to prepare chromatin. ChIP was then performed using anti-Flag antibodies. The RdDM target loci tested are indicated at the right. Cont1 and Cont2 correspond to two independent regions located 2 kb from the At4g04920 gene and represent negative controls. Values of DNA enrichment were calculated as percentage of input and were normalized to At5g52070. Error bars are SEM of three independent ChIP experiments. (C) LChIP analysis of mAGO4 binding in an E1/mAGO4-complemented line. Nonirradiated (NI) or irradiated [I (UV)] nuclei were used to prepare chromatin. ChIP was then performed using anti-myc antibodies. The RdDM target loci tested are indicated at the right. Cont1 and Cont2 represent negative controls. Values of DNA enrichment were calculated as percentage input and were normalized to At5g52070. Error bars are SEM of three independent ChIP experiments. (D) LChIP analysis of mAGO4 binding in E1/mAGO4-complemented versus e1/mAGO4-complemented lines. ChIP was then performed using anti-myc antibodies on irradiated nuclei. The RdDM target loci tested are indicated at the right. Cont1 and Cont2 correspond to two independent regions located 2 kb from the At4g04920 gene and represent negative controls. Values of DNA enrichment were calculated as the percentage of input and were normalized to Cont1. Error bars are SEM of three independent ChIP experiments. (*) P < 0.05 compared with Cont1. n = 3.
Figure 5.
Figure 5.
Revisited model for Pol V and AGO4-dependent RNA-mediated DNA methylation. The Pol V and SPT5L AGO hook platforms are essential determinants of AGO4 recruitment to chromatin at RdDM loci. (Right) The DDR complex interacts with Pol V and triggers a local DNA unwinding that is proposed to facilitate Pol V recruitment at RdDM targets. (Middle) Further stabilization of Pol V on single-stranded target DNA requires active transcription and the formation of a DNA–RNA hybrid in the transcription bubble. (Model A) As Pol V elongates, AGO4 would interact with Pol V lncRNAs, likely generating a metastable multimeric complex that would serve as the intermediate for the transfer of AGO4 to the DNA template. (Model B) Alternatively, one could imagine that the pool of AGO4–siRNA effector complexes associated with the AGO hook platforms would directly inspect both DNA strands for complementary base pairing as Pol V proceeds through transcription elongation. Following the interaction with the complementary ssDNA, AGO4–siRNA complexes would recruit DRM2 to the opposite siRNA-like DNA strand for DNA methylation.

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